[Iatrogenic electrolyte disorders]. / Iatrogene Elektrolytstörungen.

The maintenance of water and electrolyte homeostasis is of enormous importance for the functioning of cells and tissues. A number of therapeutic procedures intentionally or unintentionally influence important regulatory mechanisms of these interdependent balanced systems. Excessive salt intake doesn't only expand the extracellular volume; it can also cause a considerable increase in tonicity. Owing to its insulin-dependent duality of action, glucose can represent an effective or an ineffective osmolyte. This fact has to be considered in patients with diabetic ketoacidosis. Diuretics reduce the volume expansion via renal excretion of sodium (and water); however, in addition to hypokalemia, diuretics can also cause severe alkalosis. Nowadays, hemodialysis is a routine procedure-but even routine procedures can deliver undesirable surprises. Can dialysis cause an increase in calcium levels, or does the procedure remove therapeutically administered radioactive iodine? The current article presents a series of cases we have come across in recent years. These case reports illustrate common, but also rare iatrogenic situations. The discussion of these cases is aimed at raising awareness of the issues involved in a pathophysiological approach to clinical problems.

How anti-neutrophil cytoplasmic autoantibodies activate neutrophils.

Neutrophils are pivotal to host defence during infectious diseases. However, activated neutrophils may also cause undesired tissue damage. Ample examples include small-vessel inflammatory diseases (vasculitis) that are associated with anti-neutrophil cytoplasmic autoantibodies (ANCA) residing in the patients' plasma. In addition to being an important diagnostic tool, convincing evidence shows that ANCA are pathogenic. ANCA-neutrophil interactions induce important cellular responses that result in highly inflammatory necrotizing vascular damage. The interaction begins with ANCA binding to their target antigens on primed neutrophils, proceeds by recruiting transmembrane molecules to initiate intracellular signal transduction and culminates in activation of effector functions that ultimately mediate the tissue damage.

The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction.

Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 +/- 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0.1 microg/ml human PR3 to 10(4) NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by >70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6.25 to 100 microM as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 microM (inhibition to 42 +/- 4% with compound 27519 and to 47 +/- 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-alpha-mediated mPR3 increase on NB1(pos) neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1.

Neutrophil surface presentation of the anti-neutrophil cytoplasmic antibody-antigen proteinase 3 depends on N-terminal processing.

The neutrophil serine protease proteinase 3 (PR3) is a main autoantigen in anti-neutrophil cytoplasmic antibody-associated vasculitis. PR3 surface presentation on neutrophilic granulocytes, the main effector cells, is pathogenically important. PR3 is presented by the NB1 (CD177) glycoprotein, but how the presentation develops during neutrophil differentiation is not known. An N-terminally unprocessed PR3 (proPR3) is produced early during neutrophil development and promotes myeloid cell differentiation. We therefore investigated if PR3 presentation depended on NB1 during neutrophil differentiation and if PR3 and proPR3 could both be presented by NB1. In contrast to mature neutrophils, differentiating neutrophils showed an early NB1-independent PR3 surface display that was recognized by only two of four monoclonal anti-PR3 antibodies and occurred in parallel with proPR3, but not PR3 secretion, suggesting that the NB1-independent surface PR3 was proPR3. PR3 gene expression preceeded NB1. When the NB1 receptor was detected on the surface, a mode of PR3 surface display similar to mature neutrophils developed together with the degranulation system. Ectopic expression studies showed that NB1 was a sufficient receptor for PR3 but not proPR3. ProPR3 display on the plasma membrane may influence the bone marrow microenvironment. NB1-mediated PR3 presentation depended on PR3 N-terminal processing implicating the PR3-N-terminus as NB1-binding site.

Apoptosis, proliferation and inflammatory infiltration in ANCA-positive glomerulonephritis.

