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1.  Effectiveness and tolerability of second-line therapy with vildagliptin vs. other oral agents in type 2 diabetes: A real-life worldwide observational study (EDGE) 
Real-life studies are needed to confirm the clinical relevance of findings from randomised controlled trials (RCTs). This study aimed to assess the effectiveness and tolerability of vildagliptin add-on vs. other oral antihyperglycaemic drugs (OADs) added to OAD monotherapy in a real-life setting, and to explore the advantages and limitations of large-scale ‘pragmatic’ trials.
EDGE was a prospective, 1-year, worldwide, real-life observational study in which 2957 physicians reported on the effects of second-line OADs in 45,868 patients with T2DM not reaching glycaemic targets with monotherapy. Physicians could add any OAD, and patients entered either vildagliptin or (pooled) comparator cohort. The primary effectiveness and tolerability end-point (PEP) evaluated proportions of patients decreasing HbA1c > 0.3%, without hypoglycaemia, weight gain, peripheral oedema or gastrointestinal side effects. The most clinically relevant secondary end-point (SEP 3) was attainment of end-point HbA1c < 7% without hypoglycaemia or ≥ 3% increase in body weight.
In this large group of T2DM patients, a second OAD was added at mean HbA1c of 8.2 ± 1.3%, with no baseline HbA1c difference between cohorts. Second-line OAD therapy attained the PEP in the majority of patients, with higher attainment in those prescribed a vildagliptin-based regimen. The adjusted odds ratio was 1.49 (95% CI: 1.42, 1.55; p < 0.001). In patients with baseline HbA1c ≥ 7%, SEP 3 was achieved by 35% of patients on a vildagliptin-based combination and by 23% of those receiving comparator combinations. The adjusted odds ratio was 1.96 (95% CI: 1.85, 2.07; p < 0.001). Safety events were reported infrequently and safety profiles of vildagliptin and other OADs were consistent with previous data.
EDGE demonstrates that in a ‘real-life’ setting, vildagliptin as second OAD can lower HbA1c to target without well-recognised OAD side effects, more frequently than comparator OADs. In addition, EDGE illustrates that conducting large-scale, prospective, real-life studies poses challenges but yields valuable clinical information complementary to RCTs.
PMCID: PMC4231206  PMID: 23961850
2.  Work of breathing indices in infants with respiratory insufficiency receiving high-flow nasal cannula and nasal continuous positive airway pressure 
To compare work of breathing (WOB) indices between two nCPAP settings and two levels of HFNC in a crossover study.
Infants with a CGA 28–40 weeks, baseline of HFNC 3–5 lpm or nCPAP 5–6 cmH2O and fraction of inspired oxygen ≤40% were eligible. WOB was analyzed using respiratory inductive plethysmography (RIP) for each of the four modalities: HFNC 3 and 5 lpm, nCPAP 5 and 6 cmH2O. N = 20; Study weight 1516 g (±40 g).
Approximately 12 000 breaths were analyzed indicating a high degree of asynchronous breathing and elevated WOB indices at all four levels of support. Phase angle values (means) (P<0.01): HFNC 3 lpm (114.7°), HFNC 5 lpm (96.7°), nCPAP 5 cmH2O (87.2°), nCPAP 6 cmH2O (80.5°). The mean phase relation of total breath (PhRTB) (means) (P<0.01): HFNC 3 lpm (63.2%), HFNC 5 lpm (55.3%), nCPAP 5 cmH2O (49.3%), nCPAP 6 cmH2O (48.0%). The relative labored breathing index (LBI) (means) (P≤0.001): HFNC 3 lpm (1.39), HFNC 5 lpm (1.31), nCPAP 5 cmH2O (1.29), nCPAP 6 cmH2O (1.26). Eighty-two percent of the study subjects—respiratory mode combinations displayed clustering, in which a proportion of breaths either occurred predominantly out-of-phase (relative asynchrony) or in-phase (relative synchrony).
In this study, WOB indices were statistically different, yet clinically similar in that they were elevated with respect to normal values. These infants with mild-to-moderate respiratory insufficiency demonstrate a meaningful elevation in WOB indices and continue to require non-invasive respiratory support. Patient variability exists with regard to biphasic clustered breathing patterns and the level of supplemental fraction of inspired oxygen ≤40% alone does not provide guidance to the optimal matching of WOB indices and non-invasive respiratory support.
PMCID: PMC4141486  PMID: 24071905
preterm; infant; respiratory inductive plethysmography
3.  Vimentin is an endogenous ligand for the pattern recognition receptor Dectin-1 
Cardiovascular Research  2013;99(3):494-504.
