B-cell activation in patients with irritable bowel syndrome (IBS).

Patients with irritable bowel syndrome (IBS) may have a low grade immune activation. However, little is known about the properties of B cells of IBS patients. We therefore investigated activation level and antigen presenting phenotype of blood B cells of IBS patients. We also examined B-cell responses to lipopolysaccharide (LPS) and probiotic bacteria. Blood samples were obtained from 74 IBS patients and 30 healthy subjects. Peripheral blood mononuclear cells were isolated and stimulated with LPS or an UV-light inactivated bacterial cocktail consisting of the probiotic Gram-positive strains; Lactobacillus paracasei ssp. paracasei 19, Lactobacillus acidophilus La5, Bifidobacterium lactis B612. The phenotype of CD19(+) B cells was investigated by flow cytometry before and after 72 h cell culture. Furthermore, IBS symptom severity was assessed. B cells isolated from blood of IBS patients displayed an amplified activation level as demonstrated by increased cell surface expression of IgG, and also the costimulatory molecules CD80 and CD86. Expression of antigen presenting HLA-DR and costimulatory molecule CD40 on B cells was, however comparable in IBS patients and controls. B cells of IBS patients displayed an impaired ability to increase expression of CD80, but not CD86, in response to both LPS as well as probiotic bacteria stimulations. To conclude, blood B cells of IBS patients have an increased activation level. Bacterial component induced expression of the costimulatory molecule CD80, regarded as important for tolerance induction, is impaired. These data suggest that B-cell antigen presentation in IBS patients is associated with altered capacity of providing costimulation to T cells.

Autologous del(20q)-positive erