Topical application of glycyrrhetinic acid in the gingival sulcus inhibits attachment loss in lipopolysaccharide-induced experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Attachment loss of the junctional epithelium and alveolar bone destruction are signs of periodontitis, which is mainly caused by an inflammatory response to dental plaque. Glycyrrhetinic acid (GA), a component of the licorice herb, has been shown to have important anti-inflammatory activities; however, there are no previous reports on the ability of its inhibitory effects to prevent periodontal diseases. Hence, in this study, using our experimental periodontitis model, we attempted to evaluate whether GA had an effect on the prevention of attachment loss and alveolar bone loss. MATERIAL AND METHODS: Rats were intraperitoneally immunized with Escherichia coli lipopolysaccharide (LPS). The LPS group (n = 5) received 3 topical applications of 50 µg/µL of LPS followed by one application of the vehicle (propylene glycol:ethyl alcohol:phosphate-buffered saline [PBS] = 8:1:1) into the gingival sulcus. This protocol was repeated twice per day for 10 days. The low (n = 5) and high (n = 5) groups received topical application of LPS and 0.03% or 0.3% GA, respectively. The control group received topical application of PBS and vehicle. The rats were killed on the 10th day. Attachment loss, alveolar bone level and inflammatory cell infiltration were investigated histometrically. The formation of immune complexes and infiltration of LPS were evaluated immunohistologically. RESULTS: Attachment loss, formation of immune complexes and infiltration of inflammatory cells were increased in the LPS group compared with the control group, and were completely inhibited in the low and high groups compared with the LPS group. The LPS group showed greater alveolar bone destruction compared with the control group and GA-treated groups. In addition, invasion of LPS was detected in the LPS group, was absent in the control group and was weaker in the GA-treated groups than in the LPS group. CONCLUSION: In the present study, we showed that GA inhibits periodontal destruction in the rat experimental periodontitis model.

Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

BACKGROUND AND OBJECTIVE: Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1ß precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. MATERIAL AND METHODS: HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. RESULTS: Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental calculus was significantly inhibited by cytochalasin D, z-YVAD-fmk and glyburide, indicating NLRP3 inflammasome involvement. In permeability assays, dental calculus attenuated the barrier function of HSC-2 cell monolayers. CONCLUSION: Dental calculus induces pyroptotic cell death in human oral epithelial cells and the crystalline structure plays a major role in this process. Oral epithelial cell death induced by dental calculus might be important for the etiology of periodontitis.

The histopathological comparison on the destruction of the periodontal tissue between normal junctional epithelium and long junctional epithelium.

BACKGROUND AND OBJECTIVE: The barrier function of long junctional epithelium is thought to be important after periodontal initial therapy and periodontal surgery. Although the difference between long junctional epithelium and normal junctional epithelium regarding their resistance to destruction of periodontal tissue has been investigated, the mechanism still remains unclear. Using our rat experimental periodontitis model in which loss of attachment and resorption of alveolar bone is induced by the formation of immune complexes, we investigated the resistance of periodontal tissue containing long junctional epithelium and normal junctional epithelium to destruction. MATERIAL AND METHODS: Rats were divided into four groups. In the immunized long junctional epithelium (I-LJE) group, rats were immunized with lipopolysaccharide (LPS), and curettage and root planing procedures were performed on the palatal gingiva of the maxillary first molars to obtain reattachment by long junctional epithelium. In the immunized normal junctional epithelium (I-JE) group, rats were immunized without curettage and root planing procedures. In the nonimmunized long junctional epithelium (nI-LJE) group, rats were not immunized but curettage and root-planing procedures were performed. In the control group, neither immunization nor curettage and root-planing was performed. In all rats, periodontal inflammation was induced by topical application of LPS into the palatal gingival sulcus of maxillary first molars. The rats were killed at baseline and after the third and fifth applications of LPS. Attachment loss and the number of inflammatory cells and osteoclasts in the four groups were compared histopathologically and histometrically. RESULTS: After the third application of LPS in the I-LJE group, attachment loss showed a greater increase than in control and nI-LJE groups, and inflammatory cell infiltration and osteoclasts were increased more than in the other groups. After the fifth application of LPS, attachment loss was greater and there was a higher degree of inflammatory cell infiltration in nI-LJE and I-LJE groups than in control and I-JE groups. CONCLUSION: Our findings suggest that the destruction of periodontal tissue is increased in tissue containing long junctional epithelium compared with normal junctional epithelium and that the immunized condition accelerates the destruction by forming immune complexes.

Green tea extract inhibits the onset of periodontal destruction in rat experimental periodontitis.

BACKGROUND AND OBJECTIVE: Green tea extract exerts a variety of biological effects, including anti-inflammatory activities. However, there has been no report on the effect of green tea extract on loss of attachment, which is an important characteristic of periodontitis. Here, we examined the inhibitory effects of green tea extract on the onset of periodontitis in a rat model. MATERIAL AND METHODS: Rats were immunized intraperitoneally with Escherichia coli lipopolysaccharide (LPS). The LPS group (n = 12) received a topical application of LPS onto the palatal gingival sulcus every 24 h. The green tea extract group (n = 12) received a topical application of LPS mixed with green tea extract, sunphenon BG, every 24 h. The phosphate-buffered saline (PBS) group (n = 6) received a topical application of PBS every 24 h. The levels of anti-LPS immunoglobulin G (IgG) in serum were determined using ELISA. Rats in the LPS and green tea extract groups were killed after the 10th and 20th applications. Rats in the PBS group were killed after the 20th application. Loss of attachment, level of alveolar bone and inflammatory cell infiltration were investigated histopathologically and histometrically. RANKL-positive cells and the formation of immune complexes were evaluated immunohistologically. RESULTS: There was no significant difference in the serum levels of anti-LPS IgG between the LPS group and the green tea extract group. In contrast, loss of attachment, level of alveolar bone, inflammatory cell infiltration and RANKL expression in the green tea extract group were significantly decreased compared with those in the LPS group. CONCLUSION: These findings demonstrate that green tea extract suppresses the onset of loss of attachment and alveolar bone resorption in a rat model of experimental periodontitis.

Gram-positive bacteria as an antigen topically applied into gingival sulcus of immunized rat accelerates periodontal destruction.

BACKGROUND AND OBJECTIVE: Periodontitis is generally accepted to relate to gram-negative bacteria, and the host defense system influences its onset and progression. However, little is known about the relation between gram-positive bacteria and periodontitis. In this study, we topically applied gram-positive and gram-negative bacterial suspensions to the gingival sulcus in rats after immunization, and then histopathologically examined their influence on periodontal destruction. MATERIALS AND METHODS: Rats previously immunized with heat-treated and sonicated Staphylococcus aureus or Aggregatibacter actinomycetemcomitans were used as immunized groups. The non-immunized group received only sterile phosphate-buffered saline. In each animal, S. aureus or A. actinomycetemcomitans suspension was applied topically to the palatal gingival sulcus of first molars every 24 h for 10 d. Blood samples were collected and the serum level of anti-S. aureus or anti-A. actinomycetemcomitans immunoglobulin G (IgG) antibodies was determined by enzyme-linked immunosorbent assay. The first molar regions were resected and observed histopathologically. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). The formation of immune complexes was confirmed by immunohistological staining of C1qB. RESULTS: Serum levels of anti-S. aureus and anti-A. actinomycetemcomitans IgG antibodies in the immunized groups were significantly higher than those in the non-immunized groups were. The loss of attachment, increase in apical migration of the junctional epithelium, and decreases in alveolar bone level and number of TRAP-positive multinuclear cells in each immunized group were significantly greater than in each non-immunized group. The presence of C1qB was observed in the junctional epithelium and adjacent connective tissue in the immunized groups. CONCLUSIONS: Heat-treated and sonicated S. aureus and A. actinomycetemcomitans induced attachment loss in rats immunized with their suspensions. Our results suggest that not only gram-negative but also gram-positive bacteria are able to induce periodontal destruction.

The formation of immune complexes is involved in the acute phase of periodontal destruction in rats.

BACKGROUND AND OBJECTIVE: Loss of clinical attachment and alveolar bone destruction are major symptoms of periodontitis, caused by not only the destructive effect of periodontopathic bacteria but also the overactive response of the host immune system against periodontal pathogens. The details of the participation of the immune system in the onset and progression of periodontitis are unclear. In this study, we attempted to determine whether the host immune system, and in particular the formation of immune complexes, is involved in the periodontal destruction. MATERIAL AND METHODS: We applied ovalbumin or lipopolysaccharide (LPS) as antigens and their specific immunoglobulin G (IgG) antibodies purified from rat serum to rat gingival sulcus alternately. Loss of attachment, alveolar bone destruction and the numbers of inflammatory cells infiltrating the periodontal tissue and osteoclasts on the alveolar bone surface were investigated histometrically. The formation of immune complex was confirmed by immunohistological staining of complement C1qB. RESULTS: Loss of attachment and the presence of C1qB were observed histopathologically in both experimental groups. The group that had been treated with LPS and anti-LPS IgG showed greater loss of attachment. The number of inflammatory cells in the periodontal tissue was increased in both experimental groups, while osteoclasts at the alveolar bone crest were observed only in the group that had been treated with LPS and anti-LPS IgG. CONCLUSION: In the present study, we showed that the formation of immune complex appears to be involved in the acute phase of periodontal destruction and that the biological activity of antigens is also important.

Peptidoglycan and lipopolysaccharide synergistically enhance bone resorption and osteoclastogenesis.

BACKGROUND AND OBJECTIVE: Peptidoglycan (PGN) and lipopolysaccharide (LPS) are bacterial cell wall constituents that are able to induce bone resorption by stimulating Toll-like receptor (TLR) 2 and TLR4, respectively. The fragments of PGN also stimulate inflammatory responses via nucleotide-binding oligomerization domain (NOD) 1 and NOD2, although there are differences in the NOD-stimulatory activities between gram-positive and gram-negative PGNs. The TLR and NOD signaling pathways are known to engage in cross-talk to enhance the production of inflammatory cytokines. In the present study, we investigated the effects of gram-negative and gram-positive PGNs on bone resorption and osteoclastogenesis in the presence or absence of LPS. MATERIAL AND METHODS: We injected Escherichia coli PGN or Staphylococcus aureus PGN with or without LPS into mouse gingiva, and histopathologically assessed alveolar bone resorption by tartrate-resistant acid phosphatase staining. We also stimulated osteoclast precursors from mouse bone marrow macrophages with these PGNs in vitro and assessed osteoclastogenesis. The cells were also stimulated with synthetic ligands for NOD1; γ-D-glutamyl-meso-DAP NOD2; muramyl dipeptide or TLR2; Pam(3) CSK(4) with or without LPS to analyse the signaling cross-talk. RESULTS: S. aureus PGN, but not E. coli PGN, induced alveolar bone resorption, as did LPS. However, PGN from both sources significantly enhanced the bone resorption in the mice co-injected with LPS. Both types of PGNs induced osteoclastogenesis and accelerated osteoclastogenesis when the cells were co-stimulated with LPS in vitro. All synthetic ligands synergistically induced osteoclastogenesis by co-stimulation with LPS. CONCLUSION: Gram-positive or gram-negative PGN worked synergistically with LPS to induce bone resorption and osteoclastogenesis, possibly by co-ordinating the effects of TLR2, NOD1, NOD2 and TLR4 signaling.

Topical application of lipopolysaccharide into gingival sulcus promotes periodontal destruction in rats immunized with lipopolysaccharide.

BACKGROUND AND OBJECTIVE: The causes of periodontitis are bacteria and the host immune system, but the role of the immune system in the onset and progression of periodontal disease is still unclear. Our previous report showed that the formation of an immune complex in the gingival sulcus induces periodontal destruction. This study was carried out to investigate how the immune system, particularly immunization, is involved in periodontal destruction. MATERIAL AND METHODS: Animals immunized intraperitoneally with lipopolysaccharide (LPS) were used as the immunized group. The nonimmunized group received only phosphate-buffered saline. LPS was applied daily onto the palatal gingival sulcus in both groups 1 d after the booster injection. Serum levels of anti-LPS IgG were determined. Loss of attachment and the level of alveolar bone were histopathologically and histometrically investigated. RANKL-bearing cells and the expression of C1qB were immunohistologically evaluated. RESULTS: The serum levels of anti-LPS IgG were elevated in the early experimental period in the immunized group. There were significant increases in loss of attachment, level of alveolar bone and the number of RANKL-bearing cells in the immunized group. C1qB was observed in the junctional epithelium and adjacent connective tissue. The nonimmunized group showed similar findings at and after the time when the serum level of anti-LPS IgG was elevated. CONCLUSION: Topical application of LPS as an antigen induced periodontal destruction when the serum level of anti-LPS IgG was elevated in rats immunized with LPS. The presence of C1qB suggests that the formation of immune complexes is involved in this destruction.

Locally administered interferon-γ accelerates lipopolysaccharide-induced osteoclastogenesis independent of immunohistological RANKL upregulation.

