Marginal-Zone B-Cells Are Main Producers of IgM in Humans, and Are Reduced in Patients With Autoimmune Vasculitis

In mice, B1 and marginal zone (MZ) B-cells play an important role in prevention of autoimmunity through production of regulatory cytokines and natural antibodies. There is limited knowledge about the human counterparts of these cells. We therefore investigated functions of MZ-like B-cells and the frequency of circulating MZ-like and B1-like B-cells in healthy controls (HC), as well as in patients with autoimmune vasculitis to learn more about the role of these cells in autoimmune disease. After stimulation with CpG oligodeoxynucleotides (ODN) of class B in vitro, MZ-like B-cells were the main producers of IgM whereas switched memory B-cells primarily produced IgG and IgA. TNF and IL-10 were produced by both MZ-like and switched memory B-cells. Neither antibody nor TNF/IL-10 production by the B-cell subsets differed between patients and HC. Patients with autoimmune vasculitis, irrespective of disease activity, had lower percentage and absolute numbers of circulating MZ-like B-cells, and lower absolute numbers of B1-like B-cells. The percentage of B1-like B-cells was reduced during active disease. These findings remained significant when the analysis was confined to active treatment-naïve patients (disease onset).Our results suggest that human innate-like B-cells might have a physiological role in prevention of autoimmunity

Circulating cytokine profile in anti-neutrophilic cytoplasmatic autoantibody-associated vasculitis: prediction of outcome?

AIMS: The anti-neutrophilic cytoplasmatic autoantibody-associated vasculitides (AASV) are diseases of relapsing-remitting inflammation. Here we explore the cytokine profile in different phases of disease, looking for pathogenic clues of possible prognostic value. RESULTS: Interleukin (IL)-6, IL-8 and IL-10 were significantly elevated in plasma. Patients in the stable phase who subsequently developed adverse events had higher IL-8 values. Patients in the stable phase who relapsed within 3 months had lower IL-10 values and higher IL-6 levels. CONCLUSIONS: Patients with AASV have raised circulating cytokine levels compared with healthy controls, even during remission. Raised IL-8 seems associated with poor prognosis. Lower levels of IL-10 and higher levels of IL-6 herald a greater risk of relapse. Patients with systemic vasculitis in clinical remission have persistent disease activity, kept under control by inhibitory cytokines

Journal Staff

A myelopoiesis gene signature in circulating leucocytes, exemplified by increased myeloperoxidase (MPO) and proteinase 3 (PR3) mRNA levels, has been reported in patients with active anti-neutrophil cytoplasm antibody associated vasculitis (AAV) and to a lesser extent during remission. We hypothesized that this signature could predict disease relapse. mRNA levels of PR3, MPO, selected myelopoiesis transcription factors (CEBPA, CEBPB, SPIB, SPI1) and microRNAs (miRNAs) from patient and control peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) were analyzed and associated with clinical data. Patients in stable remission had higher mRNA levels for PR3 (PBMCs, PMNs) and MPO (PBMCs). PR3 and SPIB mRNA correlated positively in control but negatively in patient PBMCs. Statistically significant correlations existed between PR3 mRNA and several miRNAs in controls, but not in patients. PR3/MPO mRNA levels were not associated with previous or future relapses but correlated to steroid treatment. Prednisolone doses were negatively linked to SPIB and miR-155-5p, miR-339-5p (PBMCs) and to miR-221, miR-361, miR-505 (PMNs). PR3 mRNA in PBMCs correlated with time since last flare, blood leucocyte count and estimated glomerular filtration rate. Our results show that elevated leucocyte PR3 mRNA levels in AAV patients in remission do not predict relapse. The origin seems multifactorial, but to an important part explainable by prednisolone action. Gene signatures in patients with AAV undergoing steroid treatment should therefore be interpreted accordingly

