No abstract available.
Emergencies* ; Emergency Service, Hospital* ; Hospitals, General* ; Humans

Nitric oxide (NO) has been emerged as an important intracellular and intercellular regulatory molecule having functions as diverse vasodilatation, neural communication, and host defense. In the immune system, NO produced by activated macrophage or neutrophil is known to kill tumor cells as a defense molecule. In addition, recent reports demonstrated that NO could interact with superoxide to generate peroxynitrite (ONOO-), an anion and a potent oxidant, in macrophages or other cellular systems. The production of peroxynitrite has been recognized to be associated with the activation and expression of inducible NO synthase (iNOS). In this study, to evaluate the role of NO and peroxynitrite in murine bladder tumor cells, the author investigate the effect of NO and peroxynitrite on the viability, cytotoxicity, and DNA fragmentation of MBT-2 cells. The results are as followings: 1. Activated macrophages treated with INF-r, LPS, or INF-r+ LPS showed increment of nitrite (NO2) production and cytotoxicity against MBT-2 cells in a dose dependent manner. However, treatment with NGMMA, a NOS inhibitor, decreased NO2- production and cytotoxicity. 2. Treatment with SNP, a nitric oxide donor, increased NO2 production and DNA fragmentation (%), but decreased viability (%) of MBT-2 cells in a concentration dependent manner. 3. Treatment with peroxynitrite increased cytotoxicity and DNA fragmentation, but decreased viability of MBT-2 cells in a concentration dependent manner. 4. NO- and peroxynitrite-mediated increment of cytotoxicity in MBT-2 cells was corresponded to the programmed cell death, apoptosis. Taken together, these data indicate that NO and peroxynitrite elaborated from macrophages or other cellular systems may increase the cytotoxicity of MBT-2 cells via the mechanism of apoptosis.
Apoptosis* ; Cell Death ; Cell Line* ; DNA Fragmentation ; Humans ; Immune System ; Macrophages ; Neutrophils ; Nitric Oxide Synthase ; Nitric Oxide* ; Peroxynitrous Acid* ; Superoxides ; Tissue Donors ; Urinary Bladder Neoplasms ; Urinary Bladder* ; Vasodilation

The majority of nasal fractures have been managed by using closed reduction, intranasal packing and external splinting. However, in comminuted nasal bone fractures, the conventional closed reduction may be inadequate to reduce the fracture segments accurately, and insufficient to prevent secondary nasal deformity. In these cases, open reduction and interfragment wire fixation was recommended for accurate reduction, and has been mainstay of treatment modality. Furthermore, in nasoethmoid orbital fractures, anatomic reduction of fractured nasal bone, medial and inferior orbital rim segments to reconstruct nasofrontal buttress and transnasal wiring to prevent telecanthus were essential. But, the interfragment wire fixation is difficult and time-consuming procedure. The care must be taken to fix small fracture segments. It is also difficult to obtain bony support due to extensive dissection of periosteum, and to achieve rigid fixation on three- dimensional space, causing depression of bony contour. From April 1998 to August 1999, we used malleable mesh microplates for treatment of 3 comminuted nasal bone fractures and 3 nasoethmoid orbital fractures. During the follow up period of 8 months to 24 months, all of six patients had successful cosmetic result without complications. There was no recurrent depression of bony contour, no secondary nasal deformity, no displacement of microplates and no palpable, externally shown hardwares. The use of mesh microplates is reliable and useful method for the treatment of comminuted nasal fractures because it is relatively simple procedure and achieves rigid fixation on three-dimensional space without postoperative temporary nasal packing which may cause patient's discomfort. Furthermore, in nasoethmoid orbital fractures, correction of telecanthus can be done without application of transnasal wiring.
Congenital Abnormalities ; Depression ; Follow-Up Studies ; Humans ; Nasal Bone* ; Orbit ; Orbital Fractures ; Periosteum ; Splints

