Effect of transient post-transplantation hyperglycemia on the development of diabetes mellitus and transplantation outcomes in kidney transplant recipients.

BACKGROUND: Hyperglycemia occurs frequently after kidney transplantation and may be reversed when the dosage of the immunosuppressive agents is tapered. However, the effect of transient post-transplantation hyperglycemia (PTH) on transplantation outcomes is not well described. METHODS: Kidney transplant recipients without diabetes who underwent kidney transplantation between 2001 and 2012 were enrolled in the study. Transient PTH was defined as recovery from PTH without further antidiabetic therapy and the maintenance of glycated hemoglobin levels <6.5% at 1 year after transplantation. Persistent PTH until 1 year after transplantation was considered to be new-onset diabetes after transplantation (NODAT). The factors associated with increased risk of PTH were analyzed. We compared the development of diabetes mellitus, cardiovascular disease, and other transplantation outcomes among patients with no PTH, transient PTH, and NODAT. RESULTS: Among 176 kidney transplant recipients, 106 (60.2%) developed PTH and 58 (54.7%) of 106 patients with PTH had transient PTH. Older age, high body mass index (BMI), and female gender were independent risk factors for transient PTH. The incidence of diabetes was not significantly different between patients with no PTH and those with transient PTH. The incidence of cardiovascular disease was significantly increased in NODAT group compared with that in no PTH and transient PTH groups. However, the incidences of acute rejection, allograft loss, and patient death were comparable among the three groups. CONCLUSIONS: Transient hyperglycemia after kidney transplantation was found to be associated with older age, high body mass index, and female gender. Transient elevation of blood glucose level did not affect post-transplantation outcomes, including diabetes mellitus and cardiovascular disease. However, patients with NODAT should be carefully monitored for the occurrence of cardiovascular disease.

Impact of cytochrome P450 3A and ATP-binding cassette subfamily B member 1 polymorphisms on tacrolimus dose-adjusted trough concentrations among Korean renal transplant recipients.

BACKGROUND: Tacrolimus is a substrate of cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp), encoded by the CYP3A and ATP-binding cassette subfamily B member 1 (ABCB1) genes, respectively. This study was aimed to investigate the impact of CYP3A and ABCB1 polymorphisms on the tacrolimus pharmacokinetics and clinical outcomes in Korean renal transplant recipients. METHODS: We analyzed data from a cohort of 70 renal transplant recipients receiving tacrolimus. CYP3A4*4, CYP3A4*5, CYP3A4*18, CYP3A5*3, ABCB1 C1236>T, ABCB1 G2677>T/A, and ABCB1 C3435>T polymorphisms were genotyped and correlated to dose-adjusted tacrolimus trough concentration at months 1, 3, 6, and 12 after transplantation. RESULTS: Patients with the CYP3A5*3 alleles showed higher dose-adjusted tacrolimus concentrations for 12 months and higher trough levels until 6 months after transplantation. ABCB1 polymorphisms and haplotypes were not associated with tacrolimus concentrations. In a multivariate analysis, the presence of ≥1 CYP3A5*3 allele was a significant independent variable affecting dose-adjusted tacrolimus concentrations. Glomerular filtration rate, acute rejection, opportunistic infection, and graft survival were not affected by CYP3A5 polymorphisms. Calcineurin inhibitor toxicity, which showed higher tendency in patients with CYP3A5*1 alleles, might be associated with higher tacrolimus dose per kilogram. CONCLUSIONS: The CYP3A5 genotype is a major factor in determining the dose requirement of tacrolimus, and genotyping may be of value in individualization of immunosuppressive therapy of renal transplant patients.

Isolation and phylogenetic analysis of H1N1 swine influenza virus isolated in Korea.

A swine influenza H1N1 virus was isolated from a pig during a severe outbreak of respiratory disease in Korea. All genes of the H1N1 isolate, including hemagglutinin (HA), neuraminidase (NA), matrix (M), nucleoprotein (NP), non-structural (NS), PA, PB1 and PB2, were of swine origin. Also, all these genes showed a close phylogenic relationship with those of H1N1 viruses previously isolated from pigs in the United States. These results suggest that North American swine influenza virus has actually been transmitted to pigs in Korea.

Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus.