AIMS: Antineutrophil cytoplasmic antibodies (ANCA) are detected in most patients with crescentic glomerulonephritis and necrotizing small vessel vasculitis. ANCA cause renal inflammation and proliferation. Apoptosis is necessary for resolution of inflammation. We studied apoptosis, apoptosis-regulating proteins, proliferation and infiltration with ANCA target antigen containing neutrophils and monocytes in renal biopsies from ANCA patients and disease controls. METHODS: Skin biopsies from patients with leukocytoclastic vasculitis (n=6) and renal biopsies from patients with ANCA vasculitis (n=10), ANCA-negative crescentic glomerulonephritis (CGN, n=7), mesangio-proliferative GN (n=6), post-streptococcal GN (PSGN, n=4), diabetic nephropathy (n=6) and minimal change nephropathy (MCNP, n=6) were evaluated by immunohistochemistry. Biopsies were stained for apoptosis (TdT-mediated UTP nick-end labeling, TUNEL), proliferation (Ki-67), neutrophils (NP 57), and monocytes (KP 1). We also evaluated Fas and Bcl-2 expression. RESULTS: Apoptosis was common in leukocytoclastic vasculitis skin biopsies, but was rare in renal biopsies. ANCA-positive NCGN showed the lowest apoptosis rate, similar to MCNP and diabetic nephropathy. The highest apoptosis rate was seen in PSGN. The highest glomerular Bcl-2 expression was present in ANCA-positive biopsies. The Bcl-2/TUNEL ratio was significantly increased in ANCA-positive necrotizing crescentic glomerulonephritis (NCGN) compared to ANCA-negative CGN and PSGN. When proliferation (Ki-67) and apoptosis were expressed as a ratio, we observed the highest index in biopsies from patients with ANCA-positive NCGN because of their low apoptosis rates. Finally, the glomerular inflammatory infiltrate in ANCA-positive NCGN showed a high percentage of neutrophils. CONCLUSIONS: These preliminary results suggest an imbalance between apoptosis and proliferation, favoring proliferation, in renal biopsies from ANCA-positive NCGN patients.

Role of extracellular signal-regulated kinase and phosphatidylinositol-3 kinase in chemoattractant and LPS delay of constitutive neutrophil apoptosis.

The present study examined the role of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase-stimulated Akt (PI-3K/Akt) in the regulation of constitutive human neutrophil apoptosis by bacterial lipopolysaccharide (LPS) and two chemoattractants, fMLP and leukotriene B(4) (LTB(4)). LPS and LTB(4) inhibited apoptosis, while fMLP had no effect. Inhibition of extracellular signal-regulated kinase (ERK) with PD098059 significantly inhibited the anti-apoptotic effect of both LPS and LTB(4), while inhibition of p38 kinase with SB203580 had no effect. Inhibition of PI-3K with wortmannin and LY294002 significantly attenuated the anti-apoptotic effect of LTB(4), but not LPS. LPS, fMLP, and LTB(4) stimulated similar levels of ERK and Akt activation. LTB(4) and LPS inhibited neutrophil apoptosis when added simultaneously with fMLP, and LTB(4) and LPS demonstrated an additive effect. We conclude that the ERK and/or PI-3K/Akt pathways are necessary, but not sufficient, for LPS and LTB(4) to delay apoptosis, but other anti-apoptotic pathways remain to be identified.

Membrane proteinase 3 and Wegener's granulomatosis.

Proteinase 3 (PR3) is found in neutrophil and monocyte lysosomal granules. Anti-neutrophil cytoplasmatic antibodies (ANCA) with specificity for PR3 are characteristic for patients with Wegener's granulomatosis. The interaction of ANCA with neutrophilic ANCA antigens is necessary for the development of ANCA-associated diseases. ANCA bind to membrane-expressed PR3 and induce full-blown activation in primed neutrophils. We discuss two different aspects of membrane PR3 (mPR3). The first aspect is the amount of PR3 and mechanisms controlling this issue. The second aspect is the presence of two neutrophil subsets that differ in the mPR3 expression phenotype.

TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator.

We investigated intracellular signaling events involved in fibronectin-accelerated TNF-alpha-mediated PMN apoptosis by means of 2-D gel electrophoresis and western blotting. Proteins were sequenced with electrospray ionization mass spectrometry. Apoptosis was quantitated by flow cytometry. We detected a cluster of acidic, high molecular-weight proteins that were only tyrosine phosphorylated when TNF-alpha-treated PMN interacted with fibronectin. Sequence analysis revealed that one of these proteins was Ly-GDI, a regulator of Rho GTPases. Fibronectin increased the TNF-alpha-induced Ly-GDI cleavage, yielding a 23-kD fragment. At 8 h, intact Ly-GDI was decreased to 33% on fibronectin, compared with 69% on PolyHema (P<0.05). Inhibition of tyrosine phosphorylation prevented phosphorylation of Ly-GDI, fibronectin-accelerated Ly-GDI cleavage, and fibronectin-accelerated apoptosis in TNF-alpha-treated PMN. We found that Ly-GDI cleavage was dependent on caspase-3 activation and that caspase-3 inhibition decreased apoptosis. We conclude that tyrosine phosphorylation of Ly-GDI, followed by increased caspase-3-mediated Ly-GDI cleavage, is a signaling event associated with accelerated TNF-alpha-mediated apoptosis on fibronectin.

Wegener's granulomatosis masquerading as breast cancer.

We describe a patient with Wegener's granulomatosis whose disease presented as pseudotumor of the orbit and a breast mass. Both findings were misinterpreted and errors in diagnosis resulted, despite the availability of rapid and accurate diagnostic tests for this disease. We report this case to emphasize the more unusual presentations of Wegener's granulomatosis.

Spontaneous splenic hemorrhage in a patient with Wegener's granulomatosis.

We report the occurrence of spontaneous splenic hemorrhage in a patient with Wegener's granulomatosis. Pulmonary infiltrates, hemoptysis, and crescentic glomerulonephritis were accompanied by a progressive splenic enlargement with minimal abdominal symptoms. Magnetic resonance imaging was particularly helpful. The spleen was removed by minimally invasive endoscopic surgery. Subcapsular hemorrhage had occurred because of splenic vasculitis. Postoperatively, a remission was achieved by a combination of high-dose corticosteroids and cyclophosphamide.

Necrotizing small-vessel vasculitis confined to the uterine cervix.

OBJECTIVES: To report our experience with five cases of apparently isolated small-vessel vasculitis of the uterine cervix. METHODS: Case study of five patients with necrotizing vasculitis discovered incidentally in surgical specimens of the female genital tract, and a review of the pertinent literature on this subject. RESULTS: All patients lacked clinical and serological features of the well-delineated vasculitic syndromes. Comprehensive workup failed to yield any evidence of an underlying disorder. All patients were managed expectantly and did not develop systemic vasculitis during follow-up ranging from 6 months to 5 years. CONCLUSIONS: Isolated vasculitis of the female genital tract can be encountered as an innocuous finding in otherwise healthy individuals. The cause and pathogenesis of this disorder remain obscure. Rheumatologists should be familiar with this rare and vexing form of vasculitis and with its benign prognosis.

Pericardial effusion in the nephrotic syndrome.

Hydropericardium is a known cause of pericardial effusion related to severely expanded extracellular fluid volume. Nephrotic patients have expanded extracellular fluid volume but obviously may have other causes for pericardial effusion. We tested the hypothesis that pericardial effusion is related to inflammation and not to hydropericardium in patients with nephrotic syndrome. Twenty nephrotic patients with systemic lupus erythematosus (SLE) were compared to 20 patients with nephrotic syndrome of other causes. No patient in either group had symptoms or signs of pericardial disease. Pleural effusion and ascites were equally common in SLE-nephrotic patients compared to non-SLE-nephrotic patients. However, 8 SLE patients had pericardial effusion, while none of the non-SLE-nephrotic patients had pericardial effusion. We suggest that hydropericardium is rare in nephrotic patients and that an inflammatory or other secondary cause should be considered when pericardial effusion complicates nephrotic syndrome.

Genotypic testing in clinically defined HHT: would Osler approve or turn in his grave?

Modern diagnostic possibilities pose a number of challenges. When is a precise genetic diagnosis justifiable in today's climate of cost-cutting? We would like to pose that question to Sir William Osler. Sir William was a keen observer, a master 'translator' of science into clinical medicine. Would he have required or supported genetic testing? We treated a patient whose case reminded us of Sir William's belief that clinical exactness was the ultimate aim, regardless of cost.

