Graft-Versus-Host Disease Following Liver Transplantation: Development of a High-Incidence Rat Model and a Selective Prevention Method.

Graft-versus-host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT-GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti-CD8α mAb treatment, which invariably resulted in acute LT-GvHD. Compared with those in the peripheral counterpart, graft CD4+ CD25- passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4+ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti-TCRαß mAb. T cell-depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT-GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT-GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high-risk donor-recipient combinations and for analyzing immunoregulatory mechanisms of the liver.

PD-1/B7-H1 interaction contribute to the spontaneous acceptance of mouse liver allograft.

The programmed death-1 (PD-1)/B7-H1 pathway acts as an important negative regulator of immune responses. We herein investigated the role of the PD-1/B7-H1 pathway in establishing an immunological spontaneous tolerance status in mouse liver allografting. B7-H1 is highly expressed on the donor-derived tissue cells and it is also associated with the apoptosis of infiltrating T cells in the allografts. Strikingly, a blockade of the PD-1/B7-H1 pathway via anti-B7-H1mAb or using B7-H1 knockout mice as a donor led to severe cell infiltration as well as hemorrhaging and necrosis, thus resulting in mortality within 12 days. Furthermore, the expression of the FasL, perforin, granzyme B, iNOS and OPN mRNA in the liver allografts increased in the antibody-treated group in comparison to the controls. Taken together, these data revealed that the B7-H1 upregulation on the tissue cells of liver allografts thus plays an important role in the apoptosis of infiltrating cells, which might play a critical role of the induction of the spontaneous tolerance after hepatic transplantation in mice.

Superagonistic CD28 antibody induces donor-specific tolerance in rat renal allografts.

The ultimate goal of organ transplantation is to establish graft tolerance where CD4+CD25+FOXP3+ regulatory T (Treg) cells play an important role. We examined whether a superagonistic monoclonal antibody specific for CD28 (CD28 SA), which expands Treg cells in vivo, would prevent acute rejection and induce tolerance using our established rat acute renal allograft model (Wistar to Lewis). In the untreated or mouse IgG-treated recipients, graft function significantly deteriorated with marked destruction of renal tissue, and all rats died by 13 days with severe azotemia. In contrast, 90% of recipients treated with CD28 SA survived over 100 days, and 70% survived with well-preserved graft function until graft recovery at 180 days. Analysis by flow cytometry and immunohistochemistry demonstrated that CD28 SA induced marked infiltration of FOXP3+ Treg cells into the allografts. Furthermore, these long-surviving recipients showed donor-specific tolerance, accepting secondary (donor-matched) Wistar cardiac allografts, but acutely rejecting third-party BN allografts. We further demonstrated that adoptive transfer of CD4+CD25+ Treg cells, purified from CD28 SA-treated Lewis rats, significantly prolonged allograft survival and succeeded in inducing donor-specific tolerance. In conclusion, CD28 SA treatment successfully induces donor-specific tolerance with the involvement of Treg cells, and thus the therapeutic value of this approach warrants further investigation and preclinical studies.

Exogenous expression of Fas-ligand or CrmA prolongs the survival in rat liver transplantation.

Modulation of donor organs by transfection of a gene encoding immmunosuppresive molecules has been recognized as a less toxic approach to prevent allograft rejection. Fas-ligand (FasL) plays a critical role in activation-induced cell death of activated cytotoxic lymphocytes. This may provide a potential for induction of "immune privileged sites" to escape the host immune surveillance system. Cytokine response modifier A (CrmA), a gene product of cowpox virus, blocks caspase as well as perforin/granzyme-mediated apoptotic pathways. Therefore, it may suppress intragraft apoptosis. The aim of the present study was to investigate whether transfection of FasL or CrmA genes prolonged the survival of rat liver allografts. Using the high responder rat combination of DA (RT-1(a)) donor to LEW (RT-1(1)) recipient, we performed orthotopic liver transplantation with subsequent delivery of adenoviral vectors containing FasL, CrmA, or LacZ, at a dose of 1 x 10(9) pfu via a recipient tail vein using a Cre-mediated gene expression system. Recipient survival was assessed as well as immunohistochemical examination of the grafts for anti-CD2, TUNEL, and H&E staining. Statistical analysis was performed with the Mann-Whitney U test. The therapeutic groups showed significantly prolonged recipient survival compared with the LacZ-treated control group. Histologic analysis revealed reduced hepatocyte apoptosis in the CrmA-treated group and increased apoptosis of infiltrating mononuclear cells in the FasL-treated group. These data suggested that FasL and CrmA may be potent genes to prolong rat liver allograft survival.

Return to home early days after acute aortic dissection surgery.

AIM: The length of hospital stay after acute aortic dissection surgery tends to be prolonged. The aim of this study is to assess the feasibility of our protocol for early discharge after acute aortic dissection surgery. METHODS: This study enrolled 17 consecutive acute aortic dissection patients who returned to their own home within 2 weeks of surgery. In seven patients total aortic arch replacement was performed and in 7 partial arch replacement. The main aim of the first 24 h after surgery was to achieve early extubation. Patients were encouraged to return to their own home 4 days and later after surgery. The prerequisite criteria for discharge were the following: independent mobility, stable hemodynamics, apyrexia, adequate oral intake, normal bowel function, healthy surgical wound and the patient's agreement for discharge. RESULTS: The mean age of these patients was 59. The postoperative ventilation time, length of intensive care unit stay and postoperative hospital stay were 11 h, 37 h and 6.9 days, respectively. Two (12%), 13 (76%) and 14 (82%) patients returned to their own home by postoperative day 4, 7 and 10, respectively. Three patients were readmitted to a peripheral hospital in the 4 week postoperative period. The reason for all readmissions was lack of family support. Two other patients underwent pericardiocentesis for pericardial effusion at an other hospital as outpatients. There was no complication caused by early discharge. CONCLUSIONS: Early discharge after aortic dissection surgery is safe and recommended to patients who have normal bowel function and adequate family support.

Comparative 1H-NMR studies on the physical state of water in soft contact lens and mouse lens.

The physical state of water in mouse lenses (2-, 4- or 8-wk-old) and soft contact lenses (SCLs, water content from 18.4 to 79.2%) were studied by measuring spin-lattice relaxation times (T1) and apparent intermolecular cross-relaxation times (TIS) from irradiated protein or polymer protons to water protons, using 360 MHz 1H-NMR spectrometer at 25 degrees C. (1) 1/T1 values of SCLs increased gradually with increasing dry weight (W(%)). 1/TIS values of SCLs were approximately zero at W of 20.8 and 26.8%, increased gradually from 26.8% and then steeply above approximately 50%. (2) A plot of 1/T1 vs. W(%) of mouse lenses was almost equal to that of SCLs. However, a plot of 1/TIS vs. W(%) was an approximately straight line with the intercept at W of 23% and with the slope which is almost equal to that of SCLs above W of approximately 50%. The plot of 1/TIS vs. W(%) of mouse lenses might indicate the significant change in the physical state of water and/or protein-water interactions above W of 23%.

Vascular pathogenesis of normal-tension glaucoma: a possible pathogenetic factor, other than intraocular pressure, of glaucomatous optic neuropathy.

Elevated intraocular pressure is significant in the pathogenesis of glaucomatous optic neuropathy in glaucoma, however a number of studies suggest that pressure-independent factor(s) are also associated with the pathogenesis. In this article, the significance of vascular pathogenesis in glaucoma is discussed. A brief overview of substances used to treat glaucoma, independent of an effect on intraocular pressure, such as calcium channel blockers, is also discussed.

Optic disc topographic parameters measured in the normal cynomolgus monkey by confocal scanning laser tomography.

AIM: To study optic disc topographic parameters in normal cynomolgus monkeys by Heidelberg retina tomograph (HRT). METHODS: 12 optic disc topographic parameters were investigated in 36 normal eyes in 18 male monkeys. Mean (SD) and interocular differences were obtained for each parameter from three independent measurements made during a 1 week period. Correlations among the topographic parameters were analysed, too. RESULTS: No significant differences between right and left eyes were detected for any topographic parameters. Disc area, rim area, and height variation contour showed smaller right-left differences than other parameters. The coefficients of variation for rim area, height variation contour, rim volume, mean cup depth, maximum cup depth, mean retinal nerve fibre layer (RNFL) thickness, and RNFL cross section area were less than 10% (for rim area, less than 5%). Rim area and height variation contour showed relatively weak interrelations and neither showed a correlation with disc area. CONCLUSION: For evaluating time related changes in the optic disc by HRT in monkeys, rim area and height variation contour might be useful parameters because coefficients of variation and right-left differences were lower than for other parameters and because these parameters showed weak interrelations and no correlation with disc area.

Apraclonidine effects on ocular responses to YAG laser irradiation to the rabbit iris.

