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1.  Periarteritis in Lung from a Continuous-Flow Right Ventricular Assist Device: Role of the Local Renin-Angiotensin System 
The Annals of thoracic surgery  2013;96(1):10.1016/j.athoracsur.2013.04.008.
We previously reported renal arterial periarteritis after implantation of a continuous-flow left ventricular assist device in calves. The purpose of the present study was to investigate whether the same periarteritis changes occur in the intrapulmonary arteries after implantation of a continuous-flow right ventricular assist device (CFRVAD) in calves and to determine the mechanism of those histological changes.
Ten calves were implanted with a CFRVAD for 29 ± 7 days, and we compared pulmonary artery samples and hemodynamic data pre- and post-CFRVAD implantation prospectively.
Post implantation, the pulsatility index (pulmonary arterial pulse pressure/pulmonary arterial mean pressure) significantly decreased (pre 0.88 ± 0.40 vs. post 0.51 ± 0.22; p < 0.05), with severe periarteritis of the intrapulmonary arteries in all cases. Periarterial pathology included hyperplasia and inflammatory cell infiltration. The number of inflammatory cells positive for the angiotensin II type 1 receptor was significantly higher after implantation (pre-CFRVAD 7.8 ± 6.5 vs. at autopsy 313.2 ± 145.2; p < 0.01). Serum angiotensin-converting enzyme activity significantly decreased after implantation (pre-CFRVAD 100%, week 1 49.7 ± 17.7%; p = 0.01). Tissue levels of angiotensin-converting enzyme also demonstrated a significant reduction (pre 0.381 ± 0.232 vs. at autopsy 0.123 ± 0.096; p = 0.043).
Periarteritis occurred in intrapulmonary arteries of calves after CFRVAD implantation. The local renin–angiotensin system (not the angiotensin-converting enzyme pathway) plays an important role in such changes.
PMCID: PMC3885324  PMID: 23731607
Artery/arteries (includes any peripheral arteries); Circulatory support devices (LVAD, RVAD, BVAD, TAH); Pulmonary arteries/veins (includes normal and diseased); Pathology (Lung)
2.  Antiviral innate immunity disturbs podocyte cell function 
Journal of innate immunity  2012;5(3):231-241.
IgA nephropathy is the most common form of glomerulonephritis throughout the world. A majority (~60%) of patients with IgAN experience disease exacerbations associated with acute respiratory or gastrointestinal illness that appears to represent viral infection. However, the exact mechanism of the disease exacerbation by viral infection is not understood, especially at the cellular and molecular levels. Here we report that glomerular podocytes express the major sensors for double-stranded (ds) RNA, a common byproduct of viral replication. In addition to these receptors, toll-like receptor 3 (TLR3) and RIG-I like helicases (RLH), podocytes express the collateral proteins required to support intracellular signaling. The pathways that mediate responses to dsRNA are fully functional in podocytes. The transcription factors, IRF3 and NF-κB are phosphorylated and translocate to the nucleus, and dsRNA increases synthesis of proteins driven by IRF3 (P54, P56, and P60) or NF-κB (IL-8 and A20). Furthermore, dsRNA suppresses podocyte cell migration, alters the expression of a panel of podocyte marker proteins (nephrin, podocin, and CD2-associated protein or CD2AP), and changes transepithelial albumin flux. These effects are dsRNA sensor-specific: TLR3−/− podocytes do not respond to extracellular dsRNA, while intracellular dsRNA has no effect on podocytes bearing a dominant negative form of the major activating RLH. These results demonstrate that innate responses to virus can disturb podocyte cell function in vitro.
PMCID: PMC3724426  PMID: 23296190
3.  Disruption of Smad4 Expression in T Cells Leads to IgA Nephropathy-Like Manifestations 
PLoS ONE  2013;8(11):e78736.
The link between glomerular IgA nephropathy (IgAN) and T helper 2 (Th2) response has been implicated, however, the mechanisms are poorly defined because of the lack of an appropriate model. Here we report a novel murine model characterized by lineage-restricted deletion of the gene encoding MAD homologue 4 (Smad4) in T cells (Smad4co/co;Lck-cre). Loss of Smad4 expression in T cells results in overproduction of Th2 cytokines and high serum IgA levels. We found that Smad4co/co;Lck-cre mice exhibited massive glomerular IgA deposition, increased albumin creatinine ratio, aberrant glycosylated IgA, IgA complexed with IgG1 and IgG2a, and polymeric IgA, all known features of IgAN in humans. Furthermore, we examined the β1, 4-galactosyltransferases (β4GalT) enzyme which is involved in the synthesis of glycosylated murine IgA, and we found reduced β4GalT2 and β4GalT4 mRNA levels in B cells. These findings indicate that Smad4co/co;Lck-cre mice could be a useful model for studying the mechanisms between IgAN and Th2 response, and further, disruption of Smad4-dependent signaling in T cells may play an important role in the pathogenesis of human IgAN and contributing to a Th2 T cell phenotype.
