The 11th Banff meeting was held in Paris, France, from June 5 to 10, 2011, with a focus on refining diagnostic criteria for antibody-mediated rejection (ABMR). The major outcome was the acknowledgment of C4d-negative ABMR in kidney transplants. Diagnostic criteria for ABMR have also been revisited in other types of transplants. It was recognized that ABMR is associated with heterogeneous phenotypes even within the same type of transplant. This highlights the necessity of further refining the respective diagnostic criteria, and is of particular significance for the design of randomized clinical trials. A reliable phenotyping will allow for definition of robust end-points. To address this unmet need and to allow for an evidence-based refinement of the Banff classification, Banff Working Groups presented multicenter data regarding the reproducibility of features relevant to the diagnosis of ABMR. However, the consensus was that more data are necessary and further Banff Working Group activities were initiated. A new Banff working group was created to define diagnostic criteria for ABMR in kidneys independent of C4d. Results are expected to be presented at the 12th Banff meeting to be held in 2013 in Brazil. No change to the Banff classification occurred in 2011.
Allograft rejection; allograft transplantation; antibody-mediated rejection; Banff classification
The host response and remodeling of ECM scaffolds are believed to be critical determinants of success or failure in repair or reconstructive procedures. Host response has been investigated in subcutaneous or abdominal wall implantation models. The extent to which evaluation of the host response to ECM intended for tendon or ligament repair should be performed in an orthotopic site is not known. This study compared the host response to human-derived fascia lata ECM among various implantation sites in the rat model. Results showed that a xenograft in the rat shoulder does not exhibit a different host response at 7 days from xenograft in the body wall, suggesting that either site may be appropriate to study the early host response to biologic grafts as well as the effect of various treatments aimed to modify the early host response. By 28 days, a xenograft in the rat shoulder does elicit a unique host response from that seen in the body wall. Therefore, it may be more appropriate to use an orthotopic shoulder model for investigating the long-term host response and remodeling of biologic grafts to be used for rotator cuff repair.
fascia lata ECM; quantitative real time PCR; host response; pro-inflammatory; pro-remodeling; histology
To explore immunoregulatory and anti-inflammatory pathways specifically targeted by a subcutaneous anti-TNFαdrug—adalimumab—which might be relevant for controlling refractory uveitis.
Non-randomized pilot intervention study on the effects of adalimumab on Treg populations and plasma VEGF levels in refractory uveitis patients. Inflammatory and immunological parameters were measured in 12 patients before therapy, and 1 and 6 months after therapy, and analyzed in the context of ophthalmological outcomes. The results were compared with those obtained in 10 systemic prednisone-treated uveitis patients.
After 1 month of treatment, all patients responded, with 67% of adalimumab group and 80% of the corticosteroid group achieving inactivity (P=0.5). Unlike steroid-treated patients, a significant increase in T-regulatory CD4+ CD25high Foxp3+ CD127− cells was observed in adalimumab patients after 1 month of treatment, and maintained after 6 months (P=0.003). A significant adalimumab-specific drop in plasma VEGF was observed after 1 and 6 months of treatment (P=0.019). In every single patient, Tregs but not VEGF correlated with disease activity.
In refractory uveitis patients treated with adalimumab, clinical efficacy may be mediated through upregulation of Tregs in addition to modulation of VEGF-mediated inflammatory pathways. These biological properties, which were not observed in patients treated with corticosteroids, may reflect the specificity of TNF-αtargeting.
uveitis; anti-TNFα; adalimumab; immunomodulation; Tregs; VEGF
Lymphocytic myocarditis is a clinically important condition that is difficult to diagnose and distinguish. We hypothesized that the transcriptome obtained from an endomyocardial biopsy (EMB) would yield clinically relevant and accurate molecular signatures.
Methods and results
Microarray analysis was performed on samples from patients with histologically proven lymphocytic myocarditis (n=16) and idiopathic dilated cardiomyopathy (IDCM, n=32) to develop accurate diagnostic transcriptome-based biomarkers (TBB) using multiple classification algorithms. We identified 9,878 genes differentially expressed in lymphocytic myocarditis vs. IDCM (FC>1.2, FDR<5%), from which a TBB containing 62 genes was identified, which distinguished myocarditis with 100% sensitivity (95% CI: 46-100%) and 100% specificity (95% CI: 66-100%) and which was generalizable to a broad range of secondary cardiomyopathies associated with inflammation (n=27), ischemic cardiomyopathy (n=8) and the normal heart (n=11). Multiple classification algorithms and quantitative realtime RT-PCR analysis further reduced this subset to a highly robust molecular signature of 13 genes, which still performed with 100% accuracy.
Together these findings demonstrate that transcriptomic biomarkers from a single EMB can improve the clinical detection of patients with inflammatory diseases of the heart. This approach advances the clinical management and treatment of cardiac disorders with highly variable outcome.
Gene expression; heart failure; myocarditis; transcriptome; biomarker
Naturally-occurring biomaterial scaffolds derived from extracellular matrix (ECM) have been previously investigated for soft tissue repair. We propose to enrich fascia ECM with high molecular weight tyramine substituted-hyaluronan (TS-HA) to modulate inflammation associated with implantation and enhance fibroblast infiltration. As critical determinants of constructive remodeling, the host inflammatory response and macrophage polarization to TS-HA enriched fascia were characterized in a rat abdominal wall model. TS-HA treated fascia with cross-linking had a similar lymphocyte (P = 0.11) and plasma cell (P = 0.13) densities, greater macrophage (P = 0.001) and giant cell (P < 0.0001) densities, and a lower density of fibroblast-like cells (P < 0.0001) than water treated controls. Treated fascia, with or without cross-linking, exhibited a predominantly M2 pro-remodeling macrophage profile similar to water controls (P = 0.82), which is suggestive of constructive tissue remodeling. Our findings demonstrated that HA augmentation can alter the host response to an ECM, but the appropriate concentration and molecular weight needed to minimize chronic inflammation within the scaffold remains to be determined.
The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses.
synaptic ultrastructure; high-pressure freezing; mossy fiber LTP; dendritic spine; actin cytoskeleton; dentate gyrus; granule cells
Current understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1).
In the last decade, two advances have shifted attention from cellular rejection to antibody-mediated rejection (AMR) of cardiac transplants. First, more sensitive diagnostic tests for detection of AMR have been developed. Second, improvements in immunosuppression have made severe acute cellular rejection uncommon, but have had less effect on AMR. Antibodies can contribute to graft rejection by activation of complement, by activation of vascular endothelial and smooth muscle cells, and by activation of neutrophils, macrophages or natural killer cells. Because acute rejection is a risk factor for chronic rejection in all types of organ transplants, it is has been proposed that AMR can cause chronic rejection. Small animal models need to be developed to gain further insights into AMR and the role of antibodies in chronic graft arteriopathy. This article reviews the current clinical data and existing mouse models for AMR.
Cardiac transplantation; Antibody-Mediated Rejection; C4d; Arteriopathy
Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The λ isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKCλ is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix. Word count: 249.
atypical protein kinase C; Src; transformation; migration; invasion; podosome
The relationship between income inequality and health has been widely explored. Today there is some evidence suggesting that good health is inversely related to income inequality. After the economic reforms initiated in the early 1980s, China experienced one of the fastest‐growing income inequalities in the world. The state of China in the 1990s is focussed on and possible effects of provincial income inequality on individual health status are explored.
A multilevel regression model is used to analyse the data collected in 1991, 1993 and 1997 from nine provinces included in the China Health and Nutrition Survey. The effects of provincial Gini coefficients on self‐rated health in each year are evaluated by two logistic regressions estimating the odds ratios of reporting poor or fair health. The patterns of this effect are compared among the survey years and also among different demographic groups.
The analyses show an independent effect of income inequality on self‐reported health after adjusting for individual and household variables. Furthermore, the effect of income distribution is not attenuated when household income and provincial gross domestic product per capita are included in the model. The results show that there is an increased risk of about 10–15% on average for fair or poor health for people living in provinces with greater income inequalities compared with provinces with modest income inequalities.