Irreversible Kidney Damage due to Multicentric Castleman's Disease

Castleman's Disease (CD) is a rare lymphoproliferative disorder accompanied by marked systemic inflammatory response. Morphological diagnosis of CD requires biopsy of the whole of the involved lymph node tissue. Three histologic variants have already been described in CD morphology (hyaline vascular, plasma-cell, and mixed). In this study, we report a case of a multicentric Castleman's disease of the plasma cell variant type with negative Herpes Virus 8. The clinical presentation of this patient was of systemic amyloidosis as a result of both a delayed diagnosis and medical management. Previously described cases of CD with secondary amyloidosis have been of the localized type. Regardless, long-standing clinical remission of CD by cytotoxic drugs and anti-CD20 antibody therapy was achieved, but the nephrotic syndrome remained irreversible

Monocyte Chemoattractant Protein 1 is a Prognostic Marker in ANCA-Associated Small Vessel Vasculitis

Background. The (anti neutrophil cytoplasmatic autoantibody ANCA), associated small vessel vasculitides (ASVV) are relapsing-remitting inflammatory disorders, involving various organs, such as the kidneys. (Monocyte chemoattractant protein 1 MCP-1) has been shown to be locally up regulated in glomerulonephritis and recent studies have pointed out MCP-1 as a promising marker of renal inflammation. Here we measure urinary cytokine levels in different phases of disease, exploring the possible prognostic value of MCP-1, together with (interleukin 6 IL-6), (interleukin 8 IL-8) and (immunoglobulin M IgM). Methods. MCP-1, IL-6 and IL-8 were measured using commercially available ELISA kits, whereas IgM in the urine was measured by an in-house ELISA. Results. The MCP-1 levels in urine were significantly higher in patients in stable phase of the disease, compared with healthy controls. Patients in stable phase, with subsequent adverse events; had significantly higher MCP-1 values than patients who did not. MCP-1 and IgM both tended to be higher in patients relapsing within three months, an observation, however, not reaching statistical significance. Urinary levels of IL-6 correlated with relapse tendency, and IL-8 was associated with disease outcome. Conclusions. Patients with ASVV have raised cytokine levels in the urine compared to healthy controls, even during remission. Raised MCP-1 levels are associated with poor prognosis and possibly also with relapse tendency. The association with poor prognosis was stronger for U-MCP-1 than for conventional markers of disease like CRP, BVAS, and ANCA, as well as compared to candidate markers like U-IgM and U-IL-8. We thus consider U-MCP-1 to have promising potential as a prognostic marker in ASVV

Движение газов в ротационных наклоняющихся печах

Представлены результаты исследований движения газов и процессов тепломассообмена в ротационных наклоняющихся печах (РНП) при термообработке дисперсных материалов. Исследования выполняли с помощью численного моделирования с использованием прикладных программных пакетов ANSYS CFX и Solid Works Flow Simulation. Полученные результаты были использованы при разработке РНП различной мощности и назначения и позволили повысить их технико-экономические характеристики

Expression of p300-truncated fragments results in the modulation of apoptosis in rat mesangial cells

BACKGROUND: Mesangial cell proliferation, apoptosis, and matrix deposition have pivotal roles in the pathogenesis of renal diseases such as diabetic nephropathy and glomerulonephritis. The behavior of mesangial cells depends on the integration of intracellular signals elicited by hormones and cytokines. We hypothesized that p300 is primarily involved in the integration of signal transduction pathways in rat mesangial cells (RMCs) and that interference with p300 function will alter apoptotic signals. METHODS: We established an RMC cell line expressing the Tet-activator (tTA). RMC-tTA cells were transiently transfected with vectors coding for either the N-terminal third or the C-terminal third of p300. Expression was induced by the addition of doxycycline [Dox; 1 microg/mL; 5% fetal bovine serum (FBS)]. The percentage of apoptosis was determined using the TUNEL technique. Specific protein-protein interactions were determined by Western blot analysis of immunoprecipitated complexes. Cells were treated with 5% FBS or with H2O2 (500 micromol/L, 1 h) with and without Dox. RESULTS: The expression of p300-C resulted in increased susceptibility to low serum-induced (20.0 +/- 4.6 vs. 3.0 +/- 1.7%) and to H2O2-induced apoptosis (75.3 +/- 13.3 vs. 50.8 +/- 6.5%) compared with controls. Immunoprecipitation of p300-C showed an interaction with the transcription factor c-Fos, which was enhanced by H2O2 treatment. Expression of the p300-N resulted in a rescue (34.8 +/- 6. 4 vs. 50.8 +/- 6.5%) from H2O2-induced apoptosis compared with controls. P300-N was shown to form a complex with the transcription factor nuclear factor-kappaB (NF-kappaB). CONCLUSIONS: The data indicate that endogenous p300 is involved in apoptosis in mesangial cells. We propose that interference or enhancement of endogenous p300 function, by expression of exogenous fragments, can alter interactions with c-Fos or NF-kappaB and modulate signals during cellular stress