PMCID: PMC3817077  PMID: 24223846
4.  IgG3 deficiency extends lifespan and attenuates progression of glomerulonephritis in MRL/lpr mice 
Biology Direct  2012;7:3.
Antibodies of the IgG3 subclass have been implicated in the pathogenesis of the spontaneous glomerulonephritis observed in mice of the MRL/MpJ-Tnfrsf6lpr (MRL/lpr) inbred strain which have been widely studied as a model of systemic lupus erythematosus We have produced IgG3-deficient (-/-) mice with the MRL/lpr genetic background to determine whether IgG3 antibodies are necessary for or at least contributory to MRL/lpr-associated nephritis.
The gamma3 genotype (+/+ vs. +/- vs. -/-) did not appear to significantly affect serum titers of IgG auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin. However, while substantial serum titers of IgG3 auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin were seen in gamma3 +/+ mice, somewhat lower serum titers of these IgG3 auto-antibodies were found in gamma3 +/- mice, and gamma3 -/- mice exhibited baseline concentrations of these auto-antibodies. Analysis of immunoglobulins eluted from snap-frozen kidneys obtained from mice of all three gamma3 genotypes at ~18 weeks of age revealed much higher quantities of IgG in the kidneys from gamma3 +/+ than gamma3 -/- mice, and most IgG eluted from +/+ mice was IgG3. The serum creatinine levels in gamma3 +/+ mice substantially exceeded those of age-matched gamma3 -/- mice after ~21 weeks of age. Histopathological examination of kidneys from mice sacrificed at pre-determined ages also revealed more extensive glomerulosclerosis in gamma3 +/+ or +/- mice than in -/- mice beginning at 21 weeks of age. Survival analysis for IgG3-deficient and IgG3-producing MRL/lpr mice revealed that gamma3 -/- mice lived significantly longer (p = 0.0006) than either gamma3 +/- or +/+ mice. Spontaneous death appeared to be due to irreversible renal failure, because > 85% of glomeruli in kidneys from mice that died spontaneously were obliterated by glomerulosclerosis.
The available evidence suggests that IgG3 deficiency partially protects MRL/lpr mice against glomerulonephritis-associated morbidity and mortality by slowing or arresting the progression to glomerulosclerosis.
This article was reviewed by Pushpa Pandiyan, Irun Cohen, and Etienne Joly.
PMCID: PMC3293080  PMID: 22248284
5.  CpG-B Oligodeoxynucleotides Inhibit TLR-Dependent and -Independent Induction of Type I IFN in Dendritic Cells 
CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type I IFN (IFN-αβ) in dendritic cells (DCs). CpG-A ODNs are more efficacious than CpG-B ODNs for induction of IFN-αβ. Because IFN-αβ may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFN-αβ. In our studies, CpG-B ODN inhibited induction of IFN-αβ by CpG-A ODN, whereas induction of TNF-α and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFN-αβ was observed in FLT3 ligand-induced murine DCs, purified murine myeloid DCs, plasmacytoid DCs, and human PBMCs. CpG-B ODN inhibited induction of IFN-αβ by agonists of multiple receptors, including MyD88-dependent TLRs (CpG-AODN signaling via TLR9, or R837 or Sendai virus signaling via TLR7) and MyD88-independent receptors (polyinosinic:polycytidylic acid signaling via TLR3 or ds break-DNA signaling via a cytosolic pathway). CpG-B ODN did not inhibit the IFN-αβ positive feedback loop second-wave IFN-αβ, because IFN-αβ–induced expression of IFN-αβ was unaffected, and CpG-B inhibition of IFN-αβ was manifested in IFN-αβR−/− DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early TLR-induced first wave IFN-α4 and IFN-β. Chromatin immunoprecipitation revealed that association of IFN regulatory factor 1 with the IFN-α4 and IFN-β promoters was induced by CpG-A ODN but not CpG-B ODN. Moreover, CpG-A–induced association of IFN regulatory factor 1 with these promoters was inhibited by CpG-B ODN. Our studies demonstrate a novel mechanism of transcriptional regulation of first-wave IFN-αβ that selectively inhibits induction of IFN-αβ downstream of multiple receptors and may provide targets for future therapeutic inhibition of IFN-αβ expression in vivo.
PMCID: PMC2892962  PMID: 20181884
6.  Down-regulation of core 1 β1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1 
Nephrology Dialysis Transplantation  2010;25(12):3890-3897.
Background. Patients with IgA nephropathy (IgAN) have an increased amount of abnormally O-glycosylated IgA1 in circulation, in glomerular deposits and produced by tissue cells in vitro. Although increased production of Th2 cytokines by peripheral blood lymphocytes and a functional abnormality of core 1 β1,3-galactosyltransferase (C1β3Gal-T) have been proposed as mechanisms underlying pathogenesis of IgAN, they are still obscure and are not connected.
Methods. To clarify the effect of T-cell cytokines, we analysed the mRNA levels of C1β3Gal-T and its molecular chaperone Cosmc, C1β3Gal-T activity and subsequent O-glycosylation of IgA1 in a human B-cell line stimulated with these cytokines. The surface IgA1-positive human B-cell line was cultured with recombinant human IFN-γ, IL-2, IL-4 or IL-5. The production and glycosylation of IgA1 were determined by sandwich ELISA and enzyme-linked lectin binding assay, respectively. The mRNA levels of C1β3Gal-T and Cosmc were quantitatively measured by real-time PCR. C1β3Gal-T activity was analysed using high-performance liquid chromatography.
Results. IgA1 production by IL-4-stimulated cells was significantly higher than controls or after IFN-γ or IL-5. The terminal glycosylation of secreted IgA1 was altered in response to IL-4. IL-4 stimulation significantly decreased the mRNA levels of both C1β3Gal-T and Cosmc and of C1β3Gal-T activity. IL-4 stimulation was clearly blocked by recombinant human IL-4 soluble receptor.
Conclusions. It appears that Th2 cytokine IL-4 may play a key role in controlling glycosylation of the IgA1 hinge region.
PMCID: PMC2989791  PMID: 20551088
IgA1 hinge region; T-cell cytokines; the surface IgA1-positive human B-cell line; Th2 response
7.  Reduced Pulsatility Induces Periarteritis in Kidney: Role of the Local Renin-Angiotensin System 
The need for pulsatility in the circulation during long-term mechanical support has been a subject of debate. We compared histological changes in calf renal arteries subjected to various degrees of pulsatile circulation in vivo. We addressed the hypothesis that the local reninangiotensin system (RAS) may be implicated in these histological changes.
Methods and Results
Sixteen calves were implanted with devices giving differing degrees of pulsatile circulation: six had a continuous flow left ventricular assist device (LVAD); six had a continuous flow right ventricular assist device (RVAD); and four had a pulsatile total artificial heart (TAH). Six other calves were histological and immunohistochemical controls. In the LVAD group, the pulsatility index was significantly lower (0.28 ± 0.07 LVAD vs 0.56 ± 0.08 RVAD, vs 0.53 ± 0.10 TAH; p < 0.01), and we observed severe periarteritis in all cases in the LVAD group. The number of angiotensin II type 1 receptor (AT1R)-positive cells and angiotensin converting enzyme (ACE)-positive cells in periarterial areas was significantly higher in the LVAD group (AT1R: 350 ± 139 LVAD vs 8 ± 6 RVAD, vs 3 ± 2 TAH, vs 3 ± 2 in control; p < 0.001 and ACE: 325 ± 59 LVAD vs 6 ± 4 RVAD, vs 6 ± 5 TAH, vs 3 ± 1 control; p < 0.001).
The reduced pulsatility produced by a continuous flow LVAD implantation induced severe periarteritis in the kidney. The local RAS was upregulated in the inflammatory cells only in the continuous flow LVAD group.
We compared histological changes in calf renal arteries subjected to various degrees of pulsatile circulation; continuous flow left ventricular assist device (LVAD), continuous flow right ventricular assist device, pulsatile total artificial heart and control. We observed severe periarteritis, and upregulation of local renin angiotensin system only in the LVAD group. The necessity of maintaining pulsatility in the systemic circulation during long-term mechanical support has been a subject of debate. Recently, simpler and smaller continuous flow blood pumps have become more prevalent. The diminished pulsatility created by support from a continuous flow left ventricular assist device (LVAD) is physiologically abnormal, and some changes to the morphology of the aortic wall and the renal artery have been reported.1,2 Continuous flow LVAD support has been reported to cause renal cortical artery hypertrophy and inflammatory cell infiltration in the renal cortex.2 However, the mechanisms leading to those morphological changes are still unclear.
PMCID: PMC2533270  PMID: 18603068
8.  Immunocytochemical Colocalization of Specific Immunoglobulin A with Sendai Virus Protein in Infected Polarized Epithelium  
The Journal of Experimental Medicine  1998;188(7):1223-1229.
Immunoglobulin (Ig)A provides the initial immune barrier to viruses at mucosal surfaces. Specific IgA interrupts viral replication in polarized epithelium during receptor-mediated transport, probably by binding to newly synthesized viral proteins. Here, we demonstrate by immunoelectron microscopy that specific IgA monoclonal antibodies (mAbs) accumulate within Sendai virus–infected polarized cell monolayers and colocalize with the hemagglutinin– neuraminidase (HN) viral protein in a novel intracellular structure. Neither IgG specific for HN nor irrelevant IgA mAbs colocalize with viral protein. Treatment of cultures with viral-specific IgA but not with viral-specific IgG or irrelevant IgA decreases viral titers. These observations provide definitive ultrastructural evidence of a subcellular compartment in which specific IgA and viral envelope proteins interact, further strengthening our hypothesis of intracellular neutralization of virus by specific IgA antibodies. Our results have important implications for intracellular protein trafficking, viral replication, and viral vaccine development.
PMCID: PMC2212485  PMID: 9763601
immunoglobulin A; Sendai virus; mucosal immunity; colocalization; hemagglutinin–neuraminidase protein

Results 1-8 (8)