In China, societal income inequality appears to be an important determinant of population health during 1991–7.
Complement activation contributes to antibody-mediated allograft rejection, but increasing evidence also implicates complement proteins produced locally within the graft, in part by infiltrating mononuclear cells, as important mediators of tissue injury. To test this concept in transplant recipients we evaluated complement, complement regulator and Tcell/proinflammatory marker gene expression by quantitative real-time polymerase chain reaction in 71 archived heart transplant biopsies and correlated the results with the histologic grade of rejection. Significantly more transcripts encoding alternative pathway components factor B, C3 and properdin, and C3a receptor and C5a receptor, were detected in grade 3 vs. grade 0 or 1 biopsies. The grade 3 rejections also contained significantly higher amounts of CD3, interferon γ, perforin and granzyme B genes. In addition to providing supportive evidence for a pathogenic role of graft-derived complement in human heart transplant injury, these correlations suggest that molecular profiling of complement gene expression could be useful in the diagnosis of human allograft rejection.
heart transplantation; human; complement; gene expression
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent of both KS and primary effusion lymphoma (PEL). Although treatment with paclitaxel has significant antitumor activity in KS, drug resistance represents a major obstacle for improving the overall response and survival of PEL patients. The transcriptional pattern of KSHV is cell/tissue specific, as revealed by the fact that the viral latent protein LANA2 is detected exclusively in B cells. This paper focuses on the mechanism of paclitaxel resistance observed in PEL cells. Here we show that LANA2 protein modulates microtubule dynamics through its direct binding to polymerized microtubules, preventing microtubule stabilization induced by paclitaxel. This is the first demonstration of paclitaxel resistance induced by a viral protein and suggests a link between the expression of LANA2 and the resistance of PEL cells to paclitaxel.
Background: Identification of monosodium urate (MSU) and calcium pyrophosphate dehydrate (CPPD) crystals in synovial fluid samples is diagnostic of gout and CPPD crystal related arthropathy. Various studies have shown poor consistency in results of crystal analysis.
Objective: To determine whether training of the analysts increases the consistency.
Methods: An expert rheumatologist gave a course on crystal detection and identification. The four trained observers then blindly and independently examined synovial fluid samples previously classified by the expert which had been obtained from patients with both crystal arthropathies and other non-crystal related inflammatory joint conditions.
Results: 194 observations were made on 64 synovial fluid samples: 96 without crystals (49.4%), 55 with CPPD crystal (28.4%), and 43 with MSU crystals (22.2%). For crystal detection (presence or absence of crystals), sensitivity was 95.9% and specificity 86.5%. For identification of MSU crystals, sensitivity was 95.3% and specificity 97.2%. For identification of CPPD crystals, sensitivity was 92.7% and specificity 92.1%. The κ index of agreement with the reference standard between the observers was 0.84 for any crystal detection, 0.93 for MSU crystal sample identification, and 0.79 for CPPD crystal sample identification.
Conclusions: For trained observers, the detection and identification of crystals in synovial fluid is a consistent procedure.
Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56–163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant.
Antibody-mediated rejection; C3d; C4d; complement; heart transplant; macrophages
Cardiac pressure load stimulates hypertrophy, often leading to chamber dilation and dysfunction. ROS contribute to this process. Here we show that uncoupling of nitric oxide synthase–3 (NOS3) plays a major role in pressure load–induced myocardial ROS and consequent chamber remodeling/hypertrophy. Chronic transverse aortic constriction (TAC; for 3 and 9 weeks) in control mice induced marked cardiac hypertrophy, dilation, and dysfunction. Mice lacking NOS3 displayed modest and concentric hypertrophy to TAC with preserved function. NOS3–/– TAC hearts developed less fibrosis, myocyte hypertrophy, and fetal gene re-expression (B-natriuretic peptide and α–skeletal actin). ROS, nitrotyrosine, and gelatinase (MMP-2 and MMP-9) zymogen activity markedly increased in control TAC, but not in NOS3–/– TAC, hearts. TAC induced NOS3 uncoupling in the heart, reflected by reduced NOS3 dimer and tetrahydrobiopterin (BH4), increased NOS3-dependent generation of ROS, and lowered Ca2+-dependent NOS activity. Cotreatment with BH4 prevented NOS3 uncoupling and inhibited ROS, resulting in concentric nondilated hypertrophy. Mice given the antioxidant tetrahydroneopterin as a control did not display changes in TAC response. Thus, pressure overload triggers NOS3 uncoupling as a prominent source of myocardial ROS that contribute to dilatory remodeling and cardiac dysfunction. Reversal of this process by BH4 suggests a potential treatment to ameliorate the pathophysiology of chronic pressure-induced hypertrophy.