Apraclonidine (p-aminoclonidine) ophthalmic solution effectively reduces the rise in intraocular pressure (IOP) following anterior segment laser surgery. We tested the effect of topical 0.5% apraclonidine on intraocular pressure and on protein and prostaglandin (PG) E2 concentrations in aqueous humor following Q-switched Nd:YAG laser irradiation to the iris of albino rabbits, at an energy level of 2 to 200 mJ. IOP was measured prior to and for 24 hr after irradiation. Aqueous humor was withdrawn before and 1 hr after laser irradiation for determining protein (Lowry method) and PGE2 (radioimmunoassay). Four to seven rabbits were used for each experiment. The increase in IOP and protein concentration following laser irradiation was demonstrated to be dependent on the amount of laser energy. Apraclonidine completely abolished the IOP rise, and significantly reduced the elevation of protein content. Apraclonidine failed to affect the increase in PGE2.

The ocular hypotensive effect of the ETB receptor selective agonist, sarafotoxin S6c, in rabbits.

PURPOSE: Endothelin-1 (ET-1) is known to affect intraocular pressure (IOP) in rabbits, and the IOP response is likely to be mediated by the receptors ETA, ETB, or both. Sarafotoxin S6c (STX-S6c) is a selective agonist to ETB receptors. The authors attempted to clarify the role of ETB receptors in changes in IOP induced by ET-1 in rabbits using STX-S6c and to determine the relationship between the IOP response and various doses of STX-S6c. METHODS: Each concentration (10(-4) to 10(-7) M) of STX-S6c was injected intravitreally (20 microliters/eye) into one eye. The contralateral eye of each was used as a control. The IOP was measured periodically using a calibrated pneumatonometer. Indomethacin (50 mg kg-1) or vehicle (10 ml kg-1; 0.05 M phosphate buffer) was administered intraperitoneally twice, before and after intravitreal injection of STX-S6c (10(-5) M), and IOP was measured in the same protocol for 24 hours. RESULTS: In the STX-S6c (10(-4) and 10(-5) M) group, the IOP reduction was significant compared with the baseline (P < 0.05 to P < 0.01), starting from 6 and 4 hours and continuing until 192 and 72 hours after injection, respectively. A solution of 10(-6) M STX-S6c also resulted in significant reduction of IOP observed from 24 to 72 hours after injection (P < 0.05). The 10(-7) M solution of STX-S6c failed to affect IOP. The area under the curve of IOP response exhibited a significant correlation with the doses of STX-S6c (r = -0.856; P = 0.0001) in the treated eyes. Treatment with indomethacin failed to affect the IOP reduction caused by STX-S6c (10(-5) M). Ciliary injection and some dilatation of the iridial vessels were observed in eyes that received higher doses of STX-S6c. CONCLUSION: STX-S6c reduces the IOP in rabbits in a dose-dependent fashion, presumably mediated through the ETB receptors.

Optic nerve and peripapillary choroidal microvasculature of the rat eye.

PURPOSE: To investigate the three-dimensional microvascular anatomy of the optic nerve and peripapillary choroid in the rat eye. METHODS: Gross vascular anatomy of the posterior eye segment of Wistar rats was studied in serial microsections with a light microscope. The optic nerve and peripapillary choroidal vessels were sequentially microdissected, using methylmethacrylate corrosion microvascular castings, and were examined with a scanning electron microscope to determine the three-dimensional relationships of the vessels. RESULTS: The posterior ciliary artery traveled along the inferior side of the optic nerve sheath, directly entered the optic nerve head, and divided into three branches: the central retinal artery and medial and lateral long posterior ciliary arteries, which provided several short branches to the choroid. The optic nerve head vasculature was consistently nourished by a recurrent arteriole from the central retinal artery and an arteriole from the choroidal artery at the peripapillary choroid. The central retinal vein flowed into a venous anastomosis along the optic disc border of the peripapillary choroid. Capillaries within the optic nerve drained into the central retinal vein, the marginal venous anastomosis of the peripapillary choroid, and the pial veins, all of which flowed into the posterior ciliary veins along the optic nerve sheath. CONCLUSIONS: The findings illustrate vascular anatomic differences in optic nerve and peripapillary choroidal microcirculation between rat and human. In rats, the peripapillary choroid plays a significant role in both blood supply and venous drainage of the optic nerve head. The central retinal artery also contributes to the optic nerve head circulation.

Regulation of connexin phosphorylation and cell-cell coupling in trabecular meshwork cells.

