Full scale implementation of the nutrient limited BAS process at Södra Cell Värö.

A combination of the suspended carrier biofilm process and the activated sludge process (biofilm-activated sludge--BAS) has been shown to be very successful for the treatment of different types of pulp and paper mill effluents. The robust biofilm pre-treatment in combination with activated sludge results in a stable, compact and highly efficient process. Recent findings have shown that nutrient limited operation of the biofilm process greatly improves the sludge characteristics in the following activated sludge stage, while minimising sludge production and effluent discharge of nutrients. The nutrient limited BAS process was implemented at full scale at the Södra Cell Värö kraft mill and taken into operation in July 2002. After start-up and optimisation over about 5 months, the process meets all effluent discharge limits. The removal of COD is close to 70% and the removal of EDTA greater than 90%. Typical effluent concentrations of suspended solids and nutrients during stable operations have been 20-30 mg/L TSS, 0.3-0.5 mg/L phosphorus and 3-5 mg/L nitrogen. The sludge production was 0.09 kgSS/kg COD removed and the sludge volume index was 50-100 mL/g.

Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice.

Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate of Burkholderia cepacia (genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs of Cftr (m1unc-/-) (Cftr(-/-)) and congenic Cftr(+/+) controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptible Cftr(-/-) mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from the Cftr(-/-) mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages from Cftr(-/-) mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.

Indirect role for COPI in the completion of FCgamma receptor-mediated phagocytosis.

Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of epsilonCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcgammaRIIA). In the presence of functional COPI, FcgammaRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcgammaRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus.

Cleavage of annexin I in human neutrophils is mediated by a membrane-localized metalloprotease.

A truncated form of annexin I, formed during Ca2+-induced translocation to neutrophil specific granules and secretory vesicles/plasma membranes, is generated through the action of an endogenous membrane protease. The cleavage of annexin I is inhibited by the metalloprotease inhibitor 1,10-phenanthroline as well as by Triton X-100 and dithiothreitol, classifying the protease as a membrane-bound, thiol-dependent metalloprotease. The cleavage site is located close to the N-terminal of annexin I, leaving a truncated form of the molecule, des1-8 annexin I, that contains the Ca2+-binding sites, as well as a number of phosphorylation sites of importance for the function of the protein. When assessing binding capacity to different neutrophil organelles, full-length annexin I bound to azurophil granules, specific granules, and secretory vesicles/plasma membranes, while des1-8 annexin I only bound to specific granules and secretory vesicles/plasma membranes, but not to azurophil granules (C. Sjölin, C. Dahlgren, Biochim. Biophys. Acta 1281 (1996) 227-234). This implies that there are different mechanisms of binding to neutrophil organelles of full-length annexin I and the truncated form, and that cleavage of annexin I might be of regulatory importance for the degranulation process.

v-SNARE-dependent secretion is required for phagocytosis.

Phagosomes are generally believed to form by gradual apposition of the plasma membrane of leukocytes onto the surface of invading microorganisms. The internalization of the encapsulated particle is therefore predicted to reduce the surface area of the phagocyte. Contrary to this prediction, we observed that phagocytosis is associated with a net increase in cell surface area, suggesting the concomitant occurrence of exocytosis. Selective cleavage of components of the secretory machinery by microinjection or transfection of bacterial neurotoxins induced a pronounced inhibition of phagocytosis. These observations indicate that vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis of endomembranes is essential for optimal completion of particle internalization during phagocytosis.

A rise in ionized calcium activates the neutrophil NADPH-oxidase but is not sufficient to directly translocate cytosolic p47phox or p67phox to b cytochrome containing membranes.

Neutrophil production of reactive oxygen species is dependent on an assembly process that involves a translocation of the cytosolic NADPH-oxidase components (p47phox; p67phox; Rac2) to a b cytochrome containing membrane. Based on the fact that an intracellular Ca2+ rise can activate the oxidase without any extracellular release of reactive oxygen species, we suggest that the oxidase can be assembled in a membrane distinct from the plasma membrane. Disintegrated cells were used to monitor Ca2+ dependent membrane binding of neutrophil cytosolic proteins. Membranes containing the b cytochrome part of the oxidase, i.e., specific granules and plasma membranes/secretory vesicles, were used in the translocation experiments. Several cytosolic proteins were found to translocate to specific granules as well as the plasma membranes/secretory vesicles, one of them being annexin I. Using antibodies in the blotting assay against the cytosolic oxidase components p47phox and p67phox, we could show that no Ca2+ dependent translocation of these cytosolic proteins occur to neither of the b cytochrome containing membranes.

Translocation of annexin XI to neutrophil subcellular organelles.

In an earlier study, annexin XI was found to be present in the cytosol of neutrophil granulocytes (Blood (1996) 87, 4817). The protein was isolated by calcium-dependent translocation to specific granules and was found to be a 42-kDa truncated form of annexin XI. Using human autoantibodies directed against annexin XI we have now reinvestigated the ability of full size annexin XI to translocate to different neutrophil organelles isolated by subcellular fractionation. The autoantisera used recognised a protein of 55-kDa in neutrophil cytosol and comparison with a whole cell lysate indicated that the larger portion of the cellular content of this protein is localised to the cytosol. Azurophil granules, specific granules and secretory vesicles/plasma membrane were isolated by subcellular fractionation on Percoll gradients, mixed respectively with neutrophil cytosol and the calcium concentration was raised. Immunoblotting showed that annexin XI translocated to specific granules and secretory vesicles/plasma membrane at 100 micromol/l calcium. When raising the concentration of calcium to 1 mmol/l, annexin XI translocated to the azurophil granules as well. Periphagosomal translocation of annexin XI occurred during phagocytosis of yeast particles, implying that this protein plays a role in the events associated with the phagocytic process.

Isolation by calcium-dependent translation to neutrophil-specific granules of a 42-kD cytosolic protein, identified as being a fragment of annexin XI.

Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti-cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.

Diverse effects of different neutrophil organelles on truncation and membrane-binding characteristics of annexin I.

A neutrophil annexin I-related protein, detected after translocation of cytosolic proteins to specific granules and secretory vesicles/plasma membrane (Sjölin et al. (1994) Biochem. J. 300, 325-330), has been characterized with respect to origin and organelle-binding properties. The annexin I-related protein is formed as a result of annexin I cleavage, and this occurs during translocation of annexin I to the specific granules and secretory vesicles/plasma membrane, but not when annexin I is translocated to azurophil granules. The cleavage required calcium and it was facilitated in the presence of specific granules or secretory vesicles/plasma membrane, but not in the presence of azurophil granules. We conclude that the membranes of specific granules and secretory vesicles/plasma membrane contain a protease which is able to cleave annexin I into a truncated 38 kDa fragment, which retains the ability to bind to these organelles. The azurophil granules lack the capacity to cleave annexin I as well as the ability to bind the 38 kDa fragment. These findings may implicate a role for annexin I in the divergent regulation of exocytosis of the different neutrophil granules.

The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils.

The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.

Different subcellular localization of cytochrome b and the dormant NADPH-oxidase in neutrophils and macrophages: effect on the production of reactive oxygen species during phagocytosis.

When neutrophils and macrophages phagocytose a prey, e.g., complement (C3b)-opsonized yeast particles, the oxygen radical generating NADPH-oxidase is activated. In neutrophils, most of the production of oxygen metabolites occurred in an intracellular compartment, possibly in the phagolysosome. In contrast, no intracellular production could be detected in human macrophages. In these cells, the subcellular localization of the superoxide-generating NADPH-oxidase and associated cytochrome b was assessed in intact cells with indirect immunofluorescence and confocal laser scanning microscopy, and with subcellular fractionation, using centrifugation on Percoll density gradients. A dual localization of the cytochrome b as well as the dormant NADPH-oxidase activity in neutrophils was in agreement with earlier immunocytochemical, biochemical, and subcellular fractionation studies. Furthermore, most of the activity was recovered from the specific granules, whereas only a small fraction was retained in the plasma membrane. In contrast, the cytochrome b/NADPH-oxidase activity in macrophages localized primarily in the plasma membrane fraction. We suggest that the macrophages are incapable of producing reactive oxygen species intraphagosomally, due to an absence of a granule-localized pool of the membrane components of the NADPH-oxidase.

Calcium-induced translocation of annexins to subcellular organelles of human neutrophils.

The annexins are Ca(2+)-regulated, phospholipid-binding proteins which have been suggested to take part in cellular events such as exocytosis. The subcellular localization of annexins in human neutrophils was determined using monoclonal antibodies against annexins I, II, IV and VI and a polyclonal peptide antiserum against an annexin consensus sequence. Several annexins were translocated to the light membrane fraction enriched in plasma membranes and secretory vesicles. Annexins were associated also with the azurophil and specific granules. Whereas annexins I, IV and VI and one unidentified 35 kDa protein translocated to each of the isolated organelles, annexin II, a 66 kDa annexin IV-like protein, and a 38 kDa annexin I-like protein exhibited organelle-related differences in their association with membranes. The 38 kDa annexin associated only with specific granules and the secretory vesicles/plasma membrane but not with azurophil granules. Annexin II and the 66 kDa annexin IV-like protein associated with each of the neutrophil organelles, but the binding to specific granules and secretory vesicles/plasma membrane showed a Ca(2+)-dependency different from that of azurophil granules. This observation suggests that these proteins may contribute to the secretory process in neutrophils.

Quantitative slot-blot chemiluminescence assay for determination of myeloperoxidase from human granulocytes.

Using slot blot, we show that myeloperoxidase (MPO), a constituent of azurophil granules of neutrophil polymorphonuclear leukocytes, can be measured quantitatively using a commercially available chemiluminescence kit, originally developed for detection of specific proteins on Western blots. MPO is determined through its ability to catalyze the oxidation of luminol, resulting in the emission of light which is recorded on a photographic film. The sensitivity of the method is high and allows MPO from less than 100 cells to be detected. This method was used to determine MPO in exudate fluid and in neutrophil fractions following disintegration and subcellular fractionation of the postnuclear supernatant on two-layer Percoll gradients.