We investigated the mechanism of the immunomodulatory action of polysaccharide (ASP) isolated from a cell culture of Acanthopanax senticosus. ASP was found to directly increase the proliferation and differentiation of B cells, and the cytokine production of macrophage, but not the proliferation and cytokine production of T cells. Since ASP cannot penetrate the cell membrane due to its large molecular mass, such cellular activation may be caused by the surface binding of ASP to receptors expressed on B cells and macrophages. The possibility that TLRs, which are known to be involved in immune-related responses, may be the receptor(s) of ASP was investigated. The immunomodulating activities of ASP on the B cells and macrophages of C3H/HeJ mice, expressing a defective toll-like receptor (TLR)-4, were decreased versus the corresponding cells from C3H/HeN mice. In addition, the activities of ASP on B cells and macrophages were significantly reduced by treating the cells with antibodies to TLR4 and TLR2 prior to ASP, suggesting that both of them are the possible receptors of ASP. The ligation of TLRs induced by ASP was able to activate mitogen-activated protein kinases (MAPKs), such as Erk1/2, p38 and JNK, and the transcription factor NF-kappaB. Although ASP was shown to activate the TLR signaling cascades in the same manner as lipopolysaccharide (LPS), these two could be differentiated by the finding that polymyxin B (PMB), a specific inhibitor of LPS, did not significantly affect the activities of ASP on B cells and macrophages. Taken together, our results demonstrate that ASP, isolated from a cell culture of A. senticosus, activates B cells and macrophages by interacting with TLRs and leading to the subsequent activation of mitogen-activated protein kinases and NF-kappaB.

Characterization of B cell membrane receptors of polysaccharide isolated from the root of Acanthopanax koreanum.

We investigated the immunomodulatory activity of polysaccharide isolated from the root of Acanthopanax koreanum (AK) at the cellular level. AK directly increased B cell proliferation and antibody production, but did not affect the expression of IL-2, IFN-gamma or IL-4 by T cells, or T cell proliferation in vitro. Since AK cannot penetrate cells due to its large molecular mass, B cell activation may be caused by the surface binding of AK to B cell-specific receptors. The role of TLR4 as an AK receptor was shown by the fact that AK activity in B cells from C3H/HeJ mice, which are known to have a defective Toll-like receptor (TLR)-4, was found to be reduced compared with that in control cells from C3H/HeN mice. AK activity was also reduced by antibodies blocking TLR2, TLR4, CD19 or CD79b, but not by an antibody blocking CD38, which suggests AK receptor profiling in B cells. Two main differences between AK and lipopolysaccharide (LPS) were observed. First, LPS activity was inhibited by antibodies to either TLR2 or TLR4, but not by antibodies to CD19, CD79b or CD38. Another was that LPS-induced B cell proliferation was inhibited by polymyxin B (PMB), a specific inhibitor of LPS, whereas AK activity was not affected. Taken together, our results demonstrate that AK directly activates B cells, but not T cells, and suggest that AK has a broader receptor profile than LPS in B cells.

Ovarian expression of melatonin Mel(1a) receptor mRNA during mouse development.

Melatonin, secreted by the pineal gland, is involved in the regulation of many physiological functions of various species of animals. In the present study, the expression of gene for melatonin Mel(1a) receptor (MelR) was evaluated in the ovary, hypothalamus, and pituitary according to the developmental stages in female mice. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) and in situ PCR techniques were applied. According to the developmental stages, gene for MelR was differently expressed on ovary, hypothalamus, and pituitary. MelR gene was first expressed on pituitary prior to the expression in hypothalamus and ovary. Ovarian MelR gene started to express at birth. Unlike hypothalamic expression of MelR gene which was identified after birth, in pituitary, it was expressed at 16 days post coitum. In the ovary, the expression signal of MelR gene was identified on granulosa cells. However, the signal was not detected in the theca cells. It was weak in the primordial and atretic follicles. Taken together, it can be considered that melatonin has a pivotal role in the folliculogenesis.

Correlation of histologic 2-dimensional reconstruction and confocal scanning laser microscopic imaging of choroidal neovascularization in eyes with age-related maculopathy.