The most common etiology for female pseudohermaphroditism is congenital adrenal hyperplasia, which accounts for more than 60 percent of children with ambiguous genitalia, and is treated with cortisol replacement and surgical correction of ambiguous genitalia. Flap vaginoplasty, the inverted U-Shaped type has been applied worldwide to the patient with low vaginal entry. The most frequent complication of the operation is contraction of the new vaginal introitus as a result of ischemic and fibrotic changes in the overlapping suture line between the flap and posterior vaginal wall. Maintenance of a good blood supply for the flap and tension free anastomosis should always be kept in mind to avoid this complication. We experienced a vaginoplasty with labioscrotal flap instead of the inverted U-shaped flap and achieved a good result in a 14-year-old girl with low vaginal entry due to congenital adrenal hyperplasia. The labioscrotal flap seems to be more suitable than inverted U-shaped flap for vaginoplasty because the labioscrotal skin is more elastic and more easily elongated than the perineal skin.
46, XX Disorders of Sex Development* ; Adolescent ; Adrenal Hyperplasia, Congenital* ; Child ; Disorders of Sex Development ; Female* ; Humans ; Hydrocortisone ; Skin ; Sutures

Fibronectin(Fn) is a large, multidomain glycoprotein, which exists in a soluble form in plasma and an insoluble fibrillar form in extracellular matrices. Fn affects many aspects of cellular responses. However, it is not known whether Fn could activate macrophages for the tumor cell killing. We report that Fn induces the tumoricidal activity of macrophages for murine bladder tumor(MBT-2) cell. Tumoricidal activity was determined by 3[H]-thymidine uptake of MBT-2 cell. Fn alone had no effect, whereas recombinant interferon- r(IFN- r) weakly induced the tumoricidal activity of macrophages for MBT-2 cells. However combination of Fn with recombinant IFN-r synergized to activate macrophages to kill MBT-2 cells in a dose dependent manner. At this point nitric oxide(NO) was secreted by activated macrophages, and the secretion of NO and tumoricidal activity of macrophage were inhibited in the presence of NG-monomethyl- L-arginine(NGMMA), a competitive inhibitor of NO synthase(NOS). Fn has various cell binding sites. The Arg-Gly-Asp(RGD) sequence present in the central cell binding domain of Fn is the prototype of these sites. Engineered fibronectin(eFn) is formed by RGD-rich sequence. Combination of eFn, instead of Fn, with recombinant IFN- T resulted in more powerful activation of macrophage in tumor cell killing than Fn. In conclusion, our results demonstrate that Fn acts as a modulator of macrophage activation for tumor cell killing induced by IFN-r via a process involving L-arginine dependent NO production. Especially, RGD sequence of Fn has important role for tumoricidal activity of macrophage. Although the precise mechanism of Fn to promote NO synthesis induced by IFN-r remains to be further elucidated, Fn-mediated macrophage adhesion by specialized cell surface receptors and activation of intracellular signals might be important in the development of macrophage activation.
Arginine ; Binding Sites ; Extracellular Matrix ; Fibronectins* ; Glycoproteins ; Homicide ; Macrophage Activation ; Macrophages* ; Nitric Oxide ; Plasma ; Receptors, Cell Surface ; Urinary Bladder

