Isolated vascular "v" lesions in liver allografts: How to approach this unusual finding.

According to the Banff criteria for kidney allografts, isolated vascular or "v" lesions are defined as intimal inflammation, age-inappropriate fibro-intimal hyperplasia, or both, without the presence of associated interstitial T cell-mediated rejection (TCMR). In general, these lesions portend a worse outcome for kidney allografts, particularly in those where the "v" lesions are identified in patients with coexistent donor specific antibodies (DSA) or later after transplantation. Although affected arteries are rarely sampled in liver allograft biopsies, we identified nine patients at a mean of 1805 days posttransplantation and compared these to matched controls. Almost half (4 of 9) of the study patient biopsies showed inflammatory arteritis associated with focal or diffuse C4d positivity, which was not observed in matched controls. One "v" lesion patient progressed to rejection-related graft failure and two developed moderate/severe TCMR in subsequent biopsies, whereas only one rejection episode occurred in follow-up biopsies, and no rejection-related deaths or graft failures were detected in controls. In conclusion, patients with liver allograft isolated "v" lesions should undergo further evaluation and closer follow-up for impending TCMR and/or underlying co-existent chronic antibody-mediated rejection (AMR).

Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity-2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy-diagnosed HTx rejection (cellular and/or antibody-mediated), serum sST2 was elevated compared to rejection-free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor-α and IL-1ß as upstream activators. In total, our data indicate that alloimmune-associated pro-inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

A novel dual ex vivo lung perfusion technique improves immediate outcomes in an experimental model of lung transplantation.

The lungs are dually perfused by the pulmonary artery and the bronchial arteries. This study aimed to test the feasibility of dual-perfusion techniques with the bronchial artery circulation and pulmonary artery circulation synchronously perfused using ex vivo lung perfusion (EVLP) and evaluate the effects of dual-perfusion on posttransplant lung graft function. Using rat heart-lung blocks, we developed a dual-perfusion EVLP circuit (dual-EVLP), and compared cellular metabolism, expression of inflammatory mediators, and posttransplant graft function in lung allografts maintained with dual-EVLP, standard-EVLP, or cold static preservation. The microvasculature in lung grafts after transplant was objectively evaluated using microcomputed tomography angiography. Lung grafts subjected to dual-EVLP exhibited significantly better lung graft function with reduced proinflammatory profiles and more mitochondrial biogenesis, leading to better posttransplant function and compliance, as compared with standard-EVLP or static cold preservation. Interestingly, lung grafts maintained on dual-EVLP exhibited remarkably increased microvasculature and perfusion as compared with lungs maintained on standard-EVLP. Our results suggest that lung grafts can be perfused and preserved using dual-perfusion EVLP techniques that contribute to better graft function by reducing proinflammatory profiles and activating mitochondrial respiration. Dual-EVLP also yields better posttransplant graft function through increased microvasculature and better perfusion of the lung grafts after transplantation.

Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs.

INTRODUCTION: This study evaluates polyomavirus JC (JCV) large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication. METHODS: Recombinant JCV LTA was produced in an Escherichia coli based expression plasmid. ATPase activity was measured using the malachite green assay. A high throughput screen was completed using a brain-biased library of 75,000 drug-like compounds selected for physicochemical properties consistent with blood-brain barrier permeability. RESULTS: Five compounds showed non-competitive inhibition of ATPase activity with an EC50 ⩽ 15 µM. Modest antiviral activity was demonstrated in an immunofluorescence assay for JCV VP-1 expression in COS7 cells (EC50 15, 18, 20, 27, and 52 µM respectively). The compounds also inhibited viral replication in a real time PCR assay at comparable concentrations. LD50 in the MTS96 and Cell TiterGlo assays was >100 µM for all compounds in COS7 as well as HEK293 cells. However, two compounds inhibited cell proliferation in culture with IC50 values of 43 and 34 µM respectively. Despite substantial amino acid similarity between polyomavirus JC, BK and SV40 proteins, these compounds differ from those previously reported to inhibit SV40 LTA ATPase in chemical structure as well as a non-competitive mechanism of inhibition. CONCLUSION: LTA ATPase is a valid target for discovery. Additional screening and chemical optimization is needed to develop clinically useful compounds with less toxicity, which should be measured by metabolic as well as cell proliferation assays.

"Plasma cell hepatitis" in liver allografts: identification and characterization of an IgG4-rich cohort.