OBJECTIVE: To topographically localize vascular channels, macrophages, and retinal pigment epithelium and other components of choroidal neovascularization (CNV) associated with age-related maculopathy. METHODS: Two postmortem eyes with age-related maculopathy and CNV were evaluated. The formalin-fixed CNV complex was excised and processed for confocal scanning laser microscopy including immunostaining for factor VIII-related antigen and incubation with Ig fluorescein isothiocyanate. After confocal microscopy, the specimens were serial step sectioned, stained, and 2-dimensional topographic reconstructions were made. The confocal images were compared with the 2-dimensional reconstructions. RESULTS: Both specimens contained central disciform scars surrounded by areas of intact retinal pigment epithelium. The first specimen was more atrophic and contained fewer choroidal neovascular channels than the second specimen. The topographic arrangement of the CNV and retinal pigment epithelial changes in the confocal images corresponded with the 2-dimensional reconstructions. Macrophages were concentrated around areas of vascularization. CONCLUSION: Confocal scanning laser microscopy of excised CNV simulates fluorescein angiography and topographic localization of the components of CNV provides insight into the pathogenesis of CNV.

Pentobarbital and ketamine suppress serum concentrations of sex hormones in the female rat.

PURPOSE: To investigate the potential effects of pentobarbital and ketamine on serum concentrations of sex hormones, the present study was performed using pregnant mare serum gonadotropin (PMSG)-primed cyclic female Sprague-Dawley rats. METHODS: Pentobarbital sodium (37 mg.kg(-1), i. p.) or ketamine-hydrochloride (229 mg.kg(-1), i. m.) was injected 2 and 3 days after PMSG treatment. At 0, 1, 2, 3, 4, and 5 days after PMSG treatment, sera were collected by cardiac puncture. The serum concentrations of progesterone (P(4)), testosterone (T), and estradiol-17 beta (E(2)) were determined by radioimmunoassay. RESULTS: The serum concentrations of P(4) tended to be lower in both the pentobarbital- and the ketamine-treated groups compared with the control group. Significant differences were found on days 3 and 4 after pentobarbital and on days, 1, 4, and 5 after ketamine administration. Serum concentrations of T were also suppressed in both the pentobarbital and the ketamine-treated groups, whereas E(2) concentrations decreased only in the ketamine-treated group. CONCLUSION: Pentobarbital and ketamine decrease serum sex hormone concentrations in PMSG-primed female rats.

The effect of the haptic portion of intraocular lens on the development of posterior capsular opacification in rabbit.

Using a white rabbit model, the effect of the haptic portion of the intraocular lens (IOL) and intracapsular ring on the development of posterior capsular opacification (PCO) after extracapsular cataract extraction (ECCE) with phacoemulsification was studied. Implantation of both the intracapsular ring and IOL developed less PCO than implantation of the IOL alone. ECCE followed by implantation of the intracapsular ring alone also developed less PCO than ECCE alone. Through this experimental work in a rabbit model, it could be conceived that the haptic portion of IOL and the intracapsular ring can prevent the development of PCO.

Gamma-radiation accelerates ovarian follicular atresia in immature mice.

This study deals with the morphological degenerations of normal and atretic follicles based on artificially induced radiation apoptosis. ICR strain of 3 week-old female mice were whole body irradiated with 8.3 Gy and sacrificed by cervical dislocation. Ovaries were collected at 0 h, 6 h, 12 h, 1 d, 2 d, 4 d, and 8 d post irradiation and processed for morphological observation. Irradiated ovarian follicles showed such characteristics as faint connection between zona and cumulus thinning of granulosa layer, numerous apoptotic bodies, and increased cell debris in antrum. However, in normal ovaries about a half of the follicles were healthy. Within 6 h post irradiation, more than 95% of follicles steeply became apoptotic. At 4 d, severely damaged follicles with hypertrophed theca layer and fragmented oocytes were shown. At 8 d, normal shaped follicles were observed together with severely disrupted ones. The normal shaped follicles seemed to have grown from radioresistant ones. In conclusion, gamma-radiation had an additive or concomitant effect on atretic degeneration of mouse ovarian follicles.

Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia.