Atherosclerosis is a chronic inflammatory disorder of cholesterol deposition in monocyte-derived macrophages (MDM) within the arterial wall leading to impingement on the lumen of the vessel. In atherosclerotic lesions, MDM are the primary source of NADPH oxidase-derived superoxide anion (O2−) inducing low-density lipoprotein (LDL) oxidation leading to their unregulated uptake of oxidized LDL and foam cell formation. We recently discovered that zymosan potently activates monocyte NADPH oxidase via the non-toll pattern recognition receptor (PRR), Dectin-1. Other PRRs bind endogenous human ligands, yet no such ligands have been identified for Dectin-1. Our hypothesis was that inflammation generates endogenous ligands for Dectin-1 that activate O2− production and thereby contributes to atherogenesis.
Methods and results
Human: anti-zymosan antibodies were used to identify similar, cross-reactive epitopes in human atherosclerotic tissue extracts. Immunoblot analysis revealed consistent antibody reactive protein bands on one- and two-dimensional gel electrophoreses. Vimentin was identified by mass spectrometry in the immunoreactive bands across different tissue samples. Direct binding of vimentin to Dectin-1 was observed using BIACORE. Further data revealed that vimentin induces O2− production by human monocytes. Analysis of human atherosclerotic lesions revealed that vimentin was detected extracellularly in the necrotic core and in areas of active inflammation. Vimentin also co-localized with Dectin-1 in macrophage-rich regions where O2− is produced.
We conclude that vimentin is an endogenous, activating ligand for Dectin-1. Its presence in areas of artery wall inflammation and O2− production suggests that vimentin activates Dectin-1 and contributes to the oxidation of lipids and cholesterol accumulation in atherosclerosis.
PMCID: PMC3718321  PMID: 23674515
Dectin-1; Extracellular vimentin; Atherosclerosis; Superoxide anion; Alarmins
4.  Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis 
Current molecular medicine  2013;13(2):317-329.
The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial–associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate.
PMCID: PMC4104517  PMID: 23228132
Cell death; endoplasmic reticulum; metabolism; mitochondria
5.  Neuropilin-1 Conveys Semaphorin and VEGF Signaling during Neural and Cardiovascular Development 
Developmental cell  2003;5(1):45-57.
Neuropilin-1 (Npn-1) is a receptor that binds multiple ligands from structurally distinct families, including secreted semaphorins (Sema) and vascular endothelial growth factors (VEGF). We generated npn-1 knockin mice, which express an altered ligand binding site variant of Npn-1, and npn-1 conditional null mice to establish the cell-type- and ligand specificity of Npn-1 function in the developing cardiovascular and nervous systems. Our results show that VEGF-Npn-1 signaling in endothelial cells is required for angiogenesis. In striking contrast, Sema-Npn-1 signaling is not essential for general vascular development but is required for axonal pathfinding by several populations of neurons in the CNS and PNS. Remarkably, both Sema-Npn-1 signaling and VEGF-Npn-1 signaling are critical for heart development. Therefore, Npn-1 is a multifunctional receptor that mediates the activities of structurally distinct ligands during development of the heart, vasculature, and nervous system.
PMCID: PMC3918747  PMID: 12852851
6.  Report from a consensus conference on antibody-mediated rejection in heart transplantation 
The problem of AMR remains unsolved because standardized schemes for diagnosis and treatment remains contentious. Therefore, a consensus conference was organized to discuss the current status of antibody-mediated rejection (AMR) in heart transplantation.
The conference included 83 participants (transplant cardiologists, surgeons, immunologists and pathologists) representing 67 heart transplant centers from North America, Europe, and Asia who all participated in smaller break-out sessions to discuss the various topics of AMR and attempt to achieve consensus.
A tentative pathology diagnosis of AMR was established, however, the pathologist felt that further discussion was needed prior to a formal recommendation for AMR diagnosis. One of the most important outcomes of this conference was that a clinical definition for AMR (cardiac dysfunction and/or circulating donor-specific antibody) was no longer believed to be required due to recent publications demonstrating that asymptomatic (no cardiac dysfunction) biopsy-proven AMR is associated with subsequent greater mortality and greater development of cardiac allograft vasculopathy. It was also noted that donor-specific antibody is not always detected during AMR episodes as the antibody may be adhered to the donor heart. Finally, recommendations were made for the timing for specific staining of endomyocardial biopsy specimens and the frequency by which circulating antibodies should be assessed. Recommendations for management and future clinical trials were also provided.