Our study was designed to contribute to an understanding of the timing and conditions under which transmission of Andes hantavirus in Oligoryzomys longicaudatus reservoir populations takes place. Mice were caged in test habitats consisting of steel drums containing holding cages, where seronegative rodents were exposed to wild seropositive individuals by freely sharing the same cage or being separated by a wire mesh. Tests were also performed for potential viral transmission to mice from excrement-tainted bedding in the cages. Andes virus transmitted efficiently; from 130 attempts with direct contact, 12.3% resulted in virus transmission. However, if we consider only those rodents that proved to be infectious, from 93 attempts we obtained 16 infected animals (17.2%). Twelve of them resulted from intraspecies O. longicaudatus encounters where male mice were differentially affected and 4 resulted from O. longicaudatus to Abrothrix olivaceus. Experiments using Abrothrix longipilis as receptors were not successful. Transmission was not observed between wire mesh-separated animals, and mice were not infected from excrement-tainted bedding. Bites seemed not to be a requisite for oral transmission. Genomic viral RNA was amplified in two out of three saliva samples from seropositive rodents, but it was not detected in urine samples obtained by vesicle puncture from two other infected rodents. Immunohistochemistry, using antibodies against Andes (AND) hantavirus proteins, revealed strong reactions in the lung and salivary glands, supporting the possibility of oral transmission. Our study suggests that AND hantavirus may be principally transmitted via saliva or saliva aerosols rather than via feces and urine.
OBJECTIVES—This study examined the contribution of employment status, welfare benefits, alcohol use, and other individual, and contextual factors to physical aggression during marital conflict.
METHODS—Logistic regression models were used to analyse panel data collected in the National Survey of Families and Households in 1987 and 1992. A total of 4780 married or cohabiting persons re-interviewed in 1992 were included in the analysis. Domestic violence was defined as reporting that both partners were physically violent during arguments.
RESULTS—It was found that non-employed respondents are not at greater risk of family violence in comparison with employed respondents, after controlling for alcohol misuse, income, education, age, and other factors; however, employed persons receiving welfare benefits are at significantly higher risk. Alcohol misuse, which remains a predictor of violence even after controlling for other factors, increases the risk of family violence while satisfaction with social support from family and friends decreases it.
CONCLUSION—These results underscore the important effect of alcohol misuse on domestic violence, and the need to monitor the potential impact of welfare reform on domestic violence.
Keywords: family violence; alcohol misuse; employment status; welfare benefits
OBJECTIVES: While the unemployment rate of African-American people is more than twice that of the white population, the research on the impact of unemployment on the health of this population is scarce. This study analysed the impact of unemployment on depression and well being among African-American people, and the factors associated with well being. METHODS: Logistic and multiple regression models were used to analyse panel data collected in the National Survey of Families and Households 1987-1992. African-American (1369) and white (6660) respondents were analysed separately. Outcome variables included an index of depression and self reported health status. MAIN FINDINGS: Differences between employment and unemployment groups were less significant for African-Americans than for the white population in predicting depression and well being. Health enhancing factors such as education and wealth were significantly associated with better health and lower depression indices among the white population but not consistently so among African-Americans. Satisfaction with personal relationships was the strongest predictor of well being for both groups. CONCLUSION: Research should focus on the special needs and circumstances of African-Americans, because protective factors may not have the same impact in different groups of the population.
