Wegener's Granulomatosis Patients Research Articles

Endobronchial HDR Brachytherapy for Post-Transplant or Wegener’s Granulomatosis Associated Bronchial Stenosis

Bronchial stenosis is a clinically challenging problem following lung transplant and in Wegener’s Granulomatosis. Despite laser, stenting, balloon dilation among other interventions, worsening of stenosis often occurs. Endobronchial brachytherapy has been implemented for treatment refractory stenotic areas based on limited data. We hypothesize that high dose rate (HDR) Brachytherapy using 3Gy x1 fraction is effective in alleviating treatment refractory stenosis of bronchus. Patients who underwent endobronchial HDR Ir-192 brachytherapy from January 2016 to January 2020 for bronchial stenosis were retrospectively reviewed. Patients were selected for HDR brachytherapy if they had treatment refractory symptomatic re-stenosis. On day of brachytherapy, a flexible bronchoscopy was performed and a brachytherapy catheter was inserted. Intraoperative fluoroscopic imaging confirmed catheter positioning. Treatment length ranged from 3-5cm. The dose of 3Gy in 1 fraction was prescribed 1cm from the catheter surface. Patients were followed with bronchoscopies and pulmonary function testing (PFTs). Four patients were included with median follow-up of 5.2m (range: 0.9-16.8). One patient received brachytherapy 12Gy in 4 fractions 12 years prior to re-treatment. The same patient received a second double lung transplant due to bronchiolitis obliterans. All patients had initial clinical & bronchoscopic improvement with brachytherapy. The one Wegener’s Granulomatosis patient required an additional two courses of brachytherapy due to residual granulation tissue found proximally outside of the prior radiation treatment fields. Median time to next endobronchial intervention after brachytherapy was 5.2m [0.9-18.8]. The median number of interventions pre and post brachytherapy are listed below (Table 1). FEV1 prior to brachytherapy was 33% predicted [27-65] and PFT after brachytherapy was 40% predicted [25-72]. The one Wegener granulomatosis patient had a balloon dilation procedure induced grade 4 bronchopulmonary hemorrhage three months after 3rd brachytherapy treatment, requiring bronchial artery embolization. They were subsequently discharged 36 hours after the initial bleed and remains free of stenosis. Table 1 Endobronchial HDR brachytherapy utilizing 3Gy x 1 fraction was safe and effective in resolving refractory re-stenotic areas of bronchus caused by granulation tissue. There were no toxicities associated with lung transplant patients. The Wegener’s Granulomatosis patient had a potential G4 toxicity after a third brachytherapy treatment. Caution needs to be applied in the setting of repeated endobronchial brachytherapy procedures. More robust follow up is needed to assess long term success.Abstract 2512; TablePatientNumber of Pre-Brachytherapy InterventionNumber of Post-Brachytherapy Intervention12-6 months6-0 months0-6 months6-12 months194191221311303442042900Median [range]6.5 [2-13]6.5 [4-11]2.5 [0-19]0 [0-12] Open table in a new tab