PURPOSE: To investigate the expression of functional gap junctions and the effect of protein kinase C (PKC) on such junctions in confluent cultures of bovine trabecular meshwork (TM) cells. METHODS: Expression of the gap junction protein connexin43 in TM cells was examined by immunofluorescence microscopy. Intercellular communication by gap junctions was assessed by observing the diffusion of fluorescent dye from an individual cell injected with lucifer yellow. The phosphorylation of connexin43 was evaluated by immunoblot analysis with a monoclonal antibody to this protein. RESULTS: Immunofluorescence staining revealed that connexin43 was localized to sites of contact between adjacent TM cells. Exposure of cells to the PKC activator phorbol 12-myristate 13-acetate (PMA; 10 nM, 1 hour) had no marked effect on the pattern of connexin43 immunofluorescence. Injection of a TM cell with lucifer yellow resulted in the spread of the dye into neighboring cells. Dye coupling was inhibited by PMA in a dose- and time-dependent manner, and this inhibition was prevented by pretreatment of cells with the PKC inhibitor bisindolylmaleimide I. Immunoblot analysis of control TM cell lysates yielded connexin43 bands corresponding to the nonphosphorylated protein (43 kDa) and three phosphorylated forms (47, 48, and 49 kDa). Cells exposed to PMA (10 nM, 1 hour) yielded an additional band corresponding to a 44-kDa form of phosphorylated connexin43 and showed a decrease in the intensity of the band corresponding to the nonphosphorylated protein and an increase in the intensity of the 47-kDa band. CONCLUSIONS: TM cells communicate with each other through gap junctions, and the communication is inhibited by PKC, probably, at least in part, through phosphorylation of connexin43.

Transient ischemic injury in the rat retina caused by thrombotic occlusion-thrombolytic reperfusion.

PURPOSE: To establish a clinically relevant model of transient retinal ischemia by thrombotic occlusion-thrombolytic reperfusion of the central retinal artery of the rat. METHODS: Thrombus was photochemically induced in the central retinal artery by the combination of intravenous injection of photo-sensitive dye, rose bengal, and green laser irradiation focused on the artery. Transient retinal ischemia for 60 minutes was achieved by a subsequent systemic administration of tissue-type plasminogen activator to reperfuse the occluded vessel. Samples of retinas were excised from the animals killed 3, 9, 12, 24, 48, and 78 hours after the reperfusion. The experimental data were processed using the TdT-dUTP terminal nick-end labeling (TUNEL) method to detect apoptotic cells. RESULTS: The transient retinal ischemia caused time-sequential apoptotic changes in the retinal cells as evaluated by counting the number of TUNEL-positive cells. The most remarkable changes occurred in the central area of retina, and further on the sections taken 24 hours after reperfusion. The peripheral area was less affected, and the outer nuclear cell layer was almost unaffected throughout the observation period. CONCLUSIONS: The proposed method to cause retinal transient ischemia is highly reproducible, and it is easy to simulate the progress and topographical distribution of retinal changes observed in the clinical cases of central retinal arterial occlusion and its subsequent thrombolytic reperfusion. This may provide a useful tool for constructing the effective thrombolytic strategies against the central retinal arterial occlusion and for evaluating the effects of neuroprotective agents.

Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells.

It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.

The prognosis of corticosteroid-responsive individuals.

Response of intraocular pressure to topical corticosteroid administration is determined genetically, and the genes that determine corticosteroid responsiveness of IOP and primary open angle glaucoma are considered closely related. To elucidate the relationship between corticosteroid responsiveness and primary open angle glaucoma, 35 patients with high corticosteroid responsiveness were followed up without therapy for at least ten years. During the follow-up period, a sustained rises in IOP greater than 21 mm Hg developed in five of 22 originally normotensive subjects. In two cases, glaucomatous field changes were demonstrated along with pressure elevation. Of 13 originally ocular hypertensives, further rise of IOP associated with glaucomatous field changes developed in seven. Results strongly indicate that pressure elevation is more likely to develop in corticosteroid-responsive individuals and the glaucomatous field defects are more likely to develop in responsive are compared with nonresponsive subjects.

Trabeculectomy with mitomycin. A comparative study with fluorouracil.

We conducted a prospective, randomized study to compare mitomycin with fluorouracil for efficacy and safety as an adjunct to trabeculectomy in eyes with glaucoma with poor surgical prognosis. Thirty-two eyes of 32 patients were randomly allocated to either mitomycin (17 eyes) or fluorouracil therapy (15 eyes). Mitomycin (0.2 mg) was applied only once during trabeculectomy, and fluorouracil (5 mg) was subconjunctivally injected 10 times in the 2 weeks after surgery. Fifteen eyes (88%) in the mitomycin group and seven (47%) in the fluorouracil group achieved an intraocular pressure of less than or equal to 20 mm Hg without antiglaucoma medication. The follow-up period was 7 to 12 months. Corneal complications were less common in the group given mitomycin than in that given fluorouracil (12% vs 53%). The incidence of other complications was similar between the two groups.