The AMR Consensus Conference brought together clinicians, pathologists and immunologists to further the understanding of AMR. Progress was made toward a pathology AMR grading scale and consensus was accomplished regarding several clinical issues.
PMCID: PMC3829685  PMID: 21300295
heart transplant; antibody; rejection; treatment; outcomes
7.  Banff 2011 Meeting Report: New Concepts in Antibody-Mediated Rejection 
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.
PMCID: PMC3728651  PMID: 22300494
Allograft rejection; allograft transplantation; antibody-mediated rejection; Banff classification
8.  Effect of Implantation Site and Injury Condition on Host Response to Human-Derived Fascia Lata ECM in a Rat Model 
Journal of Orthopaedic Research  2011;30(3):461-467.
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.
PMCID: PMC3264843  PMID: 21858856
fascia lata ECM; quantitative real time PCR; host response; pro-inflammatory; pro-remodeling; histology
9.  Adalimumab specifically induces CD3+ CD4+ CD25high Foxp3+ CD127− T-regulatory cells and decreases vascular endothelial growth factor plasma levels in refractory immuno-mediated uveitis: a non-randomized pilot intervention study 
Eye  2012;26(3):468-477.
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.
PMCID: PMC3298996  PMID: 22222264
uveitis; anti-TNFα; adalimumab; immunomodulation; Tregs; VEGF
10.  Transcriptomic Biomarkers for the Accurate Diagnosis of Myocarditis 
Circulation  2011;123(11):1174-1184.
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.
PMCID: PMC3408077  PMID: 21382894
Gene expression; heart failure; myocarditis; transcriptome; biomarker
11.  Characterization of and host response to tyramine substituted-hyaluronan enriched fascia extracellular matrix 
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.
PMCID: PMC3245546  PMID: 21553156
12.  Structural plasticity of spines at giant mossy fiber synapses 
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.
PMCID: PMC3524460  PMID: 23264762
synaptic ultrastructure; high-pressure freezing; mossy fiber LTP; dendritic spine; actin cytoskeleton; dentate gyrus; granule cells
13.  Candida albicans forms biofilms on the vaginal mucosa 
Microbiology  2010;156(Pt 12):3635-3644.
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).
PMCID: PMC3068702  PMID: 20705667
14.  Immunological Challenges of Cardiac Transplantation: The need for better animal models to answer current clinical questions 
Journal of clinical immunology  2009;29(6):722-729.
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.
PMCID: PMC2975936  PMID: 19802689
Cardiac transplantation; Antibody-Mediated Rejection; C4d; Arteriopathy
15.  Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src-transformed cells 
Journal of cellular physiology  2009;221(1):171-182.
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.
PMCID: PMC2782718  PMID: 19492416
atypical protein kinase C; Src; transformation; migration; invasion; podosome
16.  Provincial income inequality and self‐reported health status in China during 1991–7 
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.
PMCID: PMC2465504  PMID: 17108303
17.  Complement gene expression in human cardiac allograft biopsies as a correlate of histologic grade of injury 
Transplantation  2008;86(9):1319-1321.
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.
PMCID: PMC2646139  PMID: 19005416
heart transplantation; human; complement; gene expression
18.  Induction of Paclitaxel Resistance by the Kaposi's Sarcoma-Associated Herpesvirus Latent Protein LANA2▿  
Journal of Virology  2007;82(3):1518-1525.
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.
PMCID: PMC2224414  PMID: 18032494
19.  Analysis for crystals in synovial fluid: training of the analysts results in high consistency 
Annals of the Rheumatic Diseases  2005;64(4):612-615.
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.
PMCID: PMC1755440  PMID: 15769916
20.  Antibody-Mediated Rejection in Human Cardiac Allografts: Evaluation of Immunoglobulins and Complement Activation Products C4d and C3d as Markers 
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.
PMCID: PMC1363343  PMID: 16212640
Antibody-mediated rejection; C3d; C4d; complement; heart transplant; macrophages
21.  Oxidant stress from nitric oxide synthase–3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load 
Journal of Clinical Investigation  2005;115(5):1221-1231.
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.
PMCID: PMC1077169  PMID: 15841206
22.  Transmission Study of Andes Hantavirus Infection in Wild Sigmodontine Rodents†  
Journal of Virology  2004;78(21):11972-11979.
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.
PMCID: PMC523238  PMID: 15479837
24.  Family violence, employment status, welfare benefits, and alcohol drinking in the United States: what is the relation? 
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
PMCID: PMC1731852  PMID: 11160171
25.  Unemployment, depression, and health: a look at the African-American community 
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.
PMCID: PMC1756891  PMID: 10396479

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