Laparoendoscopic Single-site Plus 1-port Donor Nephrectomy: Division of Roles to Shorten Warm Ischemic Time.

BACKGROUND: In this study we present our new surgical procedure, laparoendoscopic single-site surgery plus 1 for donor nephrectomy (LESS+1-DN), which shortens warm ischemic time (WIT) and improves surgical outcomes. METHODS: From January 2013 to February 2017, 15 patients who underwent LESS-DN and 41 patients who underwent LESS+1-DN at our institution were evaluated retrospectively. Patients were divided into 3 groups: group A, 15 cases of LESS-DN; group B, the first 15 patients who underwent LESS+1-DN; and group C, 26 patients who underwent subsequent LESS+1-DN. To reduce WIT, we clearly defined the roles of the surgeon and first assistant in the 26 subsequent LESS+1-DN cases. The surgeon dissected the renal pedicle and harvested the kidney graft using a recovery bag and the first assistant held the recovery bag. RESULTS: The mean operative time in group C (213.7 minutes) was significantly shorter than that in groups A (253.3 minutes) and B (253.8 minutes). The WIT in group C (195.2 seconds) was significantly shorter than that in groups A (389.8 seconds) and B (313.2 seconds). Open conversion was required in 1 case in group A. None of the donors required conversion to open surgery and no perioperative complications occurred in groups B and C. Linear regression analysis of the LESS+1-DN operative times and consecutive case numbers demonstrated a shallow learning curve (R2 = 0.392, P < .05). CONCLUSION: Our new procedure that divides the roles of the operator and the first assistant contributed significantly to a shortening of WIT. Dividing roles can facilitate a safer laparoscopic donor nephrectomy.

Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay.

INTRODUCTION: Antithrombin resistance (ATR) is a novel thrombotic risk in abnormal prothrombins. A manual ATR assay using Oxyuranus scutellatus (Ox) venom as a prothrombin activator was established for detecting antithrombin-resistant prothrombin. However, this assay was limited because of Ox snake venom availability and its throughput capacity. Here, we have improved the ATR assay using bovine factors Xa and Va (FXa/Va) as prothrombin activators and have optimised assay conditions for an automated instrument (ACL TOP 500). METHODS: Diluted plasma was incubated with a prothrombin activator mix (phospholipids, CaCl2 , and bovine FXa/Va), followed by inactivation with antithrombin for 10, 20 and 30 minutes. We added a chromogenic substrate S-2238, and assessed changes in absorbance/min at 405 nm. We also adapted assay conditions for ACL TOP 500. RESULTS: Optimum conditions for FXa/Va treatment were 6.25% phospholipids, 5 mM CaCL2 , 0.01 µg/mL FXa and 0.1 µg/mL FVa. ATR assay kinetics with the FXa/Va activator was comparable with that with the Ox activator in heterozygous reconstituted plasma with the recombinant wild-type or antithrombin-resistant prothrombin. Using ACL TOP 500, optimum conditions for the FXa/Va treatment were 10.0% phospholipids, 5 mM CaCl2 , 0.02 µg/mL FXa and 0.2 µg/mL FVa. The automated ATR assay with the FXa/Va activator demonstrated good detectability for antithrombin-resistant prothrombin in plasma from a heterozygous carrier with prothrombin Yukuhashi or Belgrade. CONCLUSION: We optimised the ATR assay with the FXa/Va activator and adapted the assay for ACL TOP 500; the assay showed the ability to clearly detect antithrombin-resistant prothrombin in manual and automated procedures.

Nitrogen balance in patients with hemiparetic stroke during the subacute rehabilitation phase.

BACKGROUND: In highly invasive diseases, metabolism commonly changes. Hypercatabolism is frequent in acute stroke, and nitrogen balance tends to be negative. However, there has been no study describing nitrogen balance in subacute and chronic stroke patients. The present study aimed to examine nitrogen balance in the subacute and chronic phases and to identify the factors related to it. METHODS: Nitrogen balance was calculated from the collected urine of 56 patients with subacute stroke [mean (SD) 53.8 (18.4) days post-stroke] who were admitted for rehabilitation for their first-ever ischaemic or nonsurgical haemorrhagic stroke. In the first experiment, their nitrogen balance was measured during the rehabilitation phase, and factors (type, severity of hemiparesis, activities of daily living, dysphagia and malnutrition status) related to it were evaluated. The second experiment was performed to describe the time course of nitrogen balance in 31 consecutive patients, with assessments made at admission and at discharge. RESULTS: Nitrogen balance was positive in all patients in the subacute phase. A significant difference was seen in nitrogen balance between high and low fat-free mass in male patients. In the chronic phase, nitrogen balance was positive in 96% of the patients. There was no significant difference in nitrogen balance between discharge and admission. CONCLUSIONS: In the subacute and chronic phases of stroke, it was confirmed that hypercatabolism had resolved and that intensive rehabilitation is possible in the convalescent period of stroke.

