Faecal freezing preservation period influences colonization ability for faecal microbiota transplantation.

AIMS: There has been growing interest in faecal microbiota transplantation (FMT) as treatment. Although, frozen donor faeces preserved at -20°C has been widely used for practical advantages, freezing at -20°C can affect bacterial viability. Adequacy evaluation of fresh and frozen faeces as the transplant is necessary for the methodological improvement of FMT. METHODS AND RESULTS: The viable bacterial compositions of faecal specimens under fresh and freezing conditions were compared by a microbiome analysis using propidium monoazide (PMA microbiome). In addition, recovery abilities from bacterial reduction by antibiotics were compared between fresh and frozen FMT using a murine model. PMA microbiome results suggested that freezing and freeze-thawing did not significantly affect in vitro faecal bacterial viability. However, the recovery effect from antimicrobial cleansing in frozen FMT was reduced in a freezing time-dependent manner, especially prominent in Actinobacteria and Bacteroidetes phyla. CONCLUSIONS: Short-term freezing preservation of faeces exhibited maintenance of enteric colonization ability in frozen FMT in comparison to 1 month -20°C-preservation. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term -20°C-preservation of transplanted faeces can result in instability of the clinical outcome in FMT therapy. The standardization of practical procedures of FMT therapy according to disease types is desirable.

Imaging blood vessels and lymphatic vessels in the zebrafish.

Blood vessels supply tissues and organs with oxygen, nutrients, cellular, and humoral factors, while lymphatic vessels regulate tissue fluid homeostasis, immune trafficking, and dietary fat absorption. Understanding the mechanisms of vascular morphogenesis has become a subject of intense clinical interest because of the close association of both types of vessels with pathogenesis of a broad spectrum of human diseases. The zebrafish provides a powerful animal model to study vascular morphogenesis because of their small, accessible, and transparent embryos. These unique features of zebrafish embryos permit sophisticated high-resolution live imaging of even deeply localized vessels during embryonic development and even in adult tissues. In this chapter, we summarize various methods for blood and lymphatic vessel imaging in zebrafish, including nonvital resin injection-based or dye injection-based vessel visualization, and alkaline phosphatase staining. We also provide protocols for vital imaging of vessels using microangiography or transgenic fluorescent reporter zebrafish lines.

Feasibility of 2nd generation STS retinal prosthesis in dogs.

We developed a 2(nd) generation suprachoroidal transretinal stimulation (STS) system with a 49 channel electrode array and implanted in 2 dogs. One month after surgery, all electrodes were functioning and the ocular fundus was normal in both dogs. The results indicate the 2(nd) generation STS retinal prosthesis is feasible and can be considered for clinical use.

Efficacy of intravitreal bevacizumab for polypoidal choroidal vasculopathy.

AIMS: The aim of the study was to assess the short-term efficacy of intravitreal injections of bevacizumab for polypoidal choroidal vasculopathy (PCV). METHODS: Intravitreal bevacizumab (1 mg) was injected into 11 eyes of 11 patients with PCV in this retrospective, interventional case series. The main outcome measure was the change in the polypoidal vessels on indocyanine green angiography (IA) 3 months after injection. The foveal height determined by optical coherence tomography and the best-corrected visual acuity (BCVA) also were evaluated before and after treatment. RESULTS: At baseline, subretinal fluid was observed in five eyes and a pigment epithelial detachment in eight eyes. The foveal height 1 month after injection decreased significantly (p = 0.023), but at 3 months, no significant decrease was observed, although an additional injection was administrated in five of 11 eyes. The IA at 3 months showed resolution of polyps in one eye but residual or enlarged lesions in the other ten eyes. The BCVA did not improve significantly, although the subjects had relatively good BCVA at baseline (mean 0.45). CONCLUSION: Intravitreal injection of bevacizumab may reduce the fluid from PCV but seems to be ineffective for diminishing its choroidal vascular changes.

Intravitreal injection of bevacizumab for choroidal neovascularisation associated with pathological myopia.

