[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.

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.

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.

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.

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.

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.

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice.

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.

Sudden death and Wegener's granulomatosis of the pituitary.

Involvement of brain parenchyma or meninges in ANCA-associated small-vessel vasculitis such as Wegener's granulomatosis (WG) is not uncommon. In contrast, involvement of the pituitary is exceedingly rare with only a few cases reported so far. The diagnosis is usually made on the basis of imaging techniques and abnormal pituitary function tests in the setting of active systemic vasculitis. However, histology-proven involvement of the pituitary by WG has not been reported so far. We report a case of WG with histology-proven granulomatous necrotizing inflammation of the pituitary and hypothalamo-pituitary stalk, disclosed at autopsy after the patient had died suddenly and unexpectedly in his sleep. In a setting of histology-proven WG, these findings were regarded as a pituitary manifestation of the disorder. A distinct cause of death could not be found, hence we speculate that hypothalamo-pituitary inflammation due to WG may have caused the sudden death in this patient.

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.

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.

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.

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways.

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.

Extracellular matrix regulates apoptosis in human neutrophils.

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.