Plasma cell hepatitis (PCH), also known as "de novo autoimmune" hepatitis, is an increasingly recognized, but suboptimally named and poorly understood, category of late allograft dysfunction strongly resembling autoimmune hepatitis (AIH): They share plasma-cell-rich necro-inflammatory activity on biopsy, autoantibodies and steroid responsiveness, but overlap with rejection is problematic. A retrospective study of clinical, serological, histopathological and IgG4 immunohistological features of PCH (n = 20) in liver allograft recipients, native liver AIH (n = 19) and plasma-cell-rich renal allograft rejection (n = 20) showed: (1) high frequency (44%) of HLA-DR15; (2) less female predominance (p = 0.03) and (3) n = 9/20 PCH recipients showed >25 IgG4+ plasma cells/high-power field (IgG4+ PCH) versus AIH (n = 1/19, p = 0.008) or plasma-cell-rich kidney rejection (n = 2/20, p = 0.03). The IgG4+ PCH (n = 9) subgroup showed lower alanine transaminase (ALT) (p < 0.01) and aspartate transaminase (AST) (p < 0.05) at index biopsy but (a) higher plasma cell number/percentage, (b) more aggressive-appearing portal/periportal and perivenular necro-inflammatory activity and (c) more severe portal/periportal fibrosis than IgG4- PCH (n = 11). Significant demographic, histopathologic and plasma cell phenotype differences between PCH and AIH suggest distinct pathogenic mechanisms for at least the IgG4+ PCH subgroup likely representing an overlap between allo- and auto-immunity. IgG4+ PCH was associated with fibrosis, but also highly responsive to increased immunosuppression.

STAT1-regulated lung MDSC-like cells produce IL-10 and efferocytose apoptotic neutrophils with relevance in resolution of bacterial pneumonia.

Bacterial pneumonia remains a significant burden worldwide. Although an inflammatory response in the lung is required to fight the causative agent, persistent tissue-resident neutrophils in non-resolving pneumonia can induce collateral tissue damage and precipitate acute lung injury. However, little is known about mechanisms orchestrated in the lung tissue that remove apoptotic neutrophils to restore tissue homeostasis. In mice infected with Klebsiella pneumoniae, a bacterium commonly associated with hospital-acquired pneumonia, we show that interleukin (IL)-10 is essential for resolution of lung inflammation and recovery of mice after infection. Although IL-10(-/-) mice cleared bacteria, they displayed increased morbidity with progressive weight loss and persistent lung inflammation in the later phase after infection. A source of tissue IL-10 was found to be resident CD11b(+)Gr1(int)F4/80(+) cells resembling myeloid-derived suppressor cells (MDSCs) that appeared with a delayed kinetics after infection. These cells efficiently efferocytosed apoptotic neutrophils, which was aided by IL-10. The lung neutrophil burden was attenuated in infected signal transducer and activator of transcription 1 (STAT1)(-/-) mice with concomitant increase in the frequency of the MDSC-like cells and lung IL-10 levels. Thus, inhibiting STAT1 in combination with antibiotics may be a novel therapeutic strategy to address inefficient resolution of bacterial pneumonia.

Re-examination of the lymphocytotoxic crossmatch in liver transplantation: can C4d stains help in monitoring?

C4d-assisted recognition of antibody-mediated rejection (AMR) in formalin-fixed paraffin-embedded tissues (FFPE) from donor-specific antibody-positive (DSA+) renal allograft recipients prompted study of DSA+ liver allograft recipients as measured by lymphocytotoxic crossmatch (XM) and/or Luminex. XM results did not influence patient or allograft survival, or cellular rejection rates, but XM+ recipients received significantly more prophylactic steroids. Endothelial C4d staining strongly correlates with XM+ (<3 weeks posttransplantation) and DSA+ status and cellular rejection, but not with worse Banff grading or treatment response. Diffuse C4d staining, XM+, DSA+ and ABO- incompatibility status, histopathology and clinical-serologic profile helped establish an isolated AMR diagnosis in 5 of 100 (5%) XM+ and one ABO-incompatible, recipients. C4d staining later after transplantation was associated with rejection and nonrejection-related causes of allograft dysfunction in DSA- and DSA+ recipients, some of whom had good outcomes without additional therapy. Liver allograft FFPE C4d staining: (a) can help classify liver allograft dysfunction; (b) substantiates antibody contribution to rejection; (c) probably represents nonalloantibody insults and/or complete absorption in DSA- recipients and (d) alone, is an imperfect AMR marker needing correlation with routine histopathology, clinical and serologic profiles. Further study in late biopsies and other tissue markers of liver AMR with simultaneous DSA measurements are needed.