Early neonatal deaths with perinatal asphyxia in very low birth weight Brazilian infants.

OBJECTIVE: The objective of this study was to assess the frequency of early deaths associated with birth asphyxia of very low birth weight infants between 2005 and 2010, in Brazil. STUDY DESIGN: This population study enrolled all live births with birth weight from 400 to 1499 g, gestational age ⩾ 22 weeks, without malformations that died up to 6 days after birth with perinatal asphyxia. Asphyxia was defined if intrauterine hypoxia, asphyxia at birth or meconium aspiration syndrome were written in any line of the death certificate. Active search was carried out in 27 Brazilian federative units. RESULT: For every 1000 live births of very low birth weight infants without congenital malformations, 40.25 and 32.38 died with birth asphyxia in the first week after birth, respectively, in 2005 and 2010 (P<0.001). The contribution of birth asphyxia to early neonatal death of these infants was approximately 10 to 12% all study years. CONCLUSION: Reduction of birth asphyxia in very low birth weight infants is essential to reducing neonatal mortality in Brazil.

Observations in the diagnosis of cervical myelopathy in patients suffering from diabetes mellitus.

OBJECTIVES: To identify any observations that could aid in the diagnosis of cervical myelopathy in patients suffering from diabetes mellitus (DM). We compared the preoperative neurological findings in patients with cervical myelopathy among non-diabetics, mild diabetics and severe diabetics. STUDY DESIGN: A retrospective comparative study. SETTING: Department of Orthopaedic Surgery, Wakayama Medical University, Japan. METHODS: We retrospectively reviewed 111 patients who had undergone laminoplasty for cervical compressive myelopathy: 56 without DM and 29 with severe diabetes more than 10 years of medication; more than 7.0% HbA1c; diabetic retinopathy; and delayed conduction velocity of peripheral nerves. For preoperative neurological assessment we compared the following among the three groups: the 10 s test whereby the myelopathy in the hand was quantified; sensory disturbance; deep tendon reflexes; Hoffmann's, Trömner's and Babinski's reflexes; and bladder dysfunction. RESULTS: There was no significant difference preoperatively in the 10 s test between the groups. Deep tendon reflexes were significantly decreased in group S. There were no significant differences in sensory disturbance and bladder dysfunction. Although Hoffmann's and Trömner's reflexes significantly disappeared in group S, there was no significant difference in positivity of Babinski's reflex between the groups. CONCLUSIONS: The 10 s test and Babinski's reflex are helpful for the diagnosis of cervical myelopathy in patients suffering from DM.

Difference in coronary artery intima and media calcification in autopsied patients with chronic kidney disease.

BACKGROUND: In patients with chronic kidney disease (CKD), coronary artery calcification occurs at two distinct sites in the vessel wall: the intima and the media. Arterial media calcification (AMC), a nonocclusive condition, affects hemodynamics differently compared to arterial intima calcification (AIC), which occurs in atherosclerotic plaques. Arterial calcification is considered a cell-regulated process resembling intramembranous bone formation. The purpose of this retrospective observational study was to clarify the morphological differences between AIC and AMC and to evaluate the role of vascular smooth muscle cells (VSMCs) and macrophages in AIC and AMC formation. METHODS: We histologically analyzed 14 tissue specimens from 14 autopsies of patients with CKD Stage 5D who underwent hemodialysis and 5 specimens from 5 patients with CKD Stage 2 - 3 (90 ml/min/1.73 m2 > estimated GFR >= 30 ml/min/1.73 m2). We performed immunohistochemical staining of osteopontin (OPN) as a marker for bone matrix protein, alpha-smooth muscle actin (alphaSMA) for VSMCs, Cbfa1/Runx2 as a marker for osteoblastic differentiation of VSMCs, and CD68 for macrophages. RESULTS: In the CKD 2/3 group, we also found AIC and AMC. OPN and CD68 expression in the CKD 2/3 group was similar to that in the CKD 5D group. Although we did not find Cbfa1/Runx2 positive cell expression in the CKD 2/3 group, we did find it in the CKD 5D group. We found CD68-positive cells predominantly in AIC and absent in AMC in both groups. CONCLUSIONS: These findings suggest that the influence of Cbfa1/Runx2 pathway in coronary artery calcification depends on the CKD Stage. Expression of CD68-positive cells depends on the location of the coronary artery calcification.