A comparison of latanoprost and timolol in primary open-angle glaucoma and ocular hypertension. A 12-week study.

OBJECTIVE: To evaluate the intraocular pressure (IOP)-reducing effect and the side effects of latanoprost (PhXA41), a new phenyl-substituted prostaglandin F2 alpha-isopropyl ester analogue, in patients with elevated IOP, using timolol maleate as the reference drug. METHODS: A total of 184 patients with primary open-angle glaucoma or ocular hypertension at 35 medical centers participated in this randomized double-masked study. The patients were randomized to receive either 0.005% latanoprost once daily or 0.5% timolol maleate twice daily, for a period of 12 weeks. Intraocular pressure was measured 24 hours after the administration of timolol, at 2, 4, 8, and 12 weeks of treatment. RESULTS: Latanoprost reduced IOP at the end of 12 weeks by 6.2 +/- 2.7 mm Hg (mean +/- SD) (26.8%), while timolol reduced IOP by 4.4 +/- 2.3 mm Hg (19.9%). At all visits latanoprost reduced IOP significantly more than timolol did. The main ocular side effects observed in both groups were conjunctival hyperemia and smarting. The main systemic side effect was a reduced pulse rate, which occurred in patients treated with timolol. CONCLUSIONS: The results of this study demonstrated that 0.005% latanoprost taken once daily is well tolerated and more effective in reducing IOP than 0.5% timolol taken twice daily. Thus, latanoprost may become an important choice for the medical treatment of glaucoma.

Untreated ocular hypertension. A long-term prospective study.

A prospective study was designed to determine the course of ocular hypertension without visual field loss if untreated and to estimate the value of various clinical findings in prediction the course of ocular hypertension. Seventy-five patients with intraocular pressure greater than 21 mm Hg but without demonstrable visual field defects were followed up without glaucoma therapy for a minimum of nine years. While under observation, seven of 75 ocular hypertensives developed typical glaucomatous visual field defects. Both the sensitivity and the specificity of each clinical finding in the individuals who later developed visual field defects were determined, and none of the clinical findings was found to be a good predictive indicator.

Clinical evaluation of UF-021 (Rescula; isopropyl unoprostone).

We have reviewed two Phase III clinical studies of isopropyl unoprostone conducted in Japan: a 12-week comparative study of 0.12% isopropyl unoprostone and 0.5% timolol, and a 52-week administration of two concentrations of isopropyl unoprostone in ocular hypertensive and primary open-angle glaucoma patients. These studies showed a similar ocular hypotensive effect of 0.12% isopropyl unoprostone to 0.5% timolol and a sustained ocular hypotensive effect of the drug for up to one year. Adverse reactions of isopropyl unoprostone were minor and similar to those of timolol. No pigmentary changes of the irides were noticed. In view of these results, isopropyl unoprostone seems to be a useful antiglaucoma medication.

The efficacy of cooling on excimer laser photorefractive keratectomy in the rabbit eye.

We cooled the surface of the cornea to reduce the thermal damage by the excimer laser ablation and referred to this method as "cooling photorefractive keratectomy" (cooling PRK). We performed conventional PRK and cooling PRK on rabbits' eyes and measured the thermal change during laser ablation. We also examined the degree of subepithelial haze and the tissues with a light microscope and an electron microscope. Normal corneal temperature of live rabbit corneas was about 32 degrees C and it rose to 41 degrees C after 300 laser shots. However, when we cooled the rabbit corneas to 24 degrees C with continuous irrigation of chilled BSS PLUS solution before laser ablation, the temperature rose only to 34 degrees C after the same ablation. Slit-lamp evaluation showed that more severe corneal haze occurred with the conventional PRK procedure. At 2 weeks postoperatively, the mean haze score was 1.77 +/- 0.87 in the conventional PRK and 0.87 +/- 0.38 in the cooling PRK (P < 0.01). Light-microscopy examination showed epithelial hyperplasia and fibroblasts, type III collagen, and heat shock protein-70 in the subepithelium of corneas with conventional PRK procedure than with cooling PRK. Under the electron microscope, more disruption of the layers of fibroblasts and collagen fibrils was observed in the conventional PRK procedure than that of the cooling PRK. In conclusion, the cooling of the corneal surface in PRK effectively reduces tissue damage related to subepithelial haze and may enable us to reduce the degree of myopic regression.