Digital transplantation pathology: combining whole slide imaging, multiplex staining and automated image analysis.

Conventional histopathology is the gold standard for allograft monitoring, but its value proposition is increasingly questioned. "-Omics" analysis of tissues, peripheral blood and fluids and targeted serologic studies provide mechanistic insights into allograft injury not currently provided by conventional histology. Microscopic biopsy analysis, however, provides valuable and unique information: (a) spatial-temporal relationships; (b) rare events/cells; (c) complex structural context; and (d) integration into a "systems" model. Nevertheless, except for immunostaining, no transformative advancements have "modernized" routine microscopy in over 100 years. Pathologists now team with hardware and software engineers to exploit remarkable developments in digital imaging, nanoparticle multiplex staining, and computational image analysis software to bridge the traditional histology-global "-omic" analyses gap. Included are side-by-side comparisons, objective biopsy finding quantification, multiplexing, automated image analysis, and electronic data and resource sharing. Current utilization for teaching, quality assurance, conferencing, consultations, research and clinical trials is evolving toward implementation for low-volume, high-complexity clinical services like transplantation pathology. Cost, complexities of implementation, fluid/evolving standards, and unsettled medical/legal and regulatory issues remain as challenges. Regardless, challenges will be overcome and these technologies will enable transplant pathologists to increase information extraction from tissue specimens and contribute to cross-platform biomarker discovery for improved outcomes.

Periductal interleukin-17 production in association with biliary innate immunity contributes to the pathogenesis of cholangiopathy in primary biliary cirrhosis.

An innate immune response to bacterial components is speculated to be involved in the pathogenesis of primary biliary cirrhosis (PBC). Recently, CD4-positive T helper type 17 (Th17) cells, characterized by the secretion of interleukin (IL)-17, have been implicated in the pathogenesis of autoimmune diseases. Human Th17 cells are generated from Th0 cells by IL-6 and IL-1 beta and maintained by IL-23. In this study, the role of IL-17 in PBC and its association with biliary innate immunity were examined. Using cultured human biliary epithelial cells (BECs), the expression of Th17-related cytokines and chemokines and changes therein on treatment with pathogen-associated molecular patterns (PAMPs) and IL-17 were examined. Immunohistochemistry for IL-17 and Th17-related cytokines was performed using tissue samples of human liver. Consequently, the expression of IL-6, IL-1 beta, IL-23p19 and IL-23/IL-12p40 mRNAs, and their up-regulation by PAMPs, were found in BECs. Moreover, BECs possessed IL-17-receptors and stimulation with IL-17 induced production of IL-6, IL-1 beta, IL-23p19 and chemokines. Several IL-17-positive cells had infiltrated damaged bile ducts and the expression of IL-6 and IL-1 beta was enhanced in the bile ducts of PBC patients. In conclusion, IL-17-positive cells are associated with the chronic inflammation of bile ducts in PBC which is associated causally with the biliary innate immune responses to PAMPs.

Interferon gamma accelerates NF-kappaB activation of biliary epithelial cells induced by Toll-like receptor and ligand interaction.

BACKGROUND: The Toll-like receptor (TLR) family recognises pathogen associated molecular patterns (PAMPs) and plays a pivotal role in the innate immune response. Biliary epithelial cells (BECs) lining the intrahepatic bile ducts are potentially exposed to bacterial components in bile, and murine BECs possess TLRs that recognise PAMPs, resulting in nuclear factor kappaB (NF-kappaB) activation. AIMS: To examine the presence of TLRs in human BECs and the influence of cytokines and PAMPs on TLR expression and NF-kappaB activation. METHODS: The expression of TLR2-5, MD-2, MyD88, and IRAK1 was examined in human liver tissue and cultured BECs by immunohistochemistry or reverse transcription polymerase chain reaction. The influence of PAMPs (peptidoglycan and lipopolysaccharide) in cultured cells preincubated with interferon gamma (IFNgamma) was evaluated by NF-kappaB activation. RESULTS: TLR2-5, MyD88, and IRAK-1 proteins were detectable in BECs of the intrahepatic biliary tree in human liver tissue. TLR2-5, MD-2, MyD88, and IRAK-1 mRNA was demonstrated in human cultured BECs. The expression of these TLRs was upregulated by IFNgamma, and TLR2 was upregulated by tumour necrosis factor alpha. Interleukins 4 and 6 failed to induce TLR upregulation. Interestingly, preincubation with IFNgamma synergistically increased the upregulation of NF-kappaB induced by PAMPs in cultured BECs. CONCLUSION: These results suggest that the TLR family is present in human biliary cells and participates in the innate immunity of the intrahepatic biliary tree. Disordered regulation of TLRs after intracellular signalling by cytokines and PAMPs may be involved in immune mediated biliary diseases.