Prognostic factors in Wegener's granulomatosis.

We analysed data from 64 patients with Wegener's granulomatosis to determine predictor variables of outcome. The mean period of observation after the diagnosis had been established was 3.2 (range 0.1-11.2) years. At the time of diagnosis, 15 (23%) patients had only local symptoms. The disease was generalized to multiple organs in 49 (77%) patients. Renal biopsies were obtained in 33 patients; 13 (39%) had extracapillary glomerulonephritis, which was the most common renal lesion. All but three patients received immunosuppressive therapy. At time of follow-up, 17 (27%) patients were in complete, and 26 (40%) in partial remission. We employed a Kaplan Meier analysis to identify predictor variables of outcome. Renal involvement, initial creatinine concentration, serum albumin or total protein concentration, leukocyte count and erythrocyturia proved to be predictor variables. These variables may be of value in guiding the intensity of treatment in patients with Wegener's granulomatosis.

[Cellular immune response and glomerulonephritis--their detection with the Tetamun intracutaneous test]. / Zellgebundene Immunantwort und Glomerulonephritis--ihre Erfassung mit dem Tetamun-Intrakutantest.

In the considerations of the pathogenesis of glomerulonephritis more importance is attributed to cellular immune reactions. The cell-mediated immune response was examined by means of the Tetamun intracutaneous test in 37 patients with histological verified glomerulonephritis, in 18 patients with non-glomerular renal diseases, and in 25 healthy probands. The influence of main disease, renal insufficiency, nephrotic syndrome and immunosuppressive therapy on the test results could be determined by proper group formation. A significant diminution of cell-mediated immune reactions by the glomerulonephritis was verified. The importance of this finding in the pathogenetic routes and a possible model are discussed.

Crosslinking of ANCA-antigens stimulates superoxide release by human neutrophils.

Anti-neutrophil cytoplasmic antibodies (ANCA) activate primed human polymorphonuclear neutrophils (PMN) in vitro, resulting in a respiratory burst and degranulation. In this study, the hypotheses that the initiation of this process requires engagement of the F(ab')2 portion of ANCA, and that crosslinking of ANCA target antigens is necessary to trigger superoxide (O2-) release, were explored. It is speculated that Fc gamma receptor engagement is a modulator of ANCA-mediated activation. Flow cytometry demonstrated that intact human ANCA immunoglobulin (Ig), their corresponding F(ab')2 and Fab fragments, as well as a murine monoclonal to human PR3 and its F(ab')2 fragment, bind to ANCA antigens on the surface of PMN primed with tumor necrosis factor (TNF) alpha. Intact Ig of patients with PR3-ANCA or with MPO-ANCA stimulate O2- release from TNF alpha-primed normal PMN (2.6 +/- 3.57 to 15.3 +/- 7.39 nmol O2-/2.5 x 10(6) PMN/30 min). Corresponding F(ab')2 fragments result in similar O2- production (10.2 +/- 4.34 to 36.9 nmol) in a dose-dependent manner. ANCA Fab fragments do not stimulate O2- generation until these fragments are crosslinked with F(ab')2 of goat anti-human Ig F(ab')2, or when fragments are biotinylated and crosslinked with avidin. In contrast with these human autoantibody data, a mouse monoclonal anti-human PR3 antibody (25.7 +/- 8.55 nmol O2-), but not its corresponding F(ab')2 fragment, activates TNF alpha-treated human PMN. When the Fc gamma IIa receptors were blocked, superoxide production was reduced by 33% using human PR3-ANCA Ig (P < 0.05). In conclusion, PMN activation by ANCA occurs when intact ANCA or ANCA F(ab')2 fragments crosslink target antigens on the neutrophil cell surface. ANCA F(ab') fragments result in PMN activation when crosslinked by secondary reagents.

Cytomegalovirus colitis during mycophenolate mofetil therapy for Wegener's granulomatosis.

Cytomegalovirus (CMV) infection of the gastrointestinal tract is an increasingly recognized cause of morbidity and mortality during the course of HIV infection and in association with immunosuppressive pharmacotherapy. Mycophenolate mofetil, a novel immunosuppressive drug, is currently used in renal transplant recipients and is under evaluation for a variety of disorders. There is preliminary evidence to suggest that CMV reactivation may be more common during treatment with mycophenolate than with other immunosuppressive drugs. We present the case of a 59-year-old male with Wegener's granulomatosis who received mycophenolate and presented with guaiac-positive diarrhea 8 weeks after recovery from Salmonella brandenburg infection. CMV serology and assays for CMV antigens were entirely negative. Colonoscopy demonstrated pancolitis and examination of the specimens disclosed CMV infection. Ganciclovir was administered and the patient made an uneventful recovery. We discuss aspects of gastrointestinal CMV infection with an emphasis on pitfalls in diagnosis and the association with mycophenolate mofetil treatment. We also speculate as to the potential role of previous Salmonella infection and proinflammatory cytokines in CMV reactivation. In summary, when using mycophenolate, clinicians should be more aware of CMV reactivation and disease.

[Wegener's granulomatosis and anti-cytoplasm antibodies]. / Wegenersche Granulomatose und antizytoplasmatische Antikörper.

20 patients with biopsy-proven Wegener's granulomatosis (WG) and 95 control patients underwent determination of anticytoplasmic antibodies (ACPA) by the indirect immunofluorescence technique to assess the specificity and sensitivity of ACPA for WG. Of 14 untreated patients with WG, 13 were ACPA-positive. All these patients became ACPA-negative under immunosuppressive treatment. 4 patients treated with immunosuppressive drugs and 2 patients in remission after termination of therapy had a negative ACPA-test. ACPA were detected in 9 patients of the control group (two patients with Henoch Schoenlein purpura, two patients with systemic vasculitis, 2 patients with systemic diseases, and 1 patient with systemic lupus erythematodes). With that we achieved a specificity of 90.5% and a sensitivity 65.0%. In conclusion the ACPA-determination is very helpful for diagnosis and follow-up of WG.

Neutrophil superoxide release is required for spontaneous and FMLP-mediated but not for TNF alpha-mediated apoptosis.

Polymorphonuclear leukocyte (PMN) lifespan is characterized by both rapid production and apoptotic cell death. The mechanisms triggering apoptosis in PMN are not completely understood. In this study, the relationship of neutrophil activation and apoptosis as related to released superoxide was investigated. PMN apoptosis was detected by DNA fragmentation, and ultraviolet and light microscopy, and was quantified by flow cytometry; superoxide release was measured by superoxide dismutase-inhibitable reduction of ferricytochrome C. Incubation of PMN with 20 ng/ml tumor necrosis factor (TNF)alpha induced superoxide release (8.8 +/- 7.5 nmol O2-/30 min, n = 7) in normal PMN and also resulted in apoptosis within 2 h, whereas a subactivating dose of 2 ng/ml TNF alpha, which did not trigger superoxide release (3.1 +/- 1.7 nmol O2-, n = 10), did facilitate apoptosis, although to a lesser degree. PMN cultured under nonstimulating conditions underwent apoptotic cell death after 8 h. Exogenous superoxide dismutase did not inhibit apoptosis induced by 20 ng/ml TNF alpha. No upregulation of endogenous manganese superoxide dismutase mRNA expression was observed in response to TNF alpha as measured by reverse transcription PCR. Formyl-methionyl-leucyl-phenylalanine (FMLP) stimulation (10(-7) M) resulting in superoxide release of 31.7 +/- 6.1 nmol O2-/30 min (n = 10) also significantly increased the percentage of apoptosis, but at 24 h (P < 0.05). Exogenous superoxide dismutase did inhibit FMLP-induced apoptosis, as well as apoptosis due to aging in culture. In conclusion, aging and FMLP-stimulated PMN undergo apoptosis by a superoxide release-dependent pathway, whereas TNF alpha-facilitated apoptosis appears to be unrelated to respiratory burst oxidase activity.