Myonuclear apoptosis is an early event in the pathology of dystrophin-deficient muscular dystrophy in the mdx mouse. However, events that initiate apoptosis in muscular dystrophy are unknown, and whether elimination of apoptosis can ameliorate subsequent muscle wasting remains a major question. We have tested the hypothesis that cytotoxic T-lymphocytes initiate myonuclear apoptosis in dystrophic muscle, and examined whether perforin-mediated cytotoxicity plays a role in the pathophysiology of muscular dystrophy. Mdx mice showed muscle invasion by cytotoxic T cells and helper T cells at the onset of histologically detectable muscle fiber pathology. At this time, perforin-expressing cells were also present at elevated concentration. Mdx mice depleted of CD8(+) cells showed a significant reduction of apoptotic myonuclei concentration and a reduction in necrosis, judged by macrophage invasion of muscle fibers. Double-mutant mice, deficient in dystrophin and perforin, showed nearly complete absence of myonuclear apoptosis, and a significant reduction in the concentration of macrophages in the connective tissue surrounding muscle fibers. However, muscle fiber invasion by macrophages was not reduced significantly in double mutant mice. Thus, cytotoxic T-lymphocytes contribute significantly to apoptosis and necrosis in mdx dystrophy, and perforin-mediated killing is primarily responsible for myonuclear apoptosis.
Although the literature on event monitoring is extensive, it does not cover all issues that we encountered while developing an event monitor at our institution. We had to resolve issues related to event detection, scalability, what topics were suitable for asynchronous decision support, and overlap of efforts of other groups at the institution attempting to improve quality and lower cost of care. In this paper, we describe our experience deploying CLEM, the clinical event monitor at the University of Pittsburgh with emphasis on these topics.
The genotypes of 63 isolates of Aspergillus fumigatus obtained from three hospitals in different geographical areas and of eight culture collection strains were determined by multilocus enzyme electrophoresis. Twelve of the 17 enzymatic loci studied were polymorphic, giving rise to 48 different electrophoretic types. The existence of fixed multilocus genotypes, significant heterozygote deficits and excesses at the different loci, and linkage disequilibria within subpopulations strongly suggests a clonal reproduction mode for A. fumigatus. Numerical analysis of the comparison and disposition of the different electrophoretic types demonstrates a significant genetic differentiation between the three sampling sites. However, no correlation could be found between geographical distances and genetic differentiation. On account of the multiple discriminatory markers, multilocus enzyme electrophoresis typing seems to be a very powerful tool for epidemiological and reproductive mode studies of A. fumigatus.
Relationships between azole susceptibility and in vivo response to antifungal therapy in a murine model of candidiasis were investigated for Candida albicans isolates sampled from human immunodeficiency virus type 1-positive patients with oropharyngeal candidiasis. The susceptibilities of seven clinical isolates and two reference strains to fluconazole (FCZ) and itraconazole (ITZ) were determined in vitro by the broth microdilution method. Four isolates were resistant to FCZ and ITZ, two were susceptible to both azoles, and three were resistant to FCZ and susceptible to ITZ (dissociated resistance). CD1 mice were inoculated with each isolate and treated with either FCZ or ITZ (drug regimen, 5 mg/kg of body weight twice daily for 5 days). Quantitative cultures of kidneys were performed at the end of the treatment. On the other hand, the survival rates of the mice were followed daily. These two parameters were clearly correlated with in vitro susceptibility. Thus, the phenomenon of a dissociation of resistance to FCZ and ITZ may be found in vivo as well as in vitro.
Improvement in the performance of reminder systems may be facilitated by the use of new representations. A decision-theoretic representation, for example, may enable a reminder system to represent and reason about the probabilities that a reminder will be a true or a false alarm and the relative utilities of these events. We extended a previously described decision-theoretic model to include such events. The model now represents explicitly the uncertainty, costs, and benefits of sending a reminder. We also extended the model to remove an assumption of reminder independence. As a step towards testing a hypothesis that this approach will support better performance than a rule-based approach, we analyzed a set of CARE rules and showed that our representation can represent these rules.