Application of complement component 4d immunohistochemistry to ABO-compatible and ABO-incompatible liver transplantation.

Antibody-mediated rejection (AMR) is difficult to diagnose after ABO-compatible or ABO-identical (ABO-C) liver transplantation. To determine whether complement component 4d (C4d) immunostaining would be useful for diagnosing AMR, we compared the results of C4d immunohistochemistry for allograft biopsy samples with assays for anti-donor antibodies performed at the time of biopsy. One hundred fourteen patients with ABO-C grafts and 29 patients with ABO-incompatible (ABO-I) grafts were included. Linear C4d endothelial staining (identifiable with a 4× objective lens) or staining seen in 50% or more of the portal tracts was considered positive. Five of the 114 patients (4%) with ABO-C grafts and 15 of the 29 patients (52%) with ABO-I grafts showed C4d positivity. In the ABO-C cases, C4d positivity in late biopsy samples (≥30 days after transplantation) was associated with stage 2 or higher fibrosis (METAVIR score; P = 0.01) and with the presence of donor-specific anti-human leukocyte antigen DR antibodies (HLA-DR DSAs) with a mean fluorescence intensity > 5000 according to the Luminex single-antigen bead assay (P = 0.04). Conversely, the presence of HLA-DR DSAs was associated with the presence of stage 2 or higher fibrosis, acute cellular rejection, and C4d positivity. During the 2-year follow-up, neither C4d positivity nor HLA-DR DSAs were related to graft loss. Among ABO-I patients, C4d positivity was not associated with allograft dysfunction or fibrosis. Only 3 of the 15 C4d-positive patients (20%) showed periportal hemorrhagic edema, which could be a histological sign of AMR in ABO-I grafts, and they were the only cases associated with elevations in anti-donor A/B antibody titers. In conclusion, C4d endothelial positivity among ABO-C patients is an uncommon event that could be associated with chronic graft damage with or without clinical AMR. C4d positivity is common among ABO-I patients and may not be associated with allograft dysfunction if alloantibody titers are not elevated

キョウカンセイ ト カンジョウ カクチ ノ カンレンセイ ニツイテノ ケンキュウ

We tested the hypothesis that the emotional awareness of counselors is correlated with their levels of empathy using two scales. The Empathic Experience Scale Revised measured counselors\u27 levels of empathy with regard to two aspects: the level of shared experiences and the level of unshared experiences. The Level of Emotional Awareness Scale was used to evaluate counselors\u27 levels of emotional awareness. Clinical psychologists and graduate students who were studying clinical and counseling psychology were instructed to respond to the utterances of hypothetical clients from the Therapist Response Questionnaire. Results revealed that counselors\u27 levels of emotional awareness were positively related to the extent of their experience in clinical practice; however, empathy was not found to be related with either of these factors. In items for which the clients\u27 utterances were complicated, the level of empathy and emotional awareness were positively related. Therefore, our hypothesis that a greater level of awareness of one\u27s own emotions is associated with a greater level of empathy was supported in situations involving complicated cases. This result suggests the need for awareness training for counselors in graduate schools

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

<p>Abstract</p> <p>Background</p> <p>Various protein expression systems, such as <it>Escherichia coli </it>(<it>E. coli</it>), <it>Saccharomyces cerevisiae </it>(<it>S. cerevisiae</it>), <it>Pichia pastoris </it>(<it>P. pastoris</it>), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β₂adrenergic receptor, adenosine A_2areceptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, <it>P. pastoris </it>and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.</p> <p>Results</p> <p>The ideal conditions for the expression of CHRM2 in <it>P. pastoris </it>were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [³H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by <it>P. pastoris </it>was lower than that of Sf9 insect cells, CHRM2 yield in <it>P. pastoris </it>was 2-fold higher than in Sf9 insect cells because <it>P. pastoris </it>was cultured at high cell density. The dissociation constant (Kd) for QNB in <it>P. pastoris </it>was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between <it>P. pastoris </it>and Sf9 insect cells.</p> <p>Conclusion</p> <p>Compared to insect cells, <it>P. pastoris </it>is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, <it>P. pastoris</it>, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in <it>P. pastoris </it>can be applied to the structural and biochemical studies of GPCRs.</p

Prognostic Factors for Post Recurrence Survival in Resected Pathological Stage I Non-small Cell Lung Cancer

[Background]Recurrence of lung cancer after surgical resection is a major obstacle in the cure and long-term survival of patients and has become the most common cause of death. However, prognostic factors and efficacy of therapy after recurrence remain controversial. We evaluated the prognostic factors of post recurrence survival (PRS) in patients of resected stage I non-small cell lung cancer (NSCLC). [Methods]Of the 551 patients who underwent surgery　for stage I NSCLC between 2005 and 2013, we reviewed 89 (16.2%) patients who had recurrence. We examined PRS using the Kaplan?Meier method and multivariate　Cox regression analyses. [Results]The median follow-up period after recurrence was 21.0 months. The median recurrence free interval (RFI) was 16.8 months. The 1-year PRS and 3-year PRS were 65.6% and 44.7%, respectively. Multivariate analysis revealed that size of primary lesion > 25 mm (P = 0.048), RFI ? 17 months (P = 0.048) and no treatment for recurrence (P < 0.001) were independent poor-prognosis factors of PRS. We further examined PRS in 66 patients who underwent any post recurrence therapy. For the patients who underwent treatment after recurrence, bone metastasis (P = 0.025) and treatment without epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) (P = 0.049) were independent poor prognostic factors. [Conclusion]PRS may be associated with characteristics of a recurrent lesion, including the biology of the recurrent tumor, RFI, recurrent site, the treatment for recurrence, rather than characteristics of primary lesion. Although further validation is needed, this information is important for the design of clinical trials for post-recurrence therapy

Crystal structure of the anion exchanger domain of human erythrocyte band 3

Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease

Thoracoscopic Resections of Bronchogenic Cysts Arising in the Posterior Mediastinum: A Report on 3 Patients

We report 3 cases of patients with bronchogenic cyst arising in the posterior mediastinum. The patients were a 19-year-old male, a 36-year-old female and a 33-year-old female, whose cysts were detected as abnormal shadows in the chest. In 2 of them, neurogenic tumors were suspected preoperatively. We performed thoracoscopic resection for the 3 tumors. Bronchogenic cysts may occur ectopically, and the posterior mediastinum must be sufficiently recognized as a rare but possible ectopic site