In the present study we have examined the presence of Fas, Fas ligand (FasL), and p53 in rat granulosa cells during follicular development and atresia, especially in relation to the granulosa cell cycle progression and the onset of granulosa cell apoptosis. Fas, FasL, and p53 proteins were immunolocalized, and their contents were determined by Western blotting. Granulosa cell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and in situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling (TUNEL) as well as by flow cytometry. Ovaries not exposed to gonadotropins (control) consisted predominantly of preantral and early (small) antral follicles, the latter of which were mostly atretic and demonstrated intense TUNEL staining in granulosa cells exhibiting positive immunoreactivities for FasL and Fas. Granulosa cells isolated from these follicles were apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell population in the A0 phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular growth to the medium to large antral stages. Cell cycle analysis showed significantly higher proportion of the cells in S and G2/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities for Fas, FasL, and p53. By days 4 and 5, widespread atresia and extensive granulosa cell apoptosis were noted in large antral and preovulatory follicles and were coincidental to increased expression of p53 and Fas, but not of FasL, as well as an apparent arrest of granulosa cell G1/S progression, as evident by an increased cell population in G0/G1 and a decrease in the S and G2/M. Granulosa cells from equine CG-primed ovaries exhibited marked increases in p53 and Fas protein contents and apoptosis after adenoviral p53-sense complementary DNA infection in vitro and were more responsive to Fas activation by an agonistic Fas monoclonal antibody challenge. Taken together, these findings are consistent with the well accepted concept that gonadotropin plays a central role as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. In addition, the control of granulosa cell apoptosis may involve two consecutive cellular/molecular events: cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of the p53 and Fas/FasL death pathways.

Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs.

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell lysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.

Effects of ceramide, the Fas signal intermediate, on apoptosis and phospholipase D activity in mouse ovarian granulosa cells in vitro.

Although recent studies have demonstrated that ovarian follicular atresia occurs by apoptosis of granulosa cells, the intracellular signaling pathways involved in apoptotic cell death are still poorly characterized. We examined the role of ceramide as a candidate intracellular mediator of Fas-mediated signaling in cultured granulosa cells. Expression of Fas antigen was demonstrated by Western blot of granulosa cell lysates and immunostaining of cultured granulosa cells. Exposure of granulosa cells to anti-Fas monoclonal antibody (anti-Fas mAb) resulted in significant sphingomyelin hydrolysis, which was accompanied by a progressive increase in endogenous levels of ceramide. The addition of exogenous C6-ceramide induced drastic morphological change, including nuclear fragmentation and typical apoptotic DNA degradation. Furthermore, both anti-Fas mAb and C6-ceramide decreased phospholipase D (PLD) activity and diacylglycerol (DAG) concentrations in a time- or a dose-dependent manner. In addition, treatment with phorbol 12-myristate 13-acetate completely attenuated the ceramide-induced inhibition of PLD activity and partially suppressed ceramide-induced apoptosis. These results indicate that the Fas/ceramide signaling pathway might play a role in granulosa cell apoptosis and suggest that the PLD/DAG pathway might be cross-linked to the Fas/ceramide pathway in apoptotic processes of granulosa cells.

Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells.

We have investigated the effects of ceramide on the progression of cell cycle and on apoptotic cell death in ovarian cultured granulosa cells. Rates of cellular proliferation were measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle analysis. We also examined for morphological and biochemical signs of apoptosis. The PCNA expression was downregulated in a dose-dependent manner after treatment with C6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C6-ceramide markedly decreased the population associated with G0/G1 DNA content and the reduction of cell numbers in G0/G1 phase was accompanied by the elevation of the A0 phase. The exposure of granulosa cells to exogenous C6-ceramide induced drastic morphological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, significantly inhibited the ceramide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G0/G1 phase and as a consequence, granulosa cells would be committed to apoptosis. Our findings also indicated that down-regulation of the PKC activity might be involved in the ceramide-induced apoptosis in cultured granulosa cells.

Inhibitory effect of bupleuri radix saponins on adhesion of some solid tumor cells and relation to hemolytic action: screening of 232 herbal drugs for anti-cell adhesion.