Anti-lactoferrin antibodies and other types of ANCA in ulcerative colitis, primary sclerosing cholangitis, and Crohn's disease.

Fifty two serum samples from patients with Crohn's disease, 24 from patients with ulcerative colitis, and 12 from patients with primary sclerosing cholangitis were analysed for the presence of anti-neutrophil cytoplasm antibodies (ANCA) of IgG and IgA class by means of enzyme linked immunosorbent assays using lactoferrin, myeloperoxidase, and antigen extracted from azurophil granules, 'alpha antigen' (that is, an antigen preparation containing proteinase 3) as substrates. A high frequency of positive tests for IgG anti-lactoferrin antibodies was found in sera from patients with ulcerative colitis (50%) and primary sclerosing cholangitis (50%). In Crohn's disease only 4 of 52 (8%) sera had anti-lactoferrin antibodies--in all four instances the sera belonged to patients with disease involving the colon. All patients with sclerosing cholangitis and positive tests for anti-lactoferrin had ulcerative colitis. Low levels of IgG antibodies against myeloperoxidase or alpha antigen were also found occasionally in sera from patients with ulcerative colitis and primary sclerosing cholangitis. IgA antibodies directed against lactoferrin and alpha antigen (but not myeloperoxidase) were seen in all three conditions.

Phagocytosis following translocation of the neutrophil b-cytochrome from the specific granule to the plasma membrane is associated with an increased leakage of reactive oxygen species.

The effect of neutrophil b-cytochrome translocation on the respiratory burst activation generated during phagocytosis of yeast particles was investigated. Secretion of neutrophil specific granules was induced by the calcium ionophore ionomycin prior to phagocytosis. The secretory process is associated with a translocation from the specific granules to the plasma membrane of the respiratory burst b-cytochrome. Respiratory burst activity was measured as release of hydrogen peroxide in the absence of azide (extracellular leakage) and in the presence of azide (total production). The subcellular localization of the b-cytochrome was found to affect the extracellular release of hydrogen peroxide in that a plasma membrane localization was associated with a significantly increased release during phagocytosis. It should be pointed out, however, that most of the hydrogen peroxide, both in control and in ionomycin-treated cells, is produced intracellularly, probably in the phagosomes.

Cigarette smoke-induced changes in guinea-pig airway responsiveness to histamine and citric acid.

The effect of 50 min cigarette smoke exposure on airway responsiveness to the bronchoconstrictor and tussive effects of histamine and citric acid has been examined in guinea-pigs. Intravenous histamine increased intratracheal pressure (ITP) in anaesthetized guinea-pigs and the dose-response curve was significantly (P less than 0.05) steeper in cigarette smoke- than in air-exposed animals. ED50 values were 11.4 nmol kg-1 (7.4-16.8, 95% confidence interval) and 42.5 nmol kg-1 (28.8-61.4, 95% confidence interval), respectively (P less than 0.05) in smoke- and air-exposed guinea-pigs indicating an enhanced reactivity. However, the sensitivity to intravenous histamine was not changed by the cigarette smoke exposure, and the maximum increase in intratracheal pressure was the same as in control animals (air: 247 +/- 21%, n = 4; smoke: 223 +/- 18%, n = 7). The cigarette smoke-induced hyperresponsiveness to intravenous histamine was not altered by pretreatment with nebulized lidocaine (0.20 M), ipratropium bromide (0.30 mM) or cromoglycate (0.06 M), suggesting that a neural reflex is unlikely to be involved in the development of hyperresponsiveness. Conscious, smoke-exposed guinea-pigs had a significantly (P less than 0.001) reduced responsiveness to citric acid (0.40 M) and cigarette smoke. Both cough and bronchoconstriction were suppressed for about 1 h, but unchanged 24 h after exposure. The hyporesponsiveness to citric acid was inhibited by atropine (1.4 mumol kg-1 i.p.) and may therefore, at least in part, be due to increased airway secretions. The present data demonstrate that inhalation of cigarette smoke may alter guinea-pig airway responsiveness to tussive and bronchoconstrictor stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA).

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of antineutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic alpha fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.

Rehabilitation after hip fracture in the elderly.

Sixty-eight patients with hip fracture, mean age 79 years, from the city of Lund, were studied with special reference to functional and social rehabilitation at follow-up 1 year later. As compared with patients coming from institutions for permanent care, patients coming from their own homes had a significantly better prognosis in terms of survival, mobility and ability to cope with activities of daily living (ADL). However, patients returning home needed increased domestic help. Patients living with someone returned home sooner than those living alone. Although many of the patients who returned home could walk without support or with a walking-stick, more than one half did not go out shopping. More active measures, e.g. early home visits by a rehabilitation team, might give the patients more self-confidence and independence. Immediate weight-bearing did not appear to impair healing of the hip fracture or increase the risk of necrosis of the femoral head.