AIM: To assess the efficacy and safety of an intravitreal injection of bevacizumab (Avastin(R)) for myopic choroidal neovascularisation (mCNV). METHODS: Intravitreal bevacizumab (1 mg) was injected into eight eyes of eight patients with mCNV in this non-randomised, interventional case series. The best-corrected visual acuity (BCVA) was measured and the optical coherence tomography (OCT) and fluorescein angiography findings were examined before and after treatment. The minimum follow-up time was 3 months. RESULTS: The mean BCVA was 0.26 before treatment and 0.51 at the last visit (p = 0.009). The BCVA improved to two or more lines in six eyes (75%) and remained the same in two eyes (25%). Leakage from the mCNV on fluorescein angiography decreased in seven eyes (87.5%). The choroidal neovascularisation area on fluorescein angiography (p = 0.049) and the foveal thickness on OCT images decreased significantly (p = 0.027) after the treatment. No major complications developed. CONCLUSION: Intravitreal injection of bevacizumab seems to be an effective and safe treatment for mCNV.

Suppression of stress-induced nNOS expression in the rat hypothalamus by biting.

Nitric oxide (.NO) modulates the activity of the endocrine system in the behavioral response to stress. The purpose of this study was to investigate the effect of restraining the body of an animal on expression of neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) of the hypothalamus, and the inhibitory effect of para-masticatory activity on restraint-induced nNOS expression. We observed an increase in nNOS mRNA expression and nNOS-positive neurons in the rat hypothalamus after 30 or 60 min of restraint. Biting on a wooden stick during bodily restraint decreased nNOS mRNA expression in the hypothalamus. In addition, the number of nNOS-positive neurons was significantly reduced in the PVN of the hypothalamus. These observations clearly suggest a possible anti-stress effect of the masticatory activity of biting, and this mechanism might be unconsciously in operation during exposure to psychological stressors.

Effect of cardiopulmonary bypass on pulmonary clearance of adrenomedullin in humans.

BACKGROUND: Adrenomedullin is a potent vasodilatory peptide and its plasma concentration increases after cardiopulmonary bypass. We analyzed the contribution of the lung to the disposition of adrenomedullin before and after cardiopulmonary bypass in humans. METHODS: Thirty-five patients undergoing cardiac surgery with cardiopulmonary bypass were studied. Bloods were sampled from the pulmonary artery and left atrium at the following times: prior to systemic heparinization, during pulmonary reperfusion and after cardiopulmonary bypass. Plasma concentrations of total and mature adrenomedullin were measured using an immunoradiometric assay kit specific for human adrenomedullin. Intermediate adrenomedullin was calculated as the difference between total adrenomedullin and mature adrenomedullin. RESULTS: Before cardiopulmonary bypass, mature and intermediate adrenomedullin concentrations were reduced by the pulmonary circulation by approximately 30% and 20%, respectively. However, these effects were not observed during pulmonary reperfusion. Mature, but not intermediate, adrenomedullin was reduced after cardiopulmonary bypass. Furthermore, pulmonary clearance quantity of mature adrenomedullin was significantly enhanced after cardiopulmonary bypass. CONCLUSION: These results indicate that cardiopulmonary bypass temporally impairs the pulmonary clearance of mature and intermediate adrenomedullin, but clearance of mature, not intermediate adrenomedullin is enhanced after cardiopulmonary bypass.

Ophthalmic artery blood flow in patients with internal carotid artery occlusion.

AIM: To evaluate the risk factors for rubeosis iridis by colour Doppler imaging (CDI) in patients with complete internal carotid artery occlusion (ICAO). METHODS: 34 eyes of 32 consecutive patients with complete ICAO were enrolled. Using CDI, blood flow direction (forward, reverse, undetectable) in the ophthalmic artery (OA), central retinal artery (CRA), and short posterior ciliary artery (SPCA) were determined. Arterial mean blood velocity (Vmean) and resistive index (RI) were calculated and correlations between the rubeosis iridis incidence and CDI parameters analysed. RESULTS: The eyes were classified into four types according to blood flow direction: forward flow in OA, CRA, and SPCA (type 1; n = 11); reverse OA and forward CRA and SPCA flow (type 2a; n = 12); reverse OA and undetectable CRA and SPCA flow (type 2b; n = 8); undetectable flow in all three arteries (type 3; n = 3). Rubeosis iridis was seen only in type 2b and 3 eyes. Type 2b showed significantly (p<0.01) higher Vmean and lower RI values in the OA, indicating more rapid reverse flow than in type 2a eyes. Although in type 1 and 2a eyes OA flow was in opposite directions, they manifested no rubeosis iridis and no difference in the Vmean and RI values of the CRA and SPCA. CONCLUSIONS: The classification of eyes from patients with ICAO into four types by CDI may facilitate the identification of the eyes at high risk for rubeosis iridis. Markedly diminished flow in both the CRA and SPCA may result in rubeosis iridis, regardless of OA flow direction.