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7–3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4–3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP. Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7–3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4–3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP. Angiotensin-converting enzyme (ACE) 3The abbreviations used are: ACEangiotensin-converting enzymeHBSSHanks' balanced salt solutionMRSAmethicillin-resistant S. aureus2-NBDG2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucoseFBSfetal bovine serumDAPI4′,6-diamidino-2-phenylindoleVDACvoltage-dependent anion channelMFFmitochondrial fission factorTCAtrichloroacetic acidFCCPcarbonyl cyanide 4-(trifluoromethoxy) phenylhydrazoneTMREtetramethylrhodamine, ethyl esterPMA4-phorbol 12-myristate 13-acetateKOknockoutFDRfalse discovery rateATMatmospheric pressureCVcolumn volumeANOVAanalysis of variance. is a zinc-dependent peptidase that is made by endothelium and many other tissues (1Bernstein K.E. Ong F.S. Blackwell W.L. Shah K.H. Giani J.F. Gonzalez-Villalobos R.A. Shen X.Z. Fuchs S. Touyz R.M. A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting enzyme.Pharmacol. Rev. 2013; 65 (23257181): 1-4610.1124/pr.112.006809Crossref PubMed Scopus (196) Google Scholar). ACE is best known for its effects on blood pressure because it produces the potent vasoconstrictor angiotensin II, and ACE inhibitors are widely used in treating hypertension, heart failure, and other clinical problems (2Mentz R.J. Bakris G.L. Waeber B. McMurray J.J. Gheorghiade M. Ruilope L.M. Maggioni A.P. Swedberg K. Piña I.L. Fiuzat M. O'Connor C.M. Zannad F. Pitt B. The past, present and future of renin-angiotensin aldosterone system inhibition.Int. J. Cardiol. 2013; 167 (23121914): 1677-168710.1016/j.ijcard.2012.10.007Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Less well-recognized is that ACE cleaves many other peptides beside angiotensin II and plays an important function in several other physiologic processes, including immunity, reproduction, and hematopoiesis (3Bernstein K.E. Khan Z. Giani J.F. Cao D.Y. Bernstein E.A. Shen X.Z. Angiotensin-converting enzyme in innate and adaptive immunity.Nat. Rev. Nephrol. 2018; 14 (29578208): 325-33610.1038/nrneph.2018.15Crossref PubMed Scopus (120) Google Scholar). angiotensin-converting enzyme Hanks' balanced salt solution methicillin-resistant S. aureus 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose fetal bovine serum 4′,6-diamidino-2-phenylindole voltage-dependent anion channel mitochondrial fission factor trichloroacetic acid carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone tetramethylrhodamine, ethyl ester 4-phorbol 12-myristate 13-acetate knockout false discovery rate atmospheric pressure column volume analysis of variance. In 1975, it was reported that patients with active sarcoidosis have elevated serum ACE levels (4Lieberman J. Elevation of serum angiotensin-converting-enzyme (ACE) level in sarcoidosis.Am. J. Med. 1975; 59 (169692): 365-37210.1016/0002-9343(75)90395-2Abstract Full Text PDF PubMed Scopus (764) Google Scholar). We now know that lesional macrophages and macrophage-derived giant cells in virtually all human granulomatous diseases express abundant ACE, including histoplasmosis, leprosy, miliary tuberculosis, granulomatosis with polyangiitis (Wegener's granulomatosis), and others (5Baudin B. New aspects on angiotensin-converting enzyme: from gene to disease.Clin. Chem. Lab. Med. 2002; 40 (12005216): 256-26510.1515/CCLM.2002.042Crossref PubMed Scopus (121) Google Scholar, 6Brice E.A. Friedlander W. Bateman E.D. Kirsch R.E. Serum angiotensin-converting enzyme activity, concentration, and specific activity in granulomatous interstitial lung disease, tuberculosis, and COPD.Chest. 1995; 107 (7874941): 706-71010.1378/chest.107.3.706Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 7Lieberman J. Rea T.H. Serum angiotensin-converting enzyme in leprosy and coccidioidomycosis.Ann. Intern. Med. 1977; 87 (199098): 423-42510.7326/0003-4819-87-4-422Crossref PubMed Scopus (100) Google Scholar, 8Olle E.W. Deogracias M.P. Messamore J.E. McClintock S.D. Barron A.G. Anderson T.D. Johnson K.J. Screening of serum samples from Wegener's granulomatosis patients using antibody microarrays.Proteomics Clin. Appl. 2007; 1 (21136620): 1212-122010.1002/prca.200600906Crossref PubMed Scopus (9) Google Scholar, 9Weinstock J.V. Production of neuropeptides by inflammatory cells within the granulomas of murine schistosomiasis mansoni.Eur. J. Clin. Invest. 1991; 21 (1905627): 145-15310.1111/j.1365-2362.1991.tb01803.xCrossref PubMed Scopus (33) Google Scholar). Macrophage ACE up-regulation is found in zebra fish granuloma (10Cronan M.R. Beerman R.W. Rosenberg A.F. Saelens J.W. Johnson M.G. Oehlers S.H. Sisk D.M. Jurcic Smith K.L. Medvitz N.A. Miller S.E. Trinh L.A. Fraser S.E. Madden J.F. Turner J. Stout J.E. et al.Macrophage epithelial reprogramming underlies mycobacterial granuloma formation and promotes infection.Immunity. 2016; 45 (27760340): 861-87610.1016/j.immuni.2016.09.014Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Macrophages in Gaucher's disease make abundant ACE, as do the macrophages found in both early and late stages of human atherosclerotic lesions (11Danilov S.M. Tikhomirova V.E. Metzger R. Naperova I.A. Bukina T.M. Goker-Alpan O. Tayebi N. Gayfullin N.M. Schwartz D.E. Samokhodskaya L.M. Kost O.A. Sidransky E. ACE phenotyping in Gaucher disease.Mol. Genet. Metab. 2018; 123 (29478818): 501-51010.1016/j.ymgme.2018.02.007Crossref PubMed Scopus (20) Google Scholar, 12Diet F. Pratt R.E. Berry G.J. Momose N. Gibbons G.H. Dzau V.J. Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease.Circulation. 1996; 94 (8941100): 2756-276710.1161/01.CIR.94.11.2756Crossref PubMed Scopus (399) Google Scholar, 13Ohishi M. Ueda M. Rakugi H. Naruko T. Kojima A. Okamura A. Higaki J. Ogihara T. Enhanced expression of angiotensin-converting enzyme is associated with progression of coronary atherosclerosis in humans.J. Hypertens. 1997; 15 (9383179): 1295-130210.1097/00004872-199715110-00014Crossref PubMed Scopus (98) Google Scholar). ACE increases when the human monocytic cell line THP-1 is differentiated into macrophages (12Diet F. Pratt R.E. Berry G.J. Momose N. Gibbons G.H. Dzau V.J. Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease.Circulation. 1996; 94 (8941100): 2756-276710.1161/01.CIR.94.11.2756Crossref PubMed Scopus (399) Google Scholar). Similarly, when human peripheral blood monocytes are differentiated to macrophages in vitro, cell ACE activity increases 9-fold (14Saijonmaa O. Nyman T. Fyhrquist F. Atorvastatin inhibits angiotensin-converting enzyme induction in differentiating human macrophages.Am. J. Physiol. Heart Circ. Physiol. 2007; 292 (17158648): H1917-H192110.1152/ajpheart.00920.2006Crossref PubMed Scopus (16) Google Scholar). In mice, ACE expression in splenic neutrophils, macrophages, and dendritic cells rapidly increases following Staphylococcus aureus or Listeria monocytogenes infection (15Khan Z. Shen X.Z. Bernstein E.A. Giani J.F. Eriguchi M. Zhao T.V. Gonzalez-Villalobos R.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme enhances the oxidative response and bactericidal activity of neutrophils.Blood. 2017; 130 (28515091): 328-33910.1182/blood-2016-11-752006Crossref PubMed Scopus (49) Google Scholar, 16Shen X.Z. Billet S. Lin C. Okwan-Duodu D. Chen X. Lukacher A.E. Bernstein K.E. The carboxypeptidase ACE shapes the MHC class I peptide repertoire.Nat. Immunol. 2011; 12 (21964607): 1078-108510.1038/ni.2107Crossref PubMed Scopus (59) Google Scholar). Despite these observations, little is known about the functional role of ACE up-regulation in myeloid cells. Recently, our group investigated two lines of mice in which genetic manipulation was used to selectively increase ACE expression by monocytes and macrophages or by neutrophils (15Khan Z. Shen X.Z. Bernstein E.A. Giani J.F. Eriguchi M. Zhao T.V. Gonzalez-Villalobos R.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme enhances the oxidative response and bactericidal activity of neutrophils.Blood. 2017; 130 (28515091): 328-33910.1182/blood-2016-11-752006Crossref PubMed Scopus (49) Google Scholar, 17Shen X.Z. Li P. Weiss D. Fuchs S. Xiao H.D. Adams J.A. Williams I.R. Capecchi M.R. Taylor W.R. Bernstein K.E. Mice with enhanced macrophage angiotensin-converting enzyme are resistant to melanoma.Am. J. Pathol. 2007; 170 (17525278): 2122-213410.2353/ajpath.2007.061205Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In these animals, termed ACE 10/10 and NeuACE mice, the cells overexpressing ACE respond to immune challenge with a consistent enhancement in immune response. For example, ACE 10/10 mice (in which monocytes/macrophages express 16–25-fold WT levels of ACE) resist the growth of B16 melanoma significantly better than WT mice and show increased resistance to infection with Listeria or methicillin-resistant S. aureus (MRSA) (17Shen X.Z. Li P. Weiss D. Fuchs S. Xiao H.D. Adams J.A. Williams I.R. Capecchi M.R. Taylor W.R. Bernstein K.E. Mice with enhanced macrophage angiotensin-converting enzyme are resistant to melanoma.Am. J. Pathol. 2007; 170 (17525278): 2122-213410.2353/ajpath.2007.061205Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Okwan-Duodu D. Datta V. Shen X.Z. Goodridge H.S. Bernstein E.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme overexpression in mouse myelomonocytic cells augments resistance to Listeria and methicillin-resistant Staphylococcus aureus.J. Biol. Chem. 2010; 285 (20937811): 39051-3906010.1074/jbc.M110.163782Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Similarly, NeuACE neutrophils (which express 12–18-fold WT levels of ACE) show increased resistance to MRSA, Klebsiella pneumoniae, or Pseudomonas aeruginosa, as measured both in vivo and in the ability to kill bacteria in vitro (15Khan Z. Shen X.Z. Bernstein E.A. Giani J.F. Eriguchi M. Zhao T.V. Gonzalez-Villalobos R.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme enhances the oxidative response and bactericidal activity of neutrophils.Blood. 2017; 130 (28515091): 328-33910.1182/blood-2016-11-752006Crossref PubMed Scopus (49) Google Scholar). Both NeuACE neutrophils and ACE 10/10 macrophages make increased levels of NADPH oxidase–dependent superoxide, which contributes to their enhanced killing of bacteria. To investigate the molecular basis of how ACE overexpression affects cells, we studied the metabolism of such cells. Unexpectedly, in both macrophages and neutrophils, ACE overexpression induces a significant increase in cellular ATP, in levels of Krebs cycle intermediates, and in proteins comprising the electron transport chain. These metabolic changes are due to ACE catalytic activity, as they are reverted to WT levels by treating mice with ACE inhibitors. Our studies find that ACE overexpression by both macrophages and neutrophils is associated with a marked change in the metabolism of cells and that this appears to underpin some of the phenotypic differences between these cells and myeloid cells expressing WT levels of ACE. Mass spectrometry was used to measure the intermediate metabolites of ACE 10/10 macrophages and NeuACE neutrophils, as well as equivalent cells from WT mice. Thioglycollate-elicited ACE 10/10 macrophages showed 27 significant differences from WT cells (defined as a false discovery rate–adjusted p value <0.01), with 26 metabolites increased in ACE 10/10 (Fig. S1 and Table S1). Only adenosine was diminished. A similar comparison of freshly isolated NeuACE bone marrow neutrophils with equivalent WT cells showed 15 significant differences, with all being increased in the ACE-overexpressing cells. Perhaps most surprising was the increased amounts of adenosine mono-, di-, and triphosphate in both ACE-overexpressing groups (Fig. 1, A and B). In ACE 10/10 macrophages, the increase in cellular ATP, ADP, and AMP was 3.0-, 2.3-, and 2.6-fold the levels in WT cells. In NeuACE neutrophils, there was an equivalent pattern, although the magnitude of the increase (1.9-, 2.1-, and 1.7-fold) was less than in macrophages. To verify the MS results, we measured levels of cellular ATP chemically in additional samples and found a 2.4- and 1.7-fold increase of ATP in ACE 10/10 macrophages and NeuACE neutrophils (p < 0.001; Fig. 1C). Macrophages from ACE heterozygous mice have roughly 2-fold higher levels of ATP as compared with WT (p < 0.05), whereas, in stark contrast, cells from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT (p < 0.01). For ACE 10/10 macrophages, the ATP/ADP ratio was 2.1 versus 1.4 for WT cells, as determined chemically (Fig. 1D). For NeuACE neutrophils, the ATP/ADP ratio was 3.0 versus 1.9 in WT cells. To examine whether differences in ATP between peritoneal macrophages from ACE 10/10 and WT mice were due to differences in overall cellular protein content, both protein and ATP were measured chemically in another cohort of mice. Whereas ACE 10/10 cells again had significantly more ATP (p < 0.001), the protein content of the cells was equivalent to peritoneal macrophages from WT mice (Fig. S2A). As discussed below, we also evaluated the size of ACE 10/10 and WT macrophages and found no differences (Fig. 2E). Finally, whereas the typical preparation of peritoneal macrophages contained 80–90% macrophages by flow cytometry, ATP was also measured in peritoneal macrophages purified to over 90% using an antibody-based negative cell selection protocol. This too showed more ATP in ACE 10/10 macrophages than equivalent WT cells (see Fig. 7A).Figure 7Decreasing ATP in ACE-overexpressing cells reduces superoxide production and phagocytosis to WT levels. A and B, ACE 10/10 macrophages and WT cells were treated with the ATP synthase inhibitor oligomycin (10 μg/ml) for 20 min. Cellular ATP levels (A) and PMA-induced production of superoxide (B) were measured. C and D, a similar experiment was performed using NeuACE neutrophils and WT cells. For A–D, n ≥ 5 mice, mean ± S.E. (error bars). A and C were studied by one-way ANOVA with the Tukey test. **, p < 0.01; ***, p < 0.001. E and F, peritoneal macrophages from WT, ACE 10/10, and ACE KO mice were cultured with fluorescein-labeled E. coli particles with or without oligomycin (10 μg/ml) for 2 h. Analysis of green fluorescence in CD11b+F4/80+ cells is shown in E, whereas the mean florescence intensity (MFI) is plotted in F. n ≥ 5 mice, mean ± S.E. Analysis was by one-way ANOVA with the Tukey test: *, p < 0.05; **, p < 0.01; NS, no significance.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the above analysis of ATP, neutrophils were obtained from the bone marrow of mice without any additional stimulation. In contrast, peritoneal macrophages were obtained following stimulation with thioglycollate. When resident peritoneal macrophages were isolated without thioglycollate, ACE 10/10 and WT macrophages showed no difference in ATP levels, as did bone marrow–derived macrophages produced in vitro (Fig. 2, B and C). Thus, macrophage stimulus affects the level of ATP in ACE 10/10 versus WT cells. The increase of cellular ATP is due to the catalytic activity of ACE. Specifically, when animals were continuously treated for 7 days with the ACE inhibitor ramipril, thioglycollate-induced peritoneal macrophages or bone marrow–derived neutrophils showed ATP levels that were now similar to the cells from WT mice also treated with ramipril (Fig. 1, E and F). In contrast, a similar experiment (7-day treatment) performed with the angiotensin II AT1 receptor antagonist losartan showed no significant effects as compared with equivalent animals not treated with the receptor antagonist. WT mice treated with ramipril had less ATP in macrophages and neutrophils than in WT cells from untreated mice, similar to the result in Fig. 1C with ACE knockout (KO) macrophages. Given the differences in intermediate metabolites observed in cells overexpressing ACE, we examined the quantity of cellular glucose transport proteins GLUT1 and GLUT4 by Western blot analysis. No differences were noted between macrophages and neutrophils overexpressing ACE and equivalent WT cells (Fig. 2A and Fig. S3A). Two means of assessing glucose uptake were used: the uptake of the fluorescent glucose analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG), as determined by flow cytometry, and an independent chemical analysis of the uptake of 2-deoxyglucose. Neither assay showed increased glucose uptake in ACE-overexpressing cells as compared with equivalent cells with WT ACE expression (Fig. 2B and Fig. S3B). Glycolytic flux ultimately produces lactic acid. We assessed lactic acid levels using two approaches. First, MS determination of cellular lactic acid showed no difference in ACE 10/10 macrophages or NeuACE neutrophils as compared with WT cells (Fig. 2C and Fig. 3C). In addition to this static determination of cellular lactic acid, we also measured the production of lactic acid by cultured macrophages. Thioglycollate-induced ACE 10/10 and WT macrophages were collected and cultured for 12 h in one of three media: medium with 10% fetal bovine serum (FBS), medium without FBS, and medium without FBS and without glucose. We then assayed the lactate concentration in the culture supernatant via the production of NADH by lactate dehydrogenase (Fig. 2D). This showed a small increase of lactate production by ACE 10/10 cells, but these differences were not significant with the number of samples measured. A possible explanation for differences in intermediate metabolites could be due to differences in cell size or mitochondrial number. We investigated this using two assays. First, ACE 10/10 macrophages and NeuACE neutrophils