Recently it is known that nitric oxide(NO) generated by an activatedmacrophage plays an important role in tumoricidal or bactericidal activity. Thisstudy was done to know the effects of NO produced by the activated macrophages onthe growth of the murine bladder tumor cell line (MBT-2). For the activation ofmacrophages, RAW 264.7 cell line ( macrophage-derived cell line) and peritonealmacrophages from Balb/c mouse were treated with interferon-r (INF-r),lipopolysaccharide ( LPS) or INF-r plus LPS and for the evaluation of growthinhibition of MBT -2 cell line, tritiated thymidine (3[H] -thymidine)incorporation was measured after coculturing of MBT-2 cell line with macrophageswhich had been activated to produce NO. The results are as follows : l.Peritoneal macrophages from Balb/c mouse could be activated to produce NO onlywhen they are stimulated by the combination of INF-r and LPS (35+/-1.0uM/L). Theycould produce a slight increase amount of NO when they had been stimulated byINF-r (11+/-2.5uM/ L) or LPS (14+/-3.5uM/L) compared to control macrophages(8+/-2.5uM/L). The induction of NO production by INF-r plus LPS could be abrogatedby the use of NG-monomethyl-L- arginine (NGMMA), a competitive inhibitor of NOsynthase (5+/-1.5 M/L). 2. RAW 264.7 cell lines could be activated to produce NOby the treatment of INF-r, LPS, and INF-r plus LPS (40+/-2.5uM/L, 37+/-3.0uM/Land 51+/-2.6uM/L respectively). When they are treated with NGMMA, they produced nomore NO even though they had been stimulated with INF-r plus LPS (14+/-4.0uM/L),and they could produce small .amount of NO(19+/-1.0uM/L) in the absence of thestimulation. 3. The incorporation of 3[H] -thymidine of the MBT-2 and peritonealmacrophages was reduced when the cells had been treated with INF-r plus LPScompared to the control (14,519+/-1,087cpm, 20,716+/-1,474cpm respectively).There was no reduction in the incorporation of 3[H] -thymidine when the cellshad been treated with INF-r or LPS alone. The incorporation of 3[H]-thymidineincreased when the cells had been treated with INF-r plus LPS in the presence ofNGMMA(19,622+/-1,341cpm). 4. The incorporation of 3[H] -thymidine of the MBT-2 and RAW 264.7 cell lines were reduced when the cells had been treated with INF-r, LPS and INF-r plus LPS (19.068+/-144cpm, 15,070+/-122cpm and 7,543+/-85cpm respectively) compared to control( 20,708+/-142cpm). When the cells had been pretreated with NGMMA, the incorporation of 3[H]- thymidine recovered to same degree (12,605+/-108cpm) compared to the cells stimulated with INF-r plus LPS. The above correlation of the NO production from macrophages which had been stimulated by INF-r and/or LPS and the inhibition of growth of the tumor cells suggests that NO produced by stimulated macrophages might be the responsible molecule in the defense system of the body against tumors.
Animals ; Arginine ; Cell Line* ; Macrophages* ; Macrophages, Peritoneal ; Metabolism* ; Mice ; Nitric Oxide ; Nitrogen* ; Thymidine ; Urinary Bladder Neoplasms

PURPOSE: Recently, in assisted reproductive technologies(ART) programs theme Is an increasing Interest in the use of agents for the enhancement of sperm motility for assisted fertilization. In an attempt to improve the motility at the cryopreserved human semen and hence the fertilizing capacity of asthenospermic semen samples, different semen preparation techniques have been attempted and the effects of chemical stimulants as nitric oxide(NO) have been studied extensively. Superoxide anions cause lipid peroxidative damage to cell membrane phospholipids, and sperm are known to be particularly susceptible to lipid peroxidation. Such sperm with damaged membranes are impaired functionally. Recently, peroxynitrite, an anion and a potent oxidant, generated by the interaction of nitric oxide and superoxide anions has been demonstrated In macrophages and other cellular systems. Also this anion cause lipid peroxidative damage to cell membrane phospholiplds. We therefore Investigated whether NO and peroxynitrite have the roles to modulate sperm motility and to affect Its viability. MATERIALS AND METHODS: Normal human semen samples(as per World health Organization (WHO) criteria) were obtained after 3day period of abstinence by donors. The samples(n=5) were incubated with either sodium nitroprusside(SNP; 0.1, 05, 1 or 2mM) or peroxynitrite (10, 50 or 100 micrometer ) and the percent viability and motility were assessed at various time inteval up to 4hr. The human semen samples were treated with N-acetyl-L-cystein(NAC;10mM), SNP (0.5mM), phorbol myristate acetate(PMA; 100nM), of SNF plus PMA. Both superoxide and peroxynitrite release were measured directly by chemiluminometer. Percent viability and motility were assessed at 4hr of Incubation. A sample of each aliquot was placed in a Mauler chamber for videomicrography Percent motility were analyzed by using the sperm analysis imaging system. The sperm vlability was assessed by flow cytometer using LIVE/DEAD sperm viability kIt. The production of superoxide and peroxynitrite were measured by the method of chemiluminescence assay. Result : All results represent a mean +/-SEM, n=5. Treatment of human semen samples for 4hr with SNP, a NO generating agent, significantly decreased sperm motility and viability in high concentration [relative motility(% of control); 38 +/-4 and 30 +/-5, relative viability; 42 +/-4 and 30 +/-3 by 1 and 2mM of SNP]. In the presence of low concentration SNP(0.5mM), the sperm viability was not significantly affected(82 +/-3), whereas the sperm motility was affected(64 2). SNP(0.5mM) also decreased sperm motility(80 +/-2 at 2hr 64 +/-3 at 4hr, 44 +/-3 at 6hr, and 38 +/-4 at 8hr) in a time dependent manner. Since it was demonstrated that superoxide anions are one of the common source of lipid peroxidation, we investigated whether superoxide anions produced by human semens could Interact wlth NO to generate peroxynitrite. Adding N-acetyl-L-cystein(NAC) to the human semen samples partially blocked spontaneous release of superoxide, whereas PMA augmented the release of superoxide from human semen samples (control:0.9 106 0.3, NAC: 0.5 106 +/-0.4, and PMA: 2.5 106 +/-0.4photons/60min). The production of superoxide was corresponded with the production of peroxynltrite(control: 1.0 104CPM, SNP: 3.8 106CPM, SNP plus PMA. 12chi106CPM). In addition, SNP in combination with PMA(65 +/-3) markedly decreased sperm motility than that of SNP alone(77 +/-2.5) at 4hr, implying that nitric oxide might inhibit sperm motility via the formation of peroxynitrite In human semen samples. Exogenous peroxynitrite also decreased sperm motility in a dose dependent manner(10 micrometer : 64 +/-2, 50rM: 53 +/-3, and 1 0 micrometer of peroxynitrite: 23 4). CONCLUSIONS: These results suggest that NO inhibits sperm motility via the formation of peroxynltrite and further demonstrate that NO-induced inhibition of sperm motility is depended on the production of superoxide from human semens because peroxynitrite is generated by the interaction of NO and superoxide.
Cell Membrane ; Fertilization ; Humans ; Lipid Peroxidation ; Luminescence ; Macrophages ; Membranes ; Microscopy, Video ; Myristic Acid ; Nitric Oxide* ; Peroxynitrous Acid* ; Phospholipids ; Semen ; Sodium ; Sperm Motility* ; Spermatozoa* ; Superoxides ; Tissue Donors ; World Health Organization