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure.

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.

Vitreous hemorrhage from a ciliary granuloma associated with Wegener granulomatosis.

PURPOSE: To report a case of vitreous hemorrhage from ciliary granuloma in Wegener granulomatosis. METHODS: Interventional case report. An 18-year-old woman with Wegener granulomatosis and episcleral granuloma in her LE had ultrasound biomicroscopy findings of a mass in the pars plana of the ciliary body in the meridian corresponding to the episcleral granuloma. RESULTS: The patient underwent vitrectomy in the LE for subsequent vitreous hemorrhage. Intraoperatively, the mass was diagnosed as a ciliary granuloma at the pars plana. Dense blood clotting around the ciliary granuloma and subretinal exudation at the ora serrata were observed, with no other changes causative for the vitreous hemorrhage. CONCLUSIONS: We report a case of vitreous hemorrhage associated with a ciliary granuloma that was revealed by ultrasound biomicroscopy. Careful observation is necessary in eyes with persistent inflammation in Wegener granulomatosis.

Cerebral vasoreactivity and internal carotid artery flow help to identify patients at risk for hyperperfusion after carotid endarterectomy.

BACKGROUND AND PURPOSE: Hyperperfusion syndrome is a rare but potentially devastating complication after carotid endarterectomy (CEA). The aim of this study was to investigate whether preoperative measurement of cerebral vasoreactivity (CVR) and intraoperative measurement of internal carotid artery (ICA) flow could identify patients at risk for hyperperfusion after CEA. METHODS: For 26 patients with unilateral ICA stenosis >/=70%, cerebral blood flow (CBF) and CVR were investigated before and 1 month after CEA, with resting and acetazolamide-challenge single-photon emission CT. CBF on the first postoperative day was also measured. ICA flow was measured before and after reconstruction by electromagnetic flowmeter during surgery. RESULTS: Ipsilateral CBF on the first postoperative day significantly increased relatively (56.6+/-53.2%) as well as absolutely (37.9+/-8.8 to 57.7+/-18.0 mL/100 g per minute) in the reduced CVR group (CVR <12%) but not in the normal CVR group (CVR >/=12%) (10.3+/-15.5% and 40.6+/-7.9 to 43.9+/-5.7 mL/100 g per minute, respectively). One month later, this difference almost disappeared. Two patients showed ipsilateral CBF increase of >/=100%. A significant association of intracerebral steal with hyperperfusion (CBF increase >/=100%) on the first postoperative day was also observed. ICA flow increase after reconstruction significantly correlated with CBF increase on the first postoperative day in the reduced CVR group but not in the normal CVR group. The threshold of ICA flow increase for hyperperfusion was estimated to be 330 mL/min in the reduced CVR group. CONCLUSIONS: Single-photon emission CT with acetazolamide challenge and ICA flow measurement during surgery could identify patients at risk for hyperperfusion after CEA, in whom careful monitoring and control of blood pressure should be initiated even intraoperatively.

Macular translocation with chorioscleral outfolding: an experimental study.

PURPOSE: Macular translocation by chorioscleral infolding has been proposed as a surgical intervention for exudative age-related macular degeneration, but the surgery is unpredictable and can be associated with severe complications. We tested a new surgical technique, macular translocation with chorioscleral outfolding secured by neurosurgical clips. METHODS: This was a prospective interventional study in two parts; the first in human cadaver eyes and the second in pigs. Chorioscleral infolding was performed on six human donor eyes, and chorioscleral outfolding was performed on an additional six. The inner surface of the eye wall was measured, and then the fold was unfolded and the distance was measured again. In the second half of the study, macular translocation surgery was performed on 33 pig eyes with one of three sclera shortening methods: 1) a circumferential chorioscleral infolding using 5-0 nylon sutures, 2) a circumferential chorioscleral outfolding using scleral clips, or 3) a radial chorioscleral outfolding using scleral clips. Foveal translocation was measured. RESULTS: The inner wall of the human cadaver eye was shortened in the chorioscleral infolding group by a mean of 1.6 mm, and in the chorioscleral outfolding group by 3.0 mm. In the pig eyes, the fovea was translocated a mean 2377 microm by circumferential suturing, 2582 microm by circumferential clipping, and 3386 microm by radial clipping. Irregular deformation of the globe was more apparent in the circumferential suture group. Undesirable retinal folds often formed after circumferential infolding but not after radial clipping. CONCLUSION: Radial chorioscleral outfolding with clips is more predictable and effective than infolding. It produces more translocation and prevents folds across the fovea, one of the most undesirable complications in macular translocation surgery.