Anti-cell adhesive activity and hemolytic action of herbal drugs were investigated. Among 232 herbal drugs tested, six showed a remarkable anti-cell adhesive activity, and the extract from the roots of Bupleurum falcatum (Umbelliferae), the semen of Psorala corylifolia (Leguminosae), and the semen of Areca catechu (Palmae) showed an anti-cell adhesive action at non-cytotoxic concentrations. Saikosaponins-a, d and e, isolated from the roots of Bupleurum falcatum, exhibited a potent anti-cell adhesive activity and a strong hemolytic action. In a structure-activity relationship for both activities, it seems that a sugar moiety and an ether linkage between C-13 and C-28 are required for good bioactivities. In addition, saikosaponin d with a beta-hydroxy group at C-16 was more potent than saikosaponin a possessing an alpha-hydroxy group. Taken together, it is suggested that the mechanism for anti-cell adhesive activity of saikosaponin may resemble that for their hemolytic action.

Osseous metaplasia in proliferative vitreoretinopathy.

PURPOSE: To report the clinicopathologic features of intraocular osseous production in association with proliferative vitreoretinopathy. METHOD: The clinical and histopathologic features of two patients with proliferative vitreoretinopathy and intraocular bone formation are reviewed. RESULTS: Preretinal osseous tissue incorporated in the proliferative vitreoretinopathy was surgically removed in one patient, and osseous tissue was present in the proliferative vitreoretinopathy in the enucleated eye of the other patient. CONCLUSIONS: Bone formation, presumably from metaplastic retinal pigment epithelium, may be present in proliferative vitreoretinopathy tissue. The intraocular bone is present internal rather than external to the neurosensory retina.

Keratocyte repopulation in epikeratoplasty specimens.

PURPOSE: To study the histology and pattern of keratocyte repopulation of surgically removed human epikeratoplasty lenticules. METHODS: Removed epikeratoplasty lenticules and penetrating keratoplasty buttons that contained epikeratoplasty lenticules were evaluated for duration of epikeratoplasty, histologic and ultrastructural features, and average number of keratocytes per high-power microscopic field. The keratocyte density was compared with age-matched controls. RESULTS: Fifteen epikeratoplasty specimens from eight penetrating keratoplasties and seven removed lenticules were reviewed. The indications for keratoplasty were myopia, keratoconus, and aphakia. The lenticules were in place for 7-120 months, and the keratocyte count ranged from 14 to 40 per high-power field. Keratocyte density increased to 30-40 per high-power field, similar to age-matched controls, at approximately 48 months postoperatively, similar to the density of the controls. Keratocytes appeared to have migrated from the periphery to the center of the lenticules. CONCLUSIONS: Normal keratocyte density in epikeratoplasty lenticules is reached by approximately 48 months after surgery.

Treatment of suprachoroidal hemorrhage with tissue plasminogen activator.

We evaluated the efficacy of tissue plasminogen activator in treating experimental suprachoroidal hemorrhage. Suprachoroidal hemorrhage was created in 30 white rabbit eyes by implanting four pieces of small, exogenously formed blood coagula into the suprachoroidal space. Animals were randomized for treatment with a surgical sponge soaked in 25, 50, or 75 microg of tissue plasminogen activator (tPA) or balanced salt solution (BSS) as a control. The time when initiation and completion of clot dissolution occurred was established, and histological examination was performed to assess damage. Clot dissolution started within 30 min in the 50- and 75-microg tPA group, whereas it took 2.75 days in the control group; complete dissolution of blood clots took 4.5 h in the 75-microg tPA group and 14 days in the control group. Histological examination revealed a minimal change in photoreceptors within 6 h after treatment with 75 microg tPA. Treatment of suprachoroidal hemorrhage with tPA seems to be effective, but further investigations for determining the effective and nontoxic dose are required.

Effects of gamma-radiation on ovarian follicles.

In order to observe the morphological and endocrinological changes of the rat and mouse ovarian follicles by gamma-radiation, rats were whole-body irradiated with doses of 3.2 Gy and 8.0 Gy and mice with 2.9 Gy and 7.2 Gy. Sections of ovaria were examined by light microscopy. Concentrations of progesterone, testosterone, and estradiol in ovarian homogenate were determined by radioimmunoassay techniques. Gamma-radiation resulted in the increased percentage of atretic follicles in the groups killed on day 0, day 4, and day 8 after irradiation. The decrease in granulosa cell viability was found in animals killed on day 4 after irradiation. The finding of the high ratio of testosterone to estradiol compared to that of progesterone to testosterone suggests that aromatase activity--steroid biosynthesis from testosterone to estradiol--in granulosa cell could be affected by gamma-radiation.