were stained with Mitotracker Green and measured by flow cytometry. This assay of mitochondria showed no significant differences between ACE-overexpressing cells and equivalent cells from mice with WT ACE levels (Fig. S4, A and B). In a more extensive analysis, ACE 10/10 macrophages were studied by confocal microscopy following staining of the nuclei, cytoplasm, and mitochondria with 4′,6-diamidino-2-phenylindole (DAPI), phalloidin green, and Mitotracker Red, respectively (Fig. 2E). Individual images of cells were then quantified for the number of pixels of cytoplasm and of mitochondria. This analysis showed no differences between ACE 10/10 macrophages and WT cells in cell size, mitochondrial number, mitochondrial size, and mitochondrial number per cell size (Fig. S4C). Further, analysis of ACE 10/10 macrophages by EM showed no significant differences in mitochondrial size or cristae structure (Fig. S4D). This conclusion was supported by using Western blotting to measure total cellular dynamin-like 120-kDa protein (OPA1), voltage-dependent anion channel (VDAC), mitochondrial fission factor (MFF), and TOM20 (Fig. 2F). OPA1, VDAC, and MFF were equivalent between ACE 10/10 macrophages and WT cells. Only TOM20 was different; in ACE 10/10 macrophages, it was 1.4-fold the level of WT cells. Thus, it appears that cell size, the number of mitochondria, and mitochondrial size are not major differentiating features between cells overexpressing ACE and equivalent cells with WT ACE levels. As part of the MS analysis of metabolites, we determined the levels of metabolic intermediates found in the TCA cycle. These data showed that ACE 10/10 macrophages have significantly higher levels of citric acid (3.9-fold), isocitrate (5.2-fold), succinate (1.9-fold), and malate (2.0-fold) (Fig. 3 and Table S1). NAD and FAD in ACE 10/10 macrophages were also 1.6- and 1.5-fold higher than in equivalent WT cells. In neutrophils from NeuACE mice, there was a similar trend, although the differences between cells overexpressing ACE and those with WT ACE levels were less pronounced. Specifically, for citrate, isocitrate, succinate, and l-malate, levels were 2.0-, 1.9-, 1.4-, and 1.5-fold higher in NeuACE neutrophils compared with neutrophils from WT mice. The NeuACE neutrophils also had increased NAD (1.3-fold) and FAD (1.4-fold). Given the increased concentration of ATP and TCA cycle intermediates in ACE-overexpressing macrophages, we measured cellular respiration to determine whether there were changes in mitochondrial function. Peritoneal macrophages were obtained after thioglycollate injection and then transferred to culture dishes. Following an overnight incubation, cells were gently detached and acutely adhered to an Agilent Seahorse XF96 plate, and respiratory parameters were measured as described previously (Fig. 4, A and B) (19Divakaruni A.S. Paradyse A. Ferrick D.A. Murphy A.N. Jastroch M. Analysis and interpretation of microplate-based oxygen consumption and pH data.Methods Enzymol. 2014; 547 (25416364): 309-35410.1016/B978-0-12-801415-8.00016-3Crossref PubMed Scopus (264) Google Scholar). The rates of ATP production by glycolysis and oxidative metabolism in ACE 10/10 cells were 31 and 27% higher than WT cells. Total ATP production rates by ACE 10/10 cells were 29% higher than WT (11.2 ± 1.00 pmol of ATP/min/1000 cells (ACE 10/10) versus 8.7 ± 1.37 pmol of ATP/min/1000 cells (WT); p < 0.05, n ≥ 11). To investigate whether the change in oxygen consumption upon ACE overexpression was due to direct mitochondrial changes or simply reflected an increased activation state of the cells, we measured maximal oxygen consumption rates with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). After blocking ATP synthase with oligomycin, the addition of FCCP measures maximal respiratory rates when mitochondria are disengaged (uncoupled) from ATP synthesis. Indeed, there was a clear difference in maximal oxygen consumption, which averaged 37% higher in ACE 10/10 cells than in WT macrophages (2.81 ± 0.18 pmol of O2/min/1000 cells (ACE 10/10) versus 2.05 ± 0.24 pmol of O2/min/1000 cells (WT), p < 0.02) (Fig. 4C). To further investigate whether the increase in respiration observed upon ACE overexpression was directly due to mitochondrial changes, we permeabilized the plasma membrane of ACE 10/10 and WT macrophages with recombinant perfringolysin O and offered in situ mitochondria various respiratory substrates (Fig. 4D) (20Divakaruni A.S. Wiley S.E. Rogers G.W. Andreyev A.Y. Petrosyan S. Loviscach M. Wall E.A. Yadava N. Heuck A.P. Ferrick D.A. Henry R.R. McDonald W.G. Colca J.R. Simon M.I. Ciaraldi T.P. Murphy A.N. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23513224): 5422-542710.1073/pnas.1303360110Crossref PubMed Scopus (202) Google Scholar, 21Divakaruni A.S. Rogers G.W. Murphy A.N. Measuring mitochondrial function in permeabilized cells using the Seahorse XF analyzer or a Clark-type oxygen electrode.Curr. Protoc. Toxicol. 2014; 60 (24865646): 25.2.1-25.21610.1002/0471140856.tx2502s60Crossref Scopus (66) Google Scholar). Indeed, ADP-supported respiration was significantly increased in ACE 10/10 in response to pyruvate/malate, glutamate/malate, and succinate/rotenone. Thus, these data indicate a significant increase in respiratory rate in cells overexpressing ACE. The finding of increased cellular ATP, increased concentrations of TCA intermediates, and increased respiration led us to ask whether ACE affects electron transport chain proteins. These were studied using two different approaches. First, analysis of ACE 10/10 and WT macrophages using MS provided information on 28 proteins associated with mitochondria (Fig. 5A and Table S2). Most striking was the increase in proteins from electron transport complex IV (COX2 and COX4) and complex V (ATP5A) found in ACE 10/10. TOM20 was also significantly increased in ACE 10/10 macrophages. As a second means of assessing electron transport chain protein, we used Western blot analysis with six antibodies directed against a protein present in each of the five electron transport chain complexes (NDUFB8, SDHB, UQCRC2, COX1, ATP5A, and ATP5β) of ACE 10/10 macrophages and NeuACE neutrophils (Fig. 5, B and C). An equivalent study used WT cells. In both macrophages and neutrophils overexpressing ACE, there was a significant increase in the proteins from electron transport complex I (NDUFB8) and complex V (ATP5A and ATP5β) as compared with WT data. This is despite the fact that neutrophils predominantly use glycolysis rather than oxidative phosphorylation to generate ATP (22Davies L.C. Rice C.M. McVicar D.W. Weiss J.M. Diversity and environmental adaptation of phagocytic cell metabolism.J. Leukoc. Biol. 2019; 105 (30247792): 37-4810.1002/JLB.4RI0518-195RCrossref PubMed Scopus (32) Google Scholar). ACE 10/10 macrophages also had an increase of COX1 from electron transport complex IV. Thus, these data showing an increase of a subset of electron chain proteins are complementary to the increased respiratory rate found in ACE 10/10 macrophages. Mitochondrial production of ATP is driven by the difference in membrane potential across the inner mitochondrial membrane. This was assessed by flow analysis of macrophages from ACE 10/10, WT, and ACE knockout mice after staining with the mitochondrial dye tetramethylrhodamine, ethyl ester (TMRE) (23Antonenkov V.D. Isomursu A. Mennerich D. Vapola M.H. Weiher H. Kietzmann T. Hiltunen J.K. The human mitochondrial DNA depletion syndrome gene MPV17 encodes a non-selective channel that modulates membrane potential.J. Biol. Chem. 2015; 290 (25861990): 13840-1386110.1074/jbc.M114.608083Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In this assay, increased membrane potential results in increased fluorescence. We saw that, as compared with WT cells, macrophages from ACE 10/10 mice had a 24% increase of mean fluorescence intensity (increased Δψm), whereas macrophages from ACE knockout mice had a 30% decrease of mean fluorescence intensity (Fig. 5D, p < 0.05). Whereas the NeuACE mice are transgenic, the ACE 10/10 mice were made by substituting the promoter of the endogenous ACE gene. T and B cells from ACE 10/10 do not make increased ACE, but to study whether the genetic manipulation had a generalized effect on all hematopoietic cells, we measured ATP levels and selective electron transport proteins in these cells (Fig. S5, A and B). This showed that ACE 10/10 T and B cells have levels of cellular ATP and mitochondrial proteins equivalent to those of WT cells. Western blotting was also used to assess the level of electron transport proteins in the spleen, heart, lung, liver, kidney, and skeletal muscle of ACE 10 (Fig. 5C). As with lymphocytes, there was no difference between ACE 10/10 and WT mice. ACE is a single polypeptide chain, but it is composed of two homologous catalytic domains termed the N-