BACKGROUND AND OBJECTIVE: Interferon-γ (IFN-γ) potently inhibits RANKL-induced osteoclastogenesis in vitro. In contrast, previous studies have shown that an increase in IFN-γ expression is correlated with an increase in lipopolysaccharide (LPS)-induced bone loss in vivo. However, it is not clear whether local IFN-γ accelerates osteoclastogenesis or not in vivo. Therefore, the aim of this study was to clarify the role of local IFN-γ in LPS-induced osteoclastogenesis. MATERIALS AND METHODS: We induced bone loss in calvaria by injecting LPS. One group of mice received an IFN-γ injection together with LPS injection, while another group received IFN-γ 2 d after LPS injection. Bone resorption was observed histologically. Next, we stimulated murine bone marrow macrophages with macrophage-colony stimulating factor and RANKL in vitro. We added different doses of IFN-γ and/or LPS at 0 or 48 h time points. Cells were stained with tartrate-resistant acid phosphatase at 72 h. RESULTS: Local administration of IFN-γ together with LPS injection did not affect osteoclast formation. However, IFN-γ injected after LPS injection accelerated osteoclast formation. Also, we confirmed that IFN-γ added at 0 h inhibited RANKL-induced osteoclastogenesis in vitro. However, inhibition by IFN-γ added at 48 h was reduced compared with that by IFN-γ added at 0 h. Interestingly, IFN-γ together with a low concentration of LPS accelerated osteoclast formation when both were added at 48 h compared with no addition of IFN-γ. CONCLUSION: The results suggest that local IFN-γ accelerates osteoclastogenesis in certain conditions of LPS-induced inflammatory bone loss.

Membrane-bound CD40 ligand on T cells from mice injected with lipopolysaccharide accelerates lipopolysaccharide-induced osteoclastogenesis.

BACKGROUND AND OBJECTIVE: T cells infiltrate the inflammatory site of periodontitis and consequently stimulate the loss of periodontal bone. We previously reported that T cells from lipopolysaccharide (LPS)-injected mice (LPS-T cells) accelerated osteoclastogenesis in the presence of LPS. Ηowever, the detailed mechanism of this acceleration is still unclear. In this study, we analyzed the mechanism of osteoclastogenesis accelerated by LPS-T cells. MATERIAL AND METHODS: We examined the mechanism of osteoclastogenesis acceleration. First, to determine the effect of cell-to-cell contact, we co-cultured T cells and bone marrow macrophages, prestimulated with RANKL for 48 h (R-BMMs), in the presence of LPS for 24 h, in a Transwell. Second, to determine the effect of CD40 ligand (CD40L), we co-cultured T cells and R-BMMs in the presence of LPS and anti-CD40L immunoglobulin. Third, we examined the effect of recombinant mouse CD40L (rCD40L) in the presence of LPS in vitro and in vivo. Lastly, we examined the expression of membrane-bound CD40L (mCD40L) by fluorescence-activated cell sorting (FACS). RESULTS: Blocking cell-to-cell contact between LPS-T cells and R-BMMs completely inhibited the acceleration of osteoclastogenesis. Anti-CD40L immunoglobulin also completely inhibited the acceleration of osteoclastogenesis. Moreover, rCD40L accelerated osteoclastogenesis in the presence of LPS in vitro and in vivo. Finally, the expression of mCD40L on LPS-T cells was higher than that on T cells isolated from mice not injected with LPS. CONCLUSION: The results demonstrate that CD40L accelerates osteoclastogenesis in the presence of RANKL and LPS. The results also suggest that mCD40L on LPS-T cells accelerates osteoclastogenesis.

Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo.

BACKGROUND AND OBJECTIVE: Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption. MATERIAL AND METHODS: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. RESULTS: The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium. CONCLUSION: These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

T cells are able to promote lipopolysaccharide-induced bone resorption in mice in the absence of B cells.

BACKGROUND AND OBJECTIVE: T cells and their cytokines are believed to be key factors in periodontal disease and bone resorption. We previously showed that T cells transferred to nude mice were related to inflammatory bone resorption in vivo. However, it has not been clarified whether T cells can induce bone resorption in the absence of B cells. In this study, we therefore investigated the ability of T cells to induce bone resorption without B cells, using both T cell- and B cell-deficient mice with severe combined immune deficiency (SCID). MATERIAL AND METHODS: Escherichia coli lipopolysaccharide (LPS) was injected into the gingivae of SCID mice reconstituted by T cells (SCID + T mice). Wild-type C.B-17 mice and SCID mice were used as control animals. Alveolar bone resorption and production of cytokines in the gingivae were then compared histopathologically and immunohistologically. RESULTS: The degree of bone resorption in SCID + T mice was significantly greater than that in SCID mice but less than that in wild-type mice. The same tendency was found for expression of receptor activator of nuclear factor kappaB ligand. The number of interferon-gamma-positive cells in SCID + T mice was the highest of the three groups. In contrast, interleukin-4-positive cells were detected in wild-type mice but not in SCID + T and SCID mice. CONCLUSION: The results suggest that T cells are able to promote LPS-induced bone resorption in the absence of B cells. The expressions of cytokines in the presence of B cells are quite different.