[Lung cancer with ground glass opacity diagnosed by transbronchial lung biopsy using an ultrathin bronchoscope and virtual bronchoscopy].

Although thoracic computed tomography (CT) screening indicated that there are many patients who have pulmonary shadow with ground glass opacity, it is sometimes difficult to obtain the appropriate specimens for histological diagnosis of such patients. We herein report a lung cancer patient with ground glass opacity who was diagnosed preoperatively by an ultrathin bronchoscope and virtual bronchoscopy. A 78-year-old female was admitted to our hospital due to bacterial pneumonia. At the admission, CT showed another abnormal small shadow in her right middle lobe. Since the shadow was not visible by fluoroscopy, we reconstructed the images of virtual bronchoscopy using the data obtained by multidetector CT. The location of the shadow was determined in the peripheral area of a dorsal branch of right B4aialpha. Then the transbronchial lung biopsy using an ultrathin bronchoscope with simultaneous CT guidance was performed. The histological findings of the biopsy specimens revealed that the shadow was highly suspicious for malignancy. Therefore, the right middle lobectomy was conducted, and the tumor was diagnosed as an adenocarcinoma. An ultrathin bronchoscope with virtual bronchoscopy is useful to diagnose a pulmonary shadow with ground glass opacity.

A novel polymorphism of the brain-derived neurotrophic factor (BDNF) gene associated with late-onset Alzheimer's disease.

Several lines of evidence have suggested altered functions of the brain-derived neurotrophic factor (BDNF) in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). In the search for polymorphisms in the 5'-flanking and 5'-noncoding regions of the BDNF gene, we found a novel nucleotide substitution (C270T) in the noncoding region. We performed an association study between this polymorphism and AD in a Japanese sample of 170 patients with sporadic AD (51 early-onset and 119 late-onset) and 498 controls. The frequency of individuals who carried the mutated type (T270) was significantly more common in patients with late-onset AD than in controls (P = 0.00004, odds ratio: 3.8, 95% CI 1.9-7.4). However, there was no significant difference in the genotype distribution between the patients with early-onset AD and the controls, although this might be due to the small sample size of the early-onset group. Our results suggest that the C270T polymorphism of the BDNF gene or other unknown polymorphisms, which are in linkage disequilibrium, give susceptibility to late-onset AD. We obtained no evidence for the possible interactions between the BDNF and apolipoprotein E (APOE) genes, suggesting that the possible effect of the BDNF gene on the development of late-onset AD might be independent of the APOE genotype.

No evidence for an association between the Glu298Asp polymorphism of the NOS3 gene and Alzheimer's disease.

Recently a significant association of a missense mutation (Glu298Asp) of the endothelial nitric oxide synthase (NOS3) gene with late-onset Alzheimer's disease (LOAD) was reported. We tried to replicate this finding in a Japanese sample of 121 patients with LOAD, 51 with early-onset AD (EOAD), and 165 medical controls. However, the genotype and allelic distributions for the Glu298Asp polymorphism were similar for these three groups, suggesting that the Glu298Asp polymorphism of the NOS3 gene has no relevance to the development of AD in Japanese.

Up-regulation of fas ligand at early stages and down-regulation of Fas at progressed stages of intrahepatic cholangiocarcinoma reflect evasion from immune surveillance.

We examined immunohistochemically the possible participation of the Fas/Fas ligand (FasL) system in intrahepatic cholangiocarcinoma (ICC) during the escape from immune surveillance, using 68 cases of ICC, 29 cases of normal intrahepatic large bile ducts, and 18 cases of biliary dysplasia. Apoptosis of tumor-infiltrating lymphocytes (TIL) was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Fas was weakly expressed in normal intrahepatic bile ducts. Almost all biliary dysplasia and well-differentiated ICCs showed moderate to marked expression of Fas, while Fas expression was variable in moderately and poorly differentiated ICCs. Down-regulation of Fas expression was significantly correlated with histologic de-differentiation, vascular invasion, the size of ICCs, and short survival of ICC patients. By in situ hybridization, FasL mRNA were frequently and strongly expressed in biliary dysplasia compared with non-neoplastic intrahepatic bile duct. In well-differentiated ICCs, FasL mRNA expression was frequent and intense. But, the expression gradually decreased in moderately and poorly differentiated ICCs. Down-regulation of FasL mRNA expression in ICCs was correlated with perineural invasion and tumor size (over 4 cm) (P <.05). Apoptotic TIL were more frequent in ICC foci than in non-neoplastic foci remote from ICC foci. These findings suggest that a tumor evasion mechanism involving Fas/FasL exists in ICC; frequent and intense expression of FasL mRNA in well-differentiated ICCs enable them to escape immune surveillance by counterattacking Fas-bearing TIL. This counterattack becomes insensitive in poorly differentiated ICCs, in which the down-regulation of Fas gives them a resistance against the FasL-expressing TIL. These mechanisms may be involved in the tumor progression.