[Three cases of multiseptate gallbladder].

Multiseptate gallbladder is an extremely rare congenital anomaly. We report three cases of this clinical entity. The third patient, a 70-year-old male who was asymptomatic with this condition, also had a gallbladder carcinoma. The three surgically removed gallbladders had multiple thin septa with a honeycomb appearance. Histologically, the septa contained a muscular layer that was continuous with the gallbladder wall. Furthermore, in the third patient, mucinous cystadenoma with focal carcinomatous change in the epithelium was observed. To our knowledge, this is the first description of a multiseptate gallbladder with carcinoma.

Effect of Ca(2+)-free and Mg(2+)-free BSS Plus solution on the retinal pigment epithelium and retina in rabbits.

PURPOSE: To determine whether intravitreal irrigation with Ca(2+)-free and Mg(2+)-free BSS Plus (Alcon Laboratory, Fort Worth, Texas) solution alters the adhesiveness between the retinal pigment epithelium and the retina of rabbits. METHODS: Thirty-four eyes of 34 Dutch pigmented rabbits underwent lensectomy and vitrectomy. Subsequently, the vitreous cavity of 24 eyes was irrigated with Ca(2+)-free and Mg(2+)-free BSS Plus solution for 10 or 20 minutes. The other 10 eyes were irrigated with BSS Plus solution for 20 minutes as controls. To determine the adhesiveness between the retinal pigment epithelium and retina, a retinal detachment was produced in 12 of the 34 eyes. The apical surface of the retinal pigment epithelium and the photoreceptor outer segments were examined by scanning electron microscopy. Retinal physiology was assessed by electroretinography and retinal morphology by light microscopy. RESULTS: After retinal detachment was produced, the number of cone sheaths on the apical surface of the retinal pigment epithelium after irrigation with Ca(2+)-free and Mg(2+)-free BSS Plus solution for 20 minutes (33 +/- 15, mean +/- SD) was significantly less than the number of cone sheaths on the apical surface of the retinal pigment epithelium of eyes after irrigation with BSS Plus solution for 20 minutes (120 +/- 50) or the number of cone sheaths on the apical surface of the retinal pigment epithelium of eyes after 10 minutes of irrigation with Ca(2+)-free and Mg(2+)-free BSS Plus solution (115 +/- 49; P =.02). The b-wave amplitudes in the eyes irrigated with Ca(2+)-free and Mg(2+)-free BSS Plus solution for 20 minutes were depressed compared with the b-waves in eyes irrigated with BSS Plus solution for 20 minutes on the first postoperative day (P =.03). After the third postoperative day, there was no significant difference in the b-waves (P >.06). Light microscopy demonstrated no morphologic abnormalities after the use of both solutions. CONCLUSIONS: Intravitreal irrigation with Ca(2+)-free and Mg(2+)-free BSS Plus solution for 20 minutes altered the adhesion between the retinal pigment epithelium microvilli and retinal outer segments and made the creation of retinal detachment less traumatic. These results suggest that Ca(2+)-free and Mg(2+)-free BSS Plus solution may be of clinical value for the creation of an intentional retinal detachment for foveal translocation surgery.

Indocyanine green facilitates removal of epiretinal and internal limiting membranes in myopic eyes with retinal detachment.

PURPOSE: To describe the use of intravitreal indocyanine green as an aid to identifying epiretinal membranes and internal-limiting membranes during surgery for a retinal detachment resulting from a macular hole. METHODS: A 62-year-old man who had a retinal detachment resulting from a macular hole underwent vitrectomy. During the surgery, intravitreal indocyanine green was injected intravitreally. RESULTS: The internal-limiting membrane was stained green, but the epiretinal membrane was unstained. Because the epiretinal membrane and internal-limiting membrane were clearly identified, they could be completely removed. The clinical observations of the epiretinal membrane and internal-limiting membrane excised were confirmed by electron microscopy. Successful reattachment was obtained without damage to the retina. CONCLUSION: Removal of epiretinal membrane and internal-limiting membrane can be facilitated by using intravitreal indocyanine green during vitrectomy. We recommend further studies to confirm the benefit of this technique.

Investigation of patients' demand for community pharmacies: relationship between pharmacy services and patient satisfaction.