Assessment of risk factors for low bone mineral density in Brazilian postmenopausal women.

OBJECTIVE: To assess risk factors associated with low bone mineral density (BMD) in postmenopausal women. METHODS: In this cross-sectional study, a total of 412 Brazilian postmenopausal women, aged 40-75 years, with BMD measured using central dual-energy X-ray absorptiometry, were included. The clinical risk factors assessed were: age, time since menopause, smoking, physical activity, use of hormone therapy (HT) or corticosteroids, personal fracture history, maternal history of fracture, and body mass index (BMI, weight/height(2)). Low BMD was considered when total spine and/or femoral neck T-score values were ≤ -2.0 standard deviations. Logistic regression was used to determine the odds ratio (OR) for low BMD in the presence of the influential variables analyzed. RESULTS: Low BMD, which occurred in 36.6% (151/412) of the participants, was observed in 22.4% of women aged 40-49 years, in 34.2% of those aged 50-59 years, and in 60.5% of those > 60 years (p < 0.001). Similarly, low BMD was observed in 21.9% of women with menopause duration ≤ 5 years, in 39.5% with a duration of 6-10 years, and in 57.7% with menopause duration of > 10 years (p < 0.001). Seventy percent of women with BMI < 20 kg/m(2) were osteopenic/osteoporotic (p < 0.001). The percentage of HT users was 37.4%; 27.7% took regular physical activity and 24.5% were smokers. The risk for low BMD detection increased significantly with age (OR 1.08; 95% confidence interval (CI) 1.02-1.14), time since menopause (OR 1.12; 95% CI 1.04-1.20), smoking (OR 3.43; 95% CI 1.67-6.96), fracture history (OR 2.05; 95% CI 1.11-3.78), and maternal history of fracture (OR 2.16; 95% CI 1.14-4.09). Physical activity, diet, corticotherapy and thyropathies did not influence risk. Contrarily, use of HT (OR 0.38; 95% CI 0.24-0.60) and high BMI (OR 0.89; 95% CI 0.84-0.96) reduced risk (p < 0.05). CONCLUSION: In postmenopausal women, age, time since menopause, smoking, and personal or maternal history of fracture were strong clinical indicators of risk for low BMD, whereas the use of hormone therapy and high BMI were shown to be protective factors.

Promotion of beta-cell differentiation by the alkaloid conophylline in porcine pancreatic endocrine cells.

We previously found that conophylline, an alkaloid isolated from the leaves of Ervatamia microphylla, induced beta-cell differentiation in rat pancreatic acinar carcinoma cells and in cultured fetal rat pancreatic tissue and that it also decreased the blood glucose level in streptozotocin-treated fetal rats. In the present research, we looked into the effect of conophylline on the differentiation of newborn pig pancreatic endocrine cells into insulin-secreting cells. Conophylline potentiated the differentiation of monolayer cells into insulin-producing cells in the presence of nicotinamide in 3 weeks. Next we prepared islet-like cell clusters (ICC). Cononophylline together with nicotinamide also increased the number of insulin-producing cells and the insulin content in ICC in 3-6 weeks. The ICC thus prepared were sensitive to the glucose concentration for the insulin secretion. Conophylline increased the mRNA expression of PDX-1, neurogenin3, neuroD/Beta2, and insulin in ICC. Thus, the vinca alkaloid conophylline potentiated beta-cell differentiation in porcine pancreatic endocrine-rich cells in cluster cultures. Pig pancreatic cells are practical candidate for use in transplantation therapy. Conophylline may thus be useful for the large-scale preparation of porcine insulin-producing cells for the regeneration therapy of type-1 diabetes mellitus.

The critical velocity and 1500-m surface performances in Finswimming.