Novel distribution of the secretory leucocyte proteinase inhibitor in kidney

THE secretory leucocyte proteinase inhibitor (SLPI) is a low molecular weight, tissue-specific inhibitor of, for example, elastase and cathepsin G, which also have antimicrobial capacity. SLPI has been localised to the respiratory, gastrointestinal and genital tracts, but so far not to the kidney. The presence of SLPI in renal tubuli cells was demonstrated using immunohistochemistry and, by means of in situ hybridisation on human renal biopsies, we were able to demonstrate SLPI production. In various inflammatory conditions in the kidneys, the protease-antiprotease balance is disturbed. For this reason, as well as the possible role in the defence against ascending urinary tract infections, it is interesting to establish a source of SLPI in renal tubuli cells. Key words: Glomerulonephritis, Kidney, Protease inhibitor, SLPI, Anti-leukoprotease Introduction Secretory leukocyte proteinase inhibitor (SLPI) is a 107 amino acid non-glycosylated single chain protein, stabilised by intrachain disulphide bonds. The estimated molecular weight of the acid-stable protein is 12 kDa. The C-terminal is responsible for the inhibitory activity against elastase, cathepsin G, chymotrypsin and trypsin, whereas the N-terminal has been reported to possess antimicrobial capacity. 1,2 SLPI is also known to inhibit HIV type 1 by blocking DNA synthesis. 3 The Kazal-type inhibitor forms complexes with the proteases and has a half-life of 10 min in the circulation. The complexes formed locally dissociate in plasma and the proteases are taken over by the plasma protease inhibitors (alpha-1-antitrypsin, alpha-2-macroglobulin and antichymotrypsin). 1 Furthermore, SLPI has been shown to inhibit elastin-bound elastase, which may be of importance for the preservation of elastic lung tissue. 7 The aim of the study presented here was to clarify whether there is a local renal production of SLPI. A possible local production would be of great interest, considering the multitude of inflammatory conditions, including different kinds of glomerulonephritis and nephritis, involving the kidneys. Anti-neutrophilic cytoplasmic antibodies (ANCA) are autoantibodies directed against constituents of polymorphonuclear neutrophils and monocytes, associated with systemic vasculitis and glomerulonephritis. One of the ANCA antigens, proteinase 3, has the capacity to attack and degrade components of the extracellular vessel wall matrix and the glomerular basement membrane. In blood, it is immediately bound, mainly by alpha-1-antitrypsin. Patients with ANCA-associated vasculitis have an over-representation of the PiZ gene of alpha-1-antitrypsin and thus a lack of the inhibitor. 8 SLPI has no direct inhibitory capacity against proteinase 3; it can, on the contrary, be degraded proteolytically by it. 1 Although SLPI has no direct inhibitory capacity against proteinase 3, the progressive binding of elastase to SLPI instead of alpha-1-antitrypsin has been shown to be paralleled by an increasing binding of Short Communication Mediators of Inflammation, 10, 347-350 (2001) proteinase 3 to alpha-1-antitrypsin. 10,11 A possible role in the defence against ascending urinary tract infections also draws attention to the point of issue. Subjects and methods Patient material Macroscopically and microscopically normal kidney preparations were selected from three different specimens obtained at the time of nephrectomy due to renal cancer. The Helsinki declaration regarding the use of human tissues was strictly observed. Materials and chemicals Normal rabbit serum was purchased from Dakopat AB (Copenhagen, Denmark). Swedish landrace goats were immunised with recombinant human SLPI, to produce anti-SLPI antiserum. Biotinylated rabbit anti-goat IgG and avidin-biotinylated horseradish peroxidase complexes were from Vector Laboratories (Burlingame, CA, USA). The AEC substrate-chromogen system and Faramount aqueous mounting medium were from Dako Corporation (Carpinteria, CA, USA). An oligonucleotide probe cocktail for SLPI was from R&D systems (Abingdon, UK). Blocking reagent and anti-digoxigenin (DIG)-AP conjugate (Fab fragments) were from Boehringer Mannheim (Mannheim, Germany). Visualising substrates (bromochloroindolylphosphate (BCIP) and nitroblue tetrazolium (NBT)) were from Bio-Rad Laboratories (Hercules, CA, USA). Immunohistochemistry The frozen renal tissue specimens were fixed in 4% buffered formaldehyde, dehydrated and embedded in paraffin wax for sectioning on sialinised slides. After rehydration, the sections were exposed to pepsin (4 mg/ml in 0.01 M HCl) for 20 min at 37°C. The sections were then washed between every step in Tris-buffered saline (TBS), 3 ´5 min. Endogenous peroxidase activity was removed by incubation of the specimens in 0.3% H 2 O 2 in