SAT0166 The Incidences of Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis in Northeastern Part of Turkey.

BackgroundThe epidemiological findings of anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) differ geographically. Until now, there has been no study about the prevalence of AAV in Turkey.ObjectivesIn our study, we aimed to...

Dysregulation of Histone Deacetylase and Histone Acetyltransferase in Development of Wegener's Granulomatosis

We investigated the function of the decreases in histone deacetylase (HDAC) and histone acetyltransferase (HAT) in Wegener's granulomatosis (WG) patients compared with healthy subjects. Seven patients with WG, diagnosed according to the American College of Rheumatology criteria, were examined. Fresh peripheral blood mononuclear cells were isolated from the WG patients and healthy subjects, and then whole-cell proteins were prepared. We measured the total HDAC and HAT activity in peripheral blood mononuclear cells from WG patients. The HDAC2 expression was analyzed by Western blot. We found that total HDAC activity was significantly decreased in WG patients compared to that in healthy subjects (p < 0.05). Furthermore, we found a negative correlation between total HDAC activity and C-reactive protein titer. Total HAT activity was significantly increased in WG patients. These results demonstrated reduced HDAC activity and an increase in HAT activity in WG. These were associated with concomitant induction of WG-related inflammation. Thus, dysregulation of HDAC and HAT may contribute to the disease pathogenesis of WG.

External Dacryocystorhinostomy Surgery in Patients With Wegener Granulomatosis

To determine surgical outcomes after external dacryocystorhinostomy (DCR) surgery in patients with Wegener granulomatosis (WG). The authors retrospectively reviewed the charts of consecutive patients with WG who underwent primary or secondary external DCR surgery between January 2001 and January 2010. Clinical data reviewed included patient demographics, systemic disease involvement and immunosuppression therapy, intraoperative biopsy findings, and postoperative outcomes and complications. Success was defined as resolution of epiphora. Sixteen primary external DCRs were performed on 9 patients with WG, and 2 secondary external DCRs were performed on 2 patients. At the time of surgery, all patients with WG were on systemic immunosuppressive agents including methotrexate, rapamycin, sirolimus, tacrolimus, azathioprine, cyclophosphamide, rituximab, and prednisone, and no patients received increased corticosteroids after surgery. Intraoperative biopsy in patients with WG revealed chronic inflammation (4 patients) and fibrosis (1 patient). Silicone stents were removed at an average of 5.8 months (range, 3-12 months). All surgeries were successful in resolving epiphora with an average follow up of 3.5 years (range, 10 months-6 years) and no complications. Primary and secondary external DCR surgery successfully treats nasolacrimal duct obstruction in patients with WG on systemic immunosuppression.

The protein tyrosine phosphatase nonreceptor 22 C1858T polymorphism and vasculitis: a meta-analysis

The aim of this study was to determine whether the functional protein tyrosine phosphatase nonreceptor 22 (PTPN22) C1858T polymorphism (rs2476601) confers susceptibility to vasculitis. A meta-analysis was conducted on the PTPN22 C1858T polymorphism across nine comparative studies containing 1,922 vasculitis patients and 11,505 normal control subjects. Meta-analysis showed no association between the T allele and vasculitis in all subjects [odds ratio (OR) 1.046, 95 % confidence interval (CI) 0.755-1.1.451, p = 0.786], and analysis after stratification by ethnicity indicated that the T allele was not associated with vasculitis in Europeans (OR 1.104, 95 % CI 0.798-1.528, p = 0.551). However, meta-analysis showed a significant association between the T allele and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) (OR 1.415, 95 % CI 1.228-1.630, p = 1.59 × 10(-6)) and Wegener's granulomatosis (WG) (OR 1.829, 95 % CI 1.377-2.431, p = 3.09 × 10(-5)). In addition, meta-analysis showed an association between the T allele and WG in ANCA-positive subjects (OR 2.042, 95 % CI 1.534-2.719, p = 1.02 × 10(-6)), but not in ANCA-negative WG patients (OR 0.595, 95 % CI 0.199-1.781, p = 0.353). This meta-analysis does not show that the PTPN22 C1858T polymorphism is associated with vasculitis susceptibility, but does show that this polymorphism is associated with susceptibility to AAV, WG, and ANCA status in WG.