Upregulation of functional beta(3)-adrenergic receptor in the failing canine myocardium.

Altered expression and functional responses to cardiac beta(3)-adrenergic receptors (ARs) may contribute to progressive cardiac dysfunction in heart failure (CHF). We compared myocyte beta(3)-AR mRNA and protein levels and myocyte contractile, [Ca(2+)](i) transient, and Ca(2+) current (I(Ca,L)) responses to BRL-37344 (BRL, 10(-8) mol/L), a selective beta(3)-AR agonist, in 9 instrumented dogs before and after pacing-induced CHF. Myocytes were isolated from left ventricular myocardium biopsy tissues. Using reverse transcription-polymerase chain reaction, we detected beta(3)-AR mRNA from myocyte total RNA in each animal. Using a cloned canine beta(3)-AR cDNA probe and myocyte poly A(+) RNA, we detected a single band about 3.4 kb in normal and CHF myocytes. beta(3)-AR protein was detected by Western blot. beta(3)-AR mRNA and protein levels were significantly greater in CHF myocytes than in normal myocytes. Importantly, these changes were associated with enhanced beta(3)-AR-mediated negative modulation on myocyte contractile response and [Ca(2+)](i) regulation. Compared with normal myocytes, CHF myocytes had much greater decreases in the velocity of shortening and relengthening with BRL accompanied by larger reductions in the peak systolic [Ca(2+)](i) transient and I(Ca,L). These responses were not modified by pretreating myocytes with metoprolol (a beta(1)-AR antagonist) or nadolol (a beta(1)- and beta(2)-AR antagonist), but were nearly prevented by bupranolol or L-748,337 (beta(3)-AR antagonists). We conclude that in dogs with pacing-induced CHF, beta(3)-AR gene expression and protein levels are upregulated, and the functional response to beta(3)-AR stimulation is increased. This may contribute to progression of cardiac dysfunction in CHF.

MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation.

MKT-077 (formerly known as FJ-776) is a newly synthesized, highly water-soluble ( > 200 mg/ml) rhodacyanine dye that exhibits significant antitumor activity in a variety of model systems. In culture, MKT-077 inhibits the growth of five human cancer cell lines (colon carcinoma CX-1, breast carcinoma MCF-7, pancreatic carcinoma (CRL 1420, bladder transitional cell carcinoma EJ, and melanoma LOX) but not monkey kidney CV-1, an indicator cell line for normal epithelial cells. In nude mice, MKT-077 inhibits the growth of s.c. implanted human renal carcinoma A498 and human prostate carcinoma DU145 and prolongs the survival of mice bearing i.p. implanted human melanoma LOX (tumor:control = 344%). Subcellular localization indicates that MKT-077 is taken up and retained by mitochondria, and flow cytometric analysis suggests that CX-1 cells take up MKT-077 to a much greater extent than CV-1 cells. Quantitation of MKT-077 uptake by ethanol extraction shows that CX-1 cells accumulate 65-fold more MKT-077 than do CV-1 cells. MKT-077 is the first delocalized lipophilic cation with a favorable pharmacological and toxicological profile in preclinical studies. MKT-077 is now being investigated in Phase I clinical trials.

Allopurinol enhances the contractile response to dobutamine and exercise in dogs with pacing-induced heart failure.