Intravesical bacillus calmette-guerin( BCG) therapy for superficial bladder carcinoma is believed to exert its antitumor effects through immune mechanisms which have yet to be more clearly defined. Recently, BCG infection is known to induce nitric oxide(NO) production by macrophages through a T cell mediated process. NO is known to be microbicidal and tumoricidal. Therefore, we studied the effects of intravesical BCG instillation on the induction of inducible NO synthase(iNOS) which is responsible for the production of NO in the vesical tissue of rat Forty Sprauge-Dawley female rats were equally divided into 5 groups. In group 1, normal saline( 0.85 ml/kg) was intravesically instilled one time. In group 2 and group 3, BCG of Pasteur strain(2 mg/kg, normal saline 0.85 ml/kg) was instilled one time and 3 times weekly respectively. In group 4, 10-fold dose of the strain( 20 mg/kg, 0.86 ml/kg) and in group 5, 1/10-fold dose of the strain (0.2 mg/kg, 0.85 ml/kg) were instilled one time respectively. We sacrificed two rats to excise the bladders in each group 1, 3, 7, and 14 days after the instillation( after the last instillation in group 3). iNOS mRNA was not detected in the vesical tissues from the rats of group 1, whereas it was strongly detected in all the vesical tissues from the rats in group 2, 3. or 4. More iNOS mRNA was detected 14 days after the instillation in group 3 than group 2 or 4. In group 5, iNOS mRNA was weakly detected 1, 3, and 7 days and not detected 14 days after the instillation. Our results indicate that intravesical BCG instillation of rat induces the expression of iNOS mRNA in the vesical tissue and suggest that the duration and degree of iNOS mRNA expression is dependent on the dose of BCG and the frequency of the instillations. On the basis of these observations, we conclude that adequate dose and frequency are required for effective treatment of superficial bladder carcinoma in the intravesical BCG therapy.
Animals ; Bacillus ; Female ; Humans ; Macrophages ; Mycobacterium bovis* ; Nitric Oxide Synthase Type II* ; Rats* ; RNA, Messenger ; Urinary Bladder