The purpose of this investigation was to determine whether the concepts of critical velocity (CV) and anaerobic swimming capacity (ASC) could be used by coaches as a reliable index in order to monitor 1500-m Surface (SF) performances in Finswimming. Thirteen Finswimmers (6 males and 7 females, 24+/-6 years), members of the Japanese national team, were instructed to swim three different swimming distances (400-, 800-, and 1500-m) at maximal effort in a 50m long course swimming pool. CV and the ASC were calculated using 400-m and 800-m swim times. Mean height and body mass were 170.2 cm and 69.7 kg in male and 160.5 and 61.0 kg in female. A highly positive correlation was found between the CV and the mean velocity of 1500-m SF (V1500) (r=0.91, P<0.01), but no correlation was found between the ASC and V1500. (r=0.46, P=0.11). However, a high correlation was found between the ASC and the residual error of V1500, calculated from the relationship between V1500 and the CV (r=0.89, P<0.01). These results suggest that the CV is a useful method for evaluating 1500-m SF performance and an aerobic performance expressed as the CV contributes to 1500-m SF performance.

Vasopressin-dependent upregulation of aquaporin-2 gene expression in aged rats with glucocorticoid deficiency.

AIM: The study was undertaken to determine whether ageing affects kidney expression of the aquaporin-2 (AQP2) water channel in glucocorticoid-deficient rats. METHODS: After adrenalectomy, 6- and 52-week-old Sprague-Dawley rats received aldosterone via osmotic minipumps (glucocorticoid-deficient rats). Aldosterone and dexamethasone were administered to control rats of the same age. RESULTS: An acute water load test verified impairment of water excretion in both young and aged rats with glucocorticoid deficiency, with a more serious impairment in the older rats. Despite the presence of hypoosmolality, non-suppressible release of arginine vasopressin (AVP) was particularly evident in the aged rats with glucocorticoid deficiency in comparison with the young rats. The expression levels of AQP2 mRNA and protein were lower in the aged rats, with a particularly large reduction in AQP2 protein expression. AQP2 expression levels were significantly augmented in the glucocorticoid-deficient rats compared with the controls under both basal and water-loaded conditions. Acute water loading did not suppress expression of AQP2 mRNA and protein, and the percentage increases in AQP2 mRNA and protein expression vs. the respective controls were more pronounced in the 52-week-old glucocorticoid-deficient rats compared with the 6-week-old rats. CONCLUSION: The findings indicate that upregulation of AQP2 expression is maintained dependent upon non-suppressible release of AVP in rats with glucocorticoid deficiency, and that AQP2 plays a crucial role in persistent impairment of water excretion in aged rats with glucocorticoid deficiency.

The origin of the premotor potential recorded from the second lumbrical.

PURPOSE: When recording with a palm electrode, a premotor potential (PMP) precedes the compound muscle action potential evoked from the second lumbrical muscle following median nerve stimulation. The origin of the premotor potential has remained uncertain. The aim of this study was to determine whether the PMP-2L is a SNAP derived from antidromically activated digital sensory branches of the median nerve. METHODS: We recorded three active electrodes were placed over the second lumbrical muscle, the third lumbrical muscle, the fourth lumbrical muscle by multi-channel recordings. RESULTS: PMPs are recorded only over the median digital sensory branches after stimulating the median nerve, while they are recorded only over the ulnar branch after stimulating the ulnar nerve. CONCLUSIONS: We conclude that the origin of the PMP is a SNAP arising from antidromically activated digital sensory branches.

A new atomic force microscope force ramp technique using digital force feedback control reveals mechanically weak protein unfolding events.

We have developed a new force ramp modification of the atomic force microscope (AFM) which can control multiple unfolding events of a multi-modular protein using software-based digital force feedback control. With this feedback the force loading rate can be kept constant regardless the length of soft elastic linkage or number of unfolded polypeptide domains. An unfolding event is detected as a sudden drop in force, immediately after which the feedback control reduces the applied force to a low value of a few pN by lowering the force set point. Hence the remaining folded domains can relax and the subsequent force ramp is applied to relaxed protein domains identically in each case. We have applied this technique to determine the kinetic parameters x(u), which is the distance between the native state and transition state, and α(0), which is the unfolding rate constant at zero force, for the mechanical unfolding of a pentamer of I27 domains of titin. In each force ramp the unfolding probability depends on the number of folded domains remaining in the system and we had to take account of this effect in the analysis of unfolding force data. We obtained values of x(u) and α(0) to be 0.28 nm and 1.02 × 10(-3) s(-1), which are in good agreement with those obtained from conventional constant velocity experiments. This method reveals unfolding data at low forces that are not seen in constant velocity experiments and corrects for the change in stiffness that occurs with most mechanical systems throughout the unfolding process to allow constant force ramp experiments to be carried out. In addition, a mechanically weak structure was detected, which formed from the fully extended polypeptide chain during a force quench. This indicates that the new technique will allow studies of the folding kinetics of previously hidden, mechanically weak species.