BACKGROUND: Superoxide (O(2)(-)) generated by enhanced xanthine oxidase (XO) activity may contribute to the increased myocardial oxidative stress in heart failure (CHF). Because blocking XO with allopurinol augments myofilament Ca(2+) sensitivity in reperfusion injury and CHF, we hypothesized that it may improve adrenergic inotropic responsiveness in CHF. METHODS AND RESULTS: We studied the effect of allopurinol on the contractile response to dobutamine and exercise in 7 chronically instrumented conscious dogs before and after producing CHF by rapid pacing. Left ventricular (LV) contractile performance was measured by the slopes of the LV end-systolic pressure-volume relation (E(ES)) and stroke work-end-diastolic volume relation (M(SW)). Before CHF, allopurinol produced no change in LV contractile performance and did not alter the response to dobutamine or exercise. After CHF, allopurinol produced significant (P:<0.05) increases in E(ES) (5.0+/-0.6 versus 3.3+/-0.6 mm Hg/mL) and M(SW). Dobutamine and allopurinol produced greater increases in E(ES) (5.4+/-0.6 versus 7.4+/-0.6 mm Hg/mL) and M(SW) (60.1+/-7.4 versus 73.7+/-4.4 mm Hg) than did dobutamine alone. After allopurinol, dP/dt(max), stroke volume, and M(SW) were higher during CHF exercise. LV diastolic pressures were lower during CHF exercise after allopurinol. CONCLUSIONS: Allopurinol has no discernable effects on LV contractile function or adrenergic responsiveness in normal, conscious animals. In pacing-induced CHF, however, allopurinol improves LV systolic function at rest and during adrenergic stimulation and exercise.

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity.

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.

Structure-activity of novel rhodacyanine dyes as antitumor agents.

We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine) was an essential moiety for antitumor activity. On the basis of our foregoing work, two types of rhodacyanine dyes, which categorized into class I and II depending on the methine length, were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused on the structure-activity study of two heteroaromatic rings. In class I, where the A rings were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a benzothiazole moiety for the A ring, exhibited the favorable biological and chemical properties. Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal moiety for the B ring (compound 13). Further, the variation of counteranion had a profound effect on solubility in water without influence on antitumor activity. Chloride anion was selected as the favorable anion with respect to synthetic method as well as solubility in water. Our study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3-ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothi azolidin-2 -ylidene]methyl]pyridinium chloride) as the candidate for clinical trials and is currently subjected to further investigation as a potent antitumor agent in phase I clinical trial for the treatment of solid tumors.

Pulmonary blood flow regulates plasma tissue plasminogen activator concentrations in patients with congenital heart defects.

OBJECTIVE: The wall shear stress generated by blood flow regulates the expression of fibrinolytic proteins by endothelial cells in vitro. In the present study, the effects of pulmonary blood flow on fibrinolytic activity were studied in patients with congenital heart defects and pulmonary hypertension. METHODS: Twenty-seven patients who underwent cardiac operation because of congenital heart defects were divided into four groups according to the severity of pulmonary hypertension. Group I consisted of seven patients with normal pulmonary artery pressure, group II consisted of nine patients with pulmonary hypertension caused by increased pulmonary blood flow, group III consisted of six patients with pulmonary hypertension caused by increased pulmonary vascular resistance, and group IV consisted of five patients with tetralogy of Fallot. Plasma concentrations of tissue plasminogen activator, plasmin, and thrombin were assayed as the inhibitor-bound forms. RESULTS: The preoperative concentration of tissue plasminogen activator was higher in group II than in all other groups (p = 0.0003). However, the postoperative concentration decreased only in patients in group II when compared with the preoperative value (p = 0.01). By Pearson's correlation analysis, pulmonary blood flow was found to correlate with the preoperative concentration of tissue plasminogen activator (95% confidence interval = 3.99 to 10.58, p = 0.0001). No definite conclusion was found for the relationship between tissue plasminogen activator and plasmin concentration. Further, the preoperative thrombin concentration was similar in all groups. CONCLUSIONS: These findings suggest that pulmonary blood flow may regulate the plasma concentration of tissue plasminogen activator in patients with congenital heart defects.

Characteristics of various film cooling jets injected in a conduit.

In the present study, film cooling characteristics by the jets through various easy-to-make straight holes and slots have been investigated. In this experiment, seven kinds of injection geometries were used. They were circular, rectangular, elliptic and oval holes and slots, respectively.

Elevation of serum pancreatic secretory trypsin inhibitor following serious injury.

Twenty-six of 31 seriously injured patients (84%) showed a marked elevation of serum pancreatic secretory trypsin inhibitor (PSTI) to more than twice the initial level within the first 2 weeks after admission. Serum PSTI rose from the second or third post-traumatic day and reached the maximum at day 5.8 on average. In uneventful cases, it returned to the level on admission within 2 weeks. The maximum serum PSTI in these patients was significantly correlated with the severity of the injury as judged at the time of admission, indicating that the elevation of serum PSTI in these patients was related to the extent of initial damage. In contrast, serum PSTI in patients with serious complications remained at high level even at 2 weeks after trauma, and it was not correlated with the initial severity of the injury.