Transforming growth factor-B1(TGF-B1) has many fundamental biological processes including cell growth, extracellular matrix deposition and degradation, and inflammatory responses. TGF-B1 is released by platelet and inflammatory cells, and it affects all phases of wound healing after injury. It contributes to the regulation of fibroblast chemotaxis and proliferation, and also controls the synthesis and degradation of extracellular matrix necessary for tissue repair. Clinically, scar tissue formation and subsequent stricture after urethral injury frequently results in troublesome problems to urologists. In the phase I study of this report, we intended to how the histological changes and the involvement of TGF-B1 in the formation of stricture in injured urethrae of rats. We injured urethrae of 24 adult male Sprauge-Dawley rats(200-250 g.) by urethrotome and Dormia basket and then observed histological changes and analysed TGF-f, mRNA levels of the injured urethrae by Northern blot. Northern b1ot analysis showed that TGF-t, mRNA was much expressed on day 1,3,5 after injury. Fibroblasts and deposition of extracellular matrix were markedly increased on day 5. Reepithelialization was completed and urethral lumen was narrowed on day 10. In the phase II study, we tried to know that antisense TGF-B1 oligodeoxynucleotides(ODNs) could inhibit TGF-B1 expression and the formation of stricture in injured urethrae of rats. We injured urethrae of rats and treated the urethral injury with the application of antisense TGF-B1 ODNs. Northern blot analysis showed that TGF-B1 mRNA was little expressed in the urethrae treated with the antisense on day 1,3 after injury. Comparing to the antisense-nontreated urethrae, the antisense-treated urethrae showed decrease of submucosal thickening and maintained normal sized urethral lumens on day 14, 21 after injury. In conclusion, increase of TGF-B1 mRNA in injured urethrae of rats suggests that TGF-B1 could play an important role in repair mechanism. With application of antisense TGF-B1 ODNs in injured urethrae of rats, the expression of TGF-B1 can be inhibited and also the formation of stricture prevented.
Adult ; Animals ; Biological Processes ; Blood Platelets ; Blotting, Northern ; Chemotaxis ; Cicatrix ; Constriction, Pathologic* ; Extracellular Matrix ; Fibroblasts ; Humans ; Male ; Oligodeoxyribonucleotides* ; Rats* ; RNA, Messenger ; Transforming Growth Factor beta1* ; Urethra ; Urethral Stricture ; Wound Healing

Recently, BCG infection is known to induce nitric oxide(NO) production by macrophages through T cell mediated process and NO is known to be microbicidal and tumoricidal. There are several strains of BCG which are commercially available and vary in the number, pathogenicity, viability, and immunogenicity of organisms. Therefore, we wanted to know if there are any differences between three different strains of BCG(Pasteur, Connaught or Tice strain) on the induction of inducible NO synthase(iNOS) and the histological changes in vesical tissue after intravesical instillation of BCG in rats. Thirty two Sprauge-Dowley female rats were equally divided into 4 groups. In group 1, normal saline(0.85 ml/kg) was intravesically instilled one time. In group 2, 3, and 4, BCG of Pasteur strain(2mg/kg, normal saline 0.85ml/kg), Connaught strain(1.35mg/kg, normal saline 0.85ml/kg), Tice strain(0.21mg/kg, normal saline 0.85ml/kg) was instilled one time, respectively. The bladders were excised from each group on day 1, 3, 7, and 14 after BCG instillation. iNOS mRNA was not detected in the vesical tissues of control group, whereas it was strongly detected in group 2, 3, or 4. Also, iNOS mRNA was more strongly detected on day 1, 3, and 7 after intravesical BCG instillation than day 14 in the vesical tissues of group 2, 3, and 4. Histologic findings were well related with expression of iNOS mRNA. Our results indicate that intravesical BCG instillation of rat induces expression of iNOS mRNA in the vesical tissue accompanying the infiltration of inflammatory cells and suggest that all of the 3 strains of BCG including Pasteur, Connaught, and Tice are good at inducing expression of iNOS mRNA without significant differences between the strains.
Administration, Intravesical* ; Animals ; Female ; Gene Expression* ; Humans ; Macrophages ; Mycobacterium bovis* ; Nitric Oxide Synthase Type II* ; Rats* ; RNA, Messenger ; Urinary Bladder ; Virulence