Lesões destrutivas da linha média induzidas por cocaína com ANCA positivo mimetizando a granulomatose de Wegener

Chronic use of cocaine by inhalation may induce midline destructive lesions (CIMDL), which can sometimes be difficult to distinguish from the ear, nose and throat lesions of Wegener's Granulomatosis (WG). We describe the case of a 43-year-old female patient admitted with a two-year history of nasal obstruction and rhinorrhea. She had been diagnosed with WG for five months, being on prednisone and cyclophosphamide. On her physical examination, perforation of her nasal septum and palate was observed. Laboratory tests showed elevated acute phase proteins and a positive p-ANCA test. ELISA assays anti-proteinase 3 and myeloperoxidase were negative. The paranasal sinus computed tomography (CT) showed destruction of the nasal septum and palate, in addition to bilateral maxillary sinusitis. Chest CT was normal. Nasal mucosal biopsy revealed an inflammatory infiltrate, with neither granuloma nor vasculitis. When questioned, she admitted being a cocaine user for five years. Medical therapy and cocaine use were withdrawn. She has been followed up for six months and no other lesion or other organ symptoms occurred. Differential diagnosis in patients with midline destructive lesions can be very challenging. Evaluation should include enquiry about intranasal use of cocaine. Although ANCA testing does not clearly differentiate the ANCA found in some patients with CIMDL from those found in WG patients, the localized involvement and the biopsy findings non-characteristic of small vessel granulomatous vasculitis should be recognized as features for cocaine-induced lesions.

ANCA-associated glomerulonephritis/systemic vasculitis in childhood: clinical features–outcome

Antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis and systemic vasculitis (AAGNV) is uncommon in childhood. This is a retrospective study of AAGNV cases diagnosed over a 13-year period in a tertiary pediatric nephrology department. Thirteen cases of AAGNV were identified: seven Wegener granulomatosis (WG) and six microscopic polyangiitis (MPA). Acute renal failure/nephrotic range proteinuria (NRP) was found in 77 % of the patients (4 with WG, all with MPA). Eleven (85 %) patients showed necrotizing glomerulonephritis (NGN), with ≥50 % crescents identified in nine patients (69 %) (4 with WG, 5 with MPA). Treatment with methylprednisolone, cyclophosphamide and plasma exchange resulted in extra-renal remission and antibody reduction in all patients and renal function improvement/stabilization in 77 % of the patients. Three patients, all without oliguria at presentation and few sclerotic lesions, had normal renal function at follow-up. Chronic kidney disease (CKD) stages 2 and 3-4 were observed in four (WG) and three (MPA) patients, respectively. Three patients (23 %) developed end stage renal disease: two were MPA patients with severe presentation (markedly impaired glomerular filtration rate, oliguria, NRP, crescentic NGN, glomerular sclerosis) and one was a WG patient with extensive interstitial fibrosis/tubular atrophy. Severe renal involvement was more common in children with MPA than WG. Treatment with methylprednisolone, cyclophosphamide and plasma exchange induced extra-renal remission/serological response and renal function improvement/stabilization. Markedly decreased GFR, oliguria, NRP, and chronic glomerular lesions at presentation were predictors of poor outcome.

Renal cancer and Wegener's granulomatosis: a case report

Wegener's granulomatosis (WG) is a systemic disorder characterized by necrotizing vasculitis involving the respiratory tract, and in most cases, the kidneys. The most common manifestation of WG in the kidneys is segmental necrotizing glomerulonephritis. The presence of a renal mass as a manifestation of WG is rare. We report a patient with WG in whom a CT scan revealed an infiltrating mass in the lower portion of the left kidney. After surgical exploration, we performed an open radical nephrectomy. Histopathology showed clear cell type renal cell carcinoma (RCC). RCC associated with WG has been reported in only a few cases, and in most of them, the diseases started simultaneously, suggesting common pathogenetic pathways. Long-term immunosuppressive treatment is a known risk factor in the development of malignancies, so occurrence of RCC in WG has been proposed as a side effect of cyclophosphamide treatment. Furthermore, it is important to make a differential diagnosis between RCC and pseudotumors in WG as they cannot be distinguished solely on basis of imaging findings. Due to the higher risk of urologic malignancies, more frequent checkups and screening of WG patients should be considered.

Fluorine-18-fluorodeoxyglucose PET/CT rare finding of a unique multiorgan involvement of Wegener's granulomatosis

Wegener's granulomatosis (WG) is an uncommon autoimmune disorder, which mainly involves the blood vessels, kidneys and respiratory tract. We report an interesting case of WG with unusual multiorgan involvement in a young male who presented with a short history of right-sided otalgia, nasal obstruction and a right parotid mass. His initial CT and MRI scans showed a large parotid mass with features suggestive of malignancy with bilateral cavitating pulmonary nodules suggesting metastatic disease. The imaging-based differential diagnosis was squamous cell carcinoma or adenoid cystic carcinoma. The microscopic findings on ultrasound-guided biopsy of the parotid mass were, surprisingly, those of acute necrotising granulomatous inflammation with some features suggestive of a vasculitic process. A multidisciplinary team discussion and further investigation resulted in the additional findings of haematuria, raised erythrocyte sedimentation rate and positive serum cytoplasmic anti-neutrophil cytoplasmic antibody test, which led to the diagnosis of WG. Subsequently, the patient developed acute urinary retention owing to gross prostatic enlargement related to further disease involvement, which was confirmed with a positive biopsy. Fluorine-18-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/CT scan showed disease distribution at the right maxillary sinus/nasal cavity, right parotid, mediastinum, lungs and prostate. To our knowledge, this is the first reported 18F-FDG PET/CT case with multiorgan involvement in a single WG patient. The patient has improved both clinically and on imaging after appropriate treatment with immunosuppressive therapy and steroids. Although 18F-FDG PET/CT imaging did not actually alter the management of this patient, it can help to establish the disease distribution and guide the biopsy.