The effects of time of first cleavage, developmental stage, and delipidation of nuclear-transferred porcine blastocysts on survival following vitrification.

The effect of removing cytoplasmic lipid droplets (delipidation) at the 2-cell and developmental stages on the survival of porcine somatic cell nuclear-transferred blastocysts developed from the enucleated oocytes receiving somatic cells from kidney of an adult female after cryopreservation was examined. Vitrification was performed using the Cryoloop method with a small volume of medium (0.5 microl). To select 2-cell embryos with a high potential to develop into blastocysts, the relationship between the timing of the first cleavage and the developmental potential was examined. The potential of nuclear-transferred oocytes to develop into blastocysts in the intermediate-cleavage group (20-24h after activation, 25%) was slightly or significantly (P<0.05) higher than that in fast-cleavage (<20 h after activation, 13%) and slow-cleavage groups (>24h after activation, 5%). Most non-delipidated blastocysts did not survive after thawing (0% for early-stage and 9% for advanced-stage blastocysts), but the survival rate of delipidated blastocysts 48 h after culture (54% and 72%, respectively) was not significantly different from that of non-vitrified blastocysts (80% and 92%, respectively). The survival rate of advanced-stage blastocysts after vitrification was slightly higher than that of early-stage blastocysts. The present study demonstrates that somatic cell nuclear-transferred porcine blastocysts developed from embryos selected at the 2-cell stage can be preserved by vitrification with a small volume of medium if the lipid droplets of the embryos are first removed.

Functional analysis of cytomegalovirus-specific T lymphocytes compared to tetramer assay in patients undergoing hematopoietic stem cell transplantation.

In order to evaluate whether we could predict reactivation of CMV by monitoring the number of CMV-specific cytotoxic T-lymphocytes (CTL), tetramer analysis was performed in 37 patients who underwent hematopoietic stem cell transplantation (HSCT). The results disclosed that the mean number of CMV-specific CTL at day 30 did not differ among patients who developed CMV antigenemia (22/microl) and those who did not (12/microl). Serial tetramer analysis showed that 21% of the patients had >10/microl CMV-specific CTL at the first detection of CMV antigenemia and 67% of the patients had more than 10/microl CMV-specific CTL at the onset of CMV disease. Intracellular staining upon stimulation by CMV lysates and peptide in patients with CMV colitis revealed that both IFN-gamma producing CD4+ and CD8+ lymphocytes were suppressed at the onset of CMV colitis (1.6 and 8/microl), which increased with recovery of the disease (19 and 47/microl). These data suggest that it is difficult to predict CMV reactivation solely by the number of CMV-specific CTL. We suggest that additional functional analysis by intracellular cytokine assay may be useful for immunomonitoring against CMV.

The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

Methotrexate-related lymphomatoid granulomatosis: a case report of spontaneous regression of large tumours in multiple organs after cessation of methotrexate therapy in rheumatoid arthritis.

We describe a 54-year-old female patient with rheumatoid arthritis (RA) and Sjögren's syndrome (SS) who presented with right chest pain and a large mass visible in the upper right field of a chest X-ray. Computed tomography (CT) showed multiple tumours in both lungs, the liver, and the spleen. The right lung tumour was 8 cm in diameter with a cavity. Biopsy of the lung and liver revealed lymphomatoid granulomatosis (LG) and diffuse large B-cell lymphoma (DLBCL). These lesions spontaneously regressed after withdrawal of methotrexate without any therapy for the lymphoma. This is the first report of self-limiting LG in a patient, complicated with methotrexate-treated RA.

Posterior endoscopic surgery for lumbar disc herniation with contralateral symptoms - a report of two cases.

We report two cases of lumbar disc herniation with contralateral nerve root involvement, surgically treated with a microendoscopic disectomy system (METRx-MED system). The nerve root of the symptomatic side (contralateral to the side of the disc herniation) had been compressed to the superior facet by herniated disc from the opposite side. Endoscopic observation revealed inflammatory findings of the nerve root on the symptomatic side, such as fibrosis, adhesion, redness and swelling. In contrast, on the non-symptomatic side (ipsilateral side of the disc herniation), the nerve root had been merely compressed by the herniated disc but did not demonstrate any inflammatory findings. Excision of the herniated disc and decompression of the non-symptomatic nerve root should be done first, approaching from the disc herniation side. After that, through the same approach, the nerve root of the opposite (symptomatic) side should be decompressed.