methanol for 30 min at room temperature. Normal rabbit serum (3% in TBS) was applied to block non-specific staining. The next step, after draining, was to incubate the specimens with primary antibody (goat anti-SLPI, 1/1000, 30 min). For detection, we used biotinylated rabbit anti-goat IgG (5 mg/ml), followed by application of avidin-biotinylated horseradish peroxidase complexes for 30 min. Visualisation was accomplished by addition of AEC (0.75 mg/ml of 3-amino-9-ethylcarbazole in 2.5% N,N-dimethylformamide and 50 mM acetate buffer) that was left to incubate for 15 min. The slides were washed and counterstained before mounting. All the incubations were carried out at room temperature. Adsorbed anti-SLPI antiserum, obtained by affinity chromatography on a SLPI-conjugated Sepharose 4B-column, was used as a negative control and applied instead of the primary antibody. In situ hybridisation A cocktail of three 30-base long oligonucleotide probes (59-TCTTAGGAGGACAGACTC CAGCTTT-GAAGG-39, 59-ATTTCCCACACATGCCCATG CAA-CACTTCA-39, 59-AAC ATCTCTTCTTCCCTGGA-CACTGCCAGT-39), based on the antisense sequence of SLPI and labelled with DIG at the 59 end, was used for the in situ hybridisation. Diethylpyrocarbonate was added to the millipore water and all solutions to block RNAse activity. All solutions were autoclaved or sterilised by filtration. Renal tissue was taken immediately after surgery, put into liquid nitrogen and stored at -70°C. After sectioning on sialinised slides, the specimens were left to dry in air. After fixation (4% paraformaldehyde in 1 ´phosphate-buffered saline (PBS), 2 h, 4°C), the slides were incubated in a sucrose solution (30% sucrose in 1 ´PBS, overnight, 4°C). Washing in 1 Ṕ BS for 2 ´5 min was followed by treatment with 0.1 M glycine, 2 ´5 min. After further washing in 1 Ṕ BS for 2 ´5 min, the sections were placed in 0.3% Triton X-100 in 1 ´PBS for 15 min. Washing in 1 ´PBS, 2 ´5 min, was followed by incubation with proteinase K (1 mg/ml) in TE buffer for 30 min at 37°C. After post-fixation (4% paraformaldehyde in 1 ´PBS, 5 min, 4°C) and PBS washing, 2 ´5 min, the sections were acetylated in a 0.1 M triethanolamine (TEA) buffer (pH 8) with 0.25% acetic anhydride. TEA blocks endogenous activity of alkaline phosphatase, and acetanhydride reduces probe stickiness. Incubation with 40 ml pre-hybridisation buffer at 37°C for 2 h preceded the pre-hybridisation, which was performed in a moist chamber. To prevent evaporation, the specimens were covered with parafilm. The probe cocktail (10 ng in 40 ml of hybridisation buffer) was added and left to hybridise overnight at 37°C, which is 23°C below T m . This temperature was chosen because of short oligonucleotides. The slides were then washed in 2 ´SSC (2 5 min), 1 ´SSC (2 ´15 min) and 0.25 SSC (2 1 5 min) in a shaking water bath at 37°C. The slides were then immersed in buffer I (100 mM Tris-HCl, 150 mM NaCl; pH 7.5) for 2 ´10 min, followed by treatment with buffer II (buffer I + 0.5% blocking reagent), to block non-specific anti-DIG binding. The sections were then placed in a moist chamber and the anti-DIG-AP conjugate (1/500 in buffer I, 0.1% Triton X-100 and 1% normal sheep serum) was applied. Repetition of buffer I washing was carried out for 2 1 0 min. The slides were then placed in buffer III (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl 2 ; pH 9.5) for 10 min. Visualisation was achieved by incubation in 10 ml buffer III, 45 ml of NBT, 35 ml of BCIP and 1 mM Levamisole (240 mg/ml) for 2-4 h. The sections were then mounted using an aqueous mounting solution. Control sections were incubated with the matched sense-probes or with hybridisation buffer without probes. Results Immunohistochemical staining for SLPI was carried out on healthy kidney biopsies with positive staining results Discussion SLPI is considered to be a major protease inhibitor in the respiratory as well as in the gastrointestinal and genital tracts. A potential role in the urinary tract could therefore be anticipated. In this study, both immunohistochemistry and in situ hybridisation (ISH) showed clearly positive results, with no background staining and adequate controls. The positive staining of SLPI in distal tubuli in immunohistochemistry could be explained by renal filtration, but ISH also show a positive signal for SLPI mRNA in tubuli. These results imply local SLPI production in normal kidney, which is something that has not been demonstrated before. Our primary antibody has been used in many previous studies with clear-cut results, and must be considered highly specific. 12 For our ISH, we used an oligonucleotide cocktail synthesised for us by R&D Systems. These probes have worked well in previous studies, and the negative results obtained with the matched sense-strand probes confirm the specificity of the reaction for SLPI