We performed an investigation on the patients' demand for community pharmacy based on the analysis of questionnaire responses on community pharmacy services from the patients at 32 pharmacies in Tokyo and Osaka. In the previous study, we developed seven evaluation indices for pharmacy services, and showed that the functions most sought by patients in the "ideal pharmacies" were "Attitude of pharmacy/pharmacist", "Convenient hours" and "Information management". The objective of this study is to determine the relationship between these pharmacy functions and patient satisfaction by analyzing responses from the same questionnaire survey. Overall satisfaction score with the "pharmacy used today" was employed as the dependent variable, while the six factors derived from the 26-item evaluation scale in the questionnaire by factor analysis were used as the independent variables. As a result of analysis, it was found that four variables had a significant positive correlation with patient satisfaction, one had a significant inverse correlation, and one showed no significant correlation (p < 0.05). These results suggest that: attitude of the pharmacists such as general attitude and specialized activities of pharmacy/pharmacist such as providing information and explanations, and convenience of hours are not only judged to be important by patients, but also influence their satisfaction; comfortable facilities and availability of OTC drugs, while rated relatively low by patients in terms of importance, do influence their satisfaction; and convenience of location does not influence patient satisfaction. It was also indicated that insufficient inventories of prescribed medications have an impact upon patient satisfaction. This investigation offers evidence to provide patient-based pharmacy services.

Visual function after foveal translocation with 360-degree retinotomy and simultaneous torsional muscle surgery in patients with myopic neovascular maculopathy.

PURPOSE: To assess functional and anatomical outcomes after foveal translocation with 360-degree retinotomy and simultaneous torsional muscle surgery in patients with myopic neovascular maculopathy. METHODS: Foveal translocation with 360-degree retinotomy was performed in 11 eyes of 11 patients with myopic neovascular maculopathy. Ten eyes had simultaneous torsional muscle surgery with recession of the superior oblique muscle and tucking of the inferior oblique muscle. Silicone oil removal with or without intraocular lens implantation was performed 2 to 8 weeks after the primary procedure. Visual acuity, binocular function, and degree of cyclotorsion were assessed preoperatively and postoperatively. Angles of retinal and globe rotation, distance of foveal shift, and surgical complications were also investigated. RESULTS: With a mean postoperative follow-up of 6.2 months (range, 3 to 13 months), vision improved (greater than 0.2 logarithm of minimal angle of resolution [logMAR] units) in eight eyes, was unchanged in two eyes, and worsened (greater than 0.2 logMAR units) in 1 eye. Seven of 11 eyes (64%) had a final visual acuity of 20/50 or better. Five patients developed or maintained binocular fusion, four patients continued to have suppression, and two patients developed diplopia that was managed by spectacles with Fresnel prisms. Subjective cyclotorsion was less than 8 degrees in 10 eyes. Mean retinal and globe rotations were 23.4 degrees and 19.8 degrees, respectively. Average size of the choroidal neovascular membrane was 0.8 disk diameter, whereas the average distance of foveal shift was 1.5 disk diameter. After the primary procedure, three eyes developed retinal detachment, one eye macular hole, and one eye proliferative vitreoretinopathy. These complications were successfully managed by additional surgery. CONCLUSION: Foveal translocation with 360-degree retinotomy is effective in restoring vision in some patients with myopic neovascular maculopathy. Although the development of torsional diplopia is generally obviated by simultaneous extraocular muscle surgery, a relatively high incidence of surgical complications should be taken into account with this procedure.

Establishment of hybridomas producing cancer specific human antibodies from B cell line derived from PBL of a patient with adult T cell leukemia.

Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of CD4(+) T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I) and concurs with an autoimmune disease and cancer due to attenuated immune response. In this study, we established ATL patient derived B-cell line TM-1 producing cancer-specific IgM antibodies, and further characterized its antigen specificity by establishing hybridomas fused with human-mouse origin hetero-myeloma cell line RF-S1. We established three hybridoma cell lines termed 2E12, 3E9, and 3E10, which continuously secreted human IgM antibodies. Immunohistochemical staining of formalin-fixed tissue section using antibodies secreted from these hybridomas showed that these antibodies specifically recognized tumor sites of human colon adenocarcinomas. Antibody produced from hybridoma 3E9 bound to some of leukemic cell lines, but not to normal human PBL, which was evidenced by the flow cytometric analysis, indicating that antibody produced from 3E9 recognizes cell surface antigen specifically expressed in the leukemic cells.