Chromogranin A — A comparison of serum and plasma results from different ELISA kits

Antineutrophil cytoplasmic antibodies (ANCA) are used as diagnostic markers for systemic vasculitis. However,the specificity and sensitivity of ANCA detection differs from centre to centre due in large part to variations in methodology. We compared 8 commercial ELISA kits and an in-house method (HM) for their specificity and sensitivity in detecting ANCA against proteinase 3 (PR3-ANCA, 7 kits) and meyloperoxidse (MPO-ANCA, 8 kits). Sera from 5 patients with systemic lupus erythematosus (SLE), 28 with Wegener's granulomatosis (WG), 22 with microscopic polyangiitis (MPA), 5 with idiopathic rapidly progressive glomerulonephritis (RPGN), and 5 healthy controls were examined by both the indirect immunofluorescence technique (IFT) and the ELISA kits. Sera from healthy controls and patients with SLE or cANCA-negative WG were shown to be PR3-ANCA negative by all 7 PR3-ANCA kits. In 25 cANCA-positive sera from WG patients, PR3-ANCA positivity ranged from 44% to 84%. An absolute concordance among the 7 kits was noted in 56% of the cANCA-positive samples. The PR3-ANCA levels in 5 of the 7 kits correlated with the cANCA titers in IFT. Sera from the healthy controls and 4 out of the 5 SLE and pANCA-negative patients were found to be MPO-ANCA negative in all 8 MPO-ANCA kits. In 20 pANCA-positive sera, MPO-ANCA positivity ranged from 25% to 75%. Thirty-five percent of MPO-ANCA-positive sera were confirmed by capture ELISA, immunoblot and inhibition assay. The concordance rate was only 30% among pANCA-positive sera in the 8 MPO-ANCA kits. No significant correlation was observed between pANCA titers and MPO-ANCA levels. The HM showed that 65% of cANCA-positive sera were PR3-ANCA positive, and 45% of pANCA-positive sera were MPO-ANCA positive. Our results indicate that the sensitivities and specificities for ANCA detection differ significantly among the commercial kits tested and underline the necessity of establishing uniform international standards for ANCA ELISA procedures in order to permit more reliable interpretation and comparison of data.

Cardiac involvement in Wegener’s granulomatosis resistant to induction therapy

ObjectivesThe aim of the study was to assess cardiac involvement in patients with Wegener’s granulomatosis (WG), who failed to achieve remission following >6 months induction therapy for life or organ threatening disease.MethodsEleven WG patients (eight males, mean age 47 ± 13 years), who failed to achieve remission despite >6 months induction therapy, underwent transthoracic echocardiography (TTE) and cardiac magnetic resonance (CMR).ResultsCardiac involvement was present in 9 (82%) patients. Regional wall motion abnormalities were found in two individuals, but none had left ventricular (LV) ejection fraction <50%. Nine patients had late gadolinium enhancement (LGE) lesions involving LV myocardium and right ventricle free wall was involved in four patients. LGE lesions were found in subepicardial, midwall and subendocardial LV myocardial layers. CMR revealed myocarditis in six patients. Patients with myocarditis had a higher number of LV segments with LGE (5.2 ± 3.4 vs 1.0 ± 1.2, p = 0.03) and more frequent diastolic dysfunction by TTE (5 vs 0, p = 0.02) than those without. Pericardial effusion was observed in five patients, while localized pericardial thickening in six patients.ConclusionsIn WG resistant to >6 months induction therapy cardiac involvement is frequent and is characterized by foci of LGE lesions and signs of myocardial inflammatory process.

Evaluation of smell and taste in patients with Wegener’s granulomatosis

Although a reduced olfactory/gustatory function affects patients in all parts of life, this problem has not received much attention in Wegener's granulomatosis (WG). The aim of this study was to assess the smell/taste function of WG patients. Demographic data of 16 WG patients (9 males, 7 females) were obtained. They all subjectively assessed their taste/smell function on visual analogue scale. Olfactory/gustatory functions of the patients were tested with 'Sniffin' Sticks and 'Taste' strips, respectively. The results were then compared with those from sex and age-matched control group (n = 16) and normative data. WG patients subjectively assessed their olfactory (p = 0.03) and gustatory (p = 0.02) function to be lower than control group. All the olfactory scores (odour identification, odour discrimination and threshold) in both genders were significantly below the scores in the control group. WG patients were hyposmic. For taste (total taste score, as well as scores for the qualities sweet, sour, salty and bitter), WG patients did not significantly differ from controls and were normogeusic. However, the gustatory scores showed the tendency of reduction as compared to the control group. In conclusion, WG patients truly suffer from olfactory/taste dysfunction, but this is worse with olfaction. It is, therefore, imperative that physicians should make their patients to be aware of these sensory dysfunctions and educate them on methods to cope with it for better quality of life.

Granulomatous endocarditis in a patient with Wegener's granulomatosis

Echocardiographic examination of patients with granulomatous endocarditis in patients with Wegener's granulomatosis (WG) reveals vegetation-like lesions that may be misdiagnosed as infective endocarditis resulting in inappropriate therapy. Three-dimensional transesophageal echocardiography aids differential diagnosis. Here, we report the case of a WG patient with associated mitral and aortic granulomatous endocarditis. Although the patient was treated with prednisolone and cyclophosphamide, serial echocardiography did not reveal any significant changes in disease course.

Wegener's granulomatosis in Finland in 1981–2000: risk of dialysis-dependent renal disease

Objective: To determine the risk of renal insufficiency requiring transient or chronic dialysis and its contributing factors in patients diagnosed with Wegener's granulomatosis (WG) between 1981 and 2000 in Finland.Patients and methods: A retrospective cohort study using hospital discharge registers with a review of hospital case reports. Data were complemented with files from the Finnish Registry for Kidney Diseases. All files were reviewed by 8 October 2005.Results: A total of 492 patients received a verified diagnosis of WG in 1981–2000. Of these, 84 (17%) needed dialysis by the end of follow-up. Of the 84 dialysed patients, 32 (38%) recovered initially, 32 (38%) needed chronic dialysis (dialysed > 3 months), and 19 (23%) received a kidney transplant. Forty-one (49%) of the dialysed patients were alive at the end of follow-up: 16 with a kidney transplant, 14 on dialysis, and 11 without dialysis. The cumulative rate of developing renal involvement leading to dialysis was 14.6% [95% confidence interval (CI) 11.6–18.2] at 5 years and 29.6% (95% CI 21.5–40.0) at 20 years after onset of WG symptoms. Elevated creatinine levels at presentation were associated with an increased rate of renal insufficiency requiring chronic dialysis. Age, gender, and involvement of any particular organ system had no significant effect.Conclusions: WG patients are at great risk of developing renal insufficiency, and this risk increases as the disease progresses. Elevated creatinine levels at presentation are associated with an increased risk of dialysis-dependent end-stage renal disease.