COVID-19 and ANCA-associated vasculitis: recommendations for vaccine preparedness and the use of rituximab.

COVID-19 and ANCA-associated vasculitis - recommendations for vaccine preparedness and the use of rituximab

Randomised controlled trial of prolonged treatment in the remission phase of ANCA-associated vasculitis.

OBJECTIVES: A prospective randomised trial to compare two different durations of maintenance immunosuppressive therapy for the prevention of relapse in anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). METHODS: Patients with AAV were recruited 18-24 months after diagnosis if they were in stable remission after cyclophosphamide/prednisolone-based induction followed by azathioprine/prednisolone maintenance therapy. They were randomised (1:1) to receive continued azathioprine/prednisolone to 48 months from diagnosis (continuation group) or to withdraw azathioprine/prednisolone by 24 months (withdrawal group). The primary endpoint was the relapse risk, from randomisation to 48 months from diagnosis. RESULTS: One hundred and seventeen patients were randomised and 110 remained to the trial end. At entry, median serum creatinine was 116 μmol/L (range 58-372), 53% were ANCA positive. The percentage of patients presenting with relapse was higher in the withdrawal than in the continuation treatment group (63% vs 22%, p<0.0001, OR 5.96, 95% CI 2.58 to 13.77). ANCA positivity at randomisation was associated with relapse risk (51% vs 29%, p=0.017, OR 2.57, 95% CI 1.16 to 5.68). Renal function, ANCA specificity, vasculitis type and age were not predictive of relapse. Severe adverse events were more frequent in the continuation than withdrawal groups (nine vs three events), but the continuation group had better renal outcome (0 vs 4 cases of end-stage renal disease), with no difference in patient survival. CONCLUSIONS: Prolonged remission maintenance therapy with azathioprine/prednisolone, beyond 24 months after diagnosis reduces relapse risk out to 48 months and improves renal survival in AAV. TRIAL REGISTRATION NUMBER: ISRCTN13739474