Alzheimer's disease and 5-HTTLPR polymorphism of the serotonin transporter gene: no evidence for an association.

Recently two independent research groups consistently reported a significant association between the serotonin transporter (5-HTT) gene and late-onset sporadic Alzheimer's disease (AD). They found that the "short" allele of the 5-HTT gene-linked polymorphic region (5-HTTLPR), which is associated with reduced transcriptional activity of the gene, increases the risk of developing late-onset AD. The present study tried to replicate this finding in a Japanese sample. We genotyped 41 patients with early-onset AD (<65 years), 82 with late-onset AD, and 336 controls. There was no significant difference in genotype or allele distribution between either patient group and controls in our sample, suggesting that the 5-HTTLPR does not play a major role in the pathogenesis of AD in Japanese.

Possible association of missense mutation (Gly[-63]Glu) of the neurotrophin-3 gene with Alzheimer's disease in Japanese.

Several lines of evidence have suggested a possible involvement of neurotrophic factors in the pathogenesis of neurodegenerative disease. We examined whether a missense mutation (Gly[-63]Glu) of the neurotrophin-3 (NT-3) gene is associated with Alzheimer's disease (AD) in a Japanese sample of 123 patients and 215 controls. We found that homozygotes or heterozygotes for the mutated type (Glu[-63]) were significantly more common among the patients than the controls (P = 0.013, odds ratio 1.77, 95% CI 1.12-2.79). The mutated type was more frequent among the patients than the controls (P = 0.011, odds ratio 1.63, 95%CI 1.11-2.38). This association between NT-3 and AD was more prominent among those who did not carry the epsilon4 allele of the apolipoprotein E gene than those who carried the epsilon4 allele. Our results suggest that the Glu(-63) allele of the NT-3 gene by itself or another mutation nearby which would be in linkage disequilibrium to the mutation is a risk factor for AD.

Basic and clinical studies on ApoE gene typing by line probe assay (LiPA) as a biological marker for Alzheimer's disease and related disorders: multicenter study in Japan.

The fundamental reagent capability and clinical significance of ApoE gene typing has been investigated. The result of each test shows that the reagent capability satisfied the expected standard. Four-hundred thirty-nine samples classified into three groups (Alzheimer's disease (AD), other types of dementia and nondementia) were measured to examine the clinical significance. The rate of epsilon 4 genotype of each group was 31.5%, 14.6%, 10.4%, respectively. The AD group had a higher rate than the other groups (p < 0.001). The measurement of ApoE genotype is suggested to be useful as one of the guidelines in the diagnosis of AD.

Efficacy of mianserin on symptoms of delirium in the aged: an open trial study.

1. The authors administered the tetracyclic antidepressant mianserin to aged patients with delirium to examine its efficacy in delirium. 2. The subjects were 62 consecutive aged patients with delirium, diagnosed according to DSM-IV. 3. The patients' symptoms of delirium were assessed once a week over the 4-week study period using the Delirium Rating Scale. 4. It was found that mianserin was effective, especially in treating behavioral and sleep-wake disturbance and that it was almost free from undesirable side-effects. It appears to be particularly useful for elderly patients with delirium, who easily develop extrapyramidal side-effects when given antipsychotic drugs. These properties of mianserin are worthy of further study.

Screening for mutations at codon 717 of the amyloid precursor protein gene in Alzheimer's disease.

Three kinds of missense mutation at codon 717 of amyloid precursor protein (APP) gene (Val --> Ile; Val --> Gly; Val --> Phe) were screened in 114 patients with familial and sporadic Alzheimer's disease (AD), using a rapid testing method for each Val --> Gly and Val --> Phe mutation and Goate's method for Val --> Ile mutation based on the polymerase chain reaction. Mutations were not found in the subjects, confirming earlier suggestions that these three mutations at codon 717 of APP gene account for only a small proportion of cases of not only familial AD but also sporadic AD.