Immune deposits in cutaneous lesions of Wegener's granulomatosis: predictor of an active disease.

A retrospective analysis was conducted of eight cases of Wegener's granulomatosis (WG), who presented with cutaneous lesions. The clinical, immunopathologic and histopathologic features of the cutaneous lesions were reviewed. Antineutrophil cytoplasmic antibody (ANCA) status of the patients was established. When possible, a comparison of immunofluorescence findings of skin biopsies was made with those of renal biopsies taken at the same time. In all except one, systemic and cutaneous disease developed concurrently. On histopathology, leukocytoclastic vasculitis was noted in five patients and features of lupus erythematosus and pyoderma gangrenosum in one case each. Four patients showed immunoglobulin deposits in subepidermal blood vessel walls, while one patient showed granular immune deposits at dermo-epidermal junction only. Immunoglobulin G was the most common immunoreactant detected. C-ANCA/proteinase 3 (PR3)-ANCA was positive in six patients, P-ANCA/myeloperoxidase (MPO)-ANCA in one patient, while one patient did not show ANCA positivity on indirect immunofluorescence. All four renal biopsies showed pauci-immune glomerulonephritis, irrespective of the presence (n=3) or absence (n=1) of immune deposits in the skin biopsy. Skin manifestations are encountered in nearly half of the patients with WG, thus it is important to be familiar with cutaneous histopathologic as well as immunofluorescence findings in WG patients.

Intestinal Involvement in Wegener's Granulomatosis Diagnosed and Followed up by Double Balloon Enteroscopy

Wegener's granulomatosis (WG) is a multisystemic disease of unknown etiology characterized by necrotizing vasculitis and granulomatous inflammation (1-3). The disease typically involves the upper airways, lungs and kidneys, and gastrointestinal involvement is uncommon. Described here is a 33-year-old man who presented at the hospital with abdominal pain. Colonoscopy revealed multiple ulcers, including round ulcers, throughout the large intestine. Small bowel ulcers were detected by double balloon enteroscopy (DBE). Further study confirmed that these ulcers were caused by gastrointestinal complications of WG. The patient was administered prednisolone and cyclophosphamide and remains in remission. This case indicates the importance of considering a gastrointestinal complication of WG as the potential cause of abdominal symptoms among WG patients as well as the use of DBE in detecting such a complication.

Improved outcome in 445 patients with Wegener's granulomatosis in a German vasculitis center over four decades

To determine the long-term outcome in patients with Wegener's granulomatosis (WG) over 4 decades in an academic hospital unit specializing in rheumatology. We included 290 patients, divided them into 2 cohorts, and compared them with the historical cohort of 155 patients. Comparisons were retrospective regarding disease manifestations, therapy, mortality, and incidence of malignancies. The historical cohort (cohort 1) included 155 patients diagnosed between 1966 and 1993, cohort 2 included 123 patients diagnosed between 1994 and 1998, and cohort 3 included 167 patients diagnosed between 1999 and 2002. Over time, the interval between first symptoms and diagnosis was reduced by half (from 8 months to 4 months). Organ manifestations were similar in the 3 cohorts, and more than 80% of patients still required cyclophosphamide (CYC); however, the median cumulative dose was reduced significantly (from 67 gm in cohort 1 to 36 gm in cohort 2 and to 24 gm in cohort 3). The standardized mortality ratios (SMRs) declined (from 2.1 in cohort 1 to 1.41 in cohort 2 and to 1.03 in cohort 3), with fewer deaths related to WG and/or therapy (86.4% in cohort 1, 76.9% in cohort 2, 50% in cohort 3), decreasing relapse rates (63.9% in cohort 1, 51.2% in cohort 2, 35.3% in cohort 3), and no increased rate of malignancies. Compared with young females, young males had a considerably higher SMR (8.87 [95% confidence interval 4.05-16.8]) and more frequent renal manifestations (54.4% versus 33.8%). Mortality of WG patients declined over the last 4 decades, probably due to improved diagnostic and therapeutic procedures and increased awareness of WG, which led to earlier diagnosis and therapy, reduction in relapse rates, and lower cumulative CYC dose with fewer deaths related to therapy.

Altered serum levels of human neutrophil peptides (HNP) and human beta-defensin 2 (hBD2) in Wegener’s granulomatosis

Defensins are highly conserved peptides with antimicrobial and immunomodulatory functions. Due to their chemotactic properties on mononuclear cells, including dendritic cells and macrophages, defensins may contribute to granuloma formation in Wegener's granulomatosis (WG). In order to explore whether these peptides might be involved in WG pathogenesis, sera of patients were screened to detect altered defensin levels. For this purpose, serum and EDTA-blood of patients with WG (n = 17; aged 54.8 ± 15.5 years) and age- and sex-matched healthy controls (n = 24; aged 55.5 ± 16.8 years) were collected. Levels of neutrophil α-defensins (human neutrophil peptides, HNP) and β-defensin (hBD) 2 and 3 in serum were measured by ELISA. By this means, WG patients displayed higher serum levels of hBD2 and HNP when compared to controls. Furthermore, serum hBD2 was raised in patients with meningeal granulomas (n = 4) or in those undergoing treatment with cyclophosphamide (n = 4). In order to detect whether increased gene expression in polymorphonuclear cells is responsible for the elevated defensin levels, real-time polymerase chain reaction with gene-specific primers was performed. Expression of specific mRNA in polymorphonuclear cells was observed for HNP only, but did not parallel HNP serum levels, suggesting that degranulation rather than increased gene expression may be responsible for increased HNP serum levels in WG. In conclusion, elevated serum levels of HNP and hBD2 in WG patients suggest a role for both defensins in WG pathogenesis.

Intralesional corticosteroid injection and dilatation provides effective management of subglottic stenosis in Wegener's granulomatosis

To describe our experience with the use of intralesional corticosteroid injection and dilatation (ILCD) in the management of subglottic stenosis (SGS). Retrospective chart review. A retrospective chart review was performed of all patients with SGS requiring ILCD, from 2003 to 2008, at the Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital, Toronto, Canada. Twelve patients with SGS underwent 36 ILCD operations with a mean of three procedures per patient. We identified eight patients with Wegener's granulomatosis (WG) and four patients without WG. The eight WG patients received an average of 3.37 procedures, whereas non-WG patients required an average of 2.25 procedures. This maintained airway patency and symptom control for an average of 11.9 and 8.1 months, respectively. Only one complication was identified, and no long-term sequelae were found. No patients required new tracheotomies and one patient with a previous tracheotomy was successfully decannulated. Our data supports the use of ILCD as a safe and effective treatment of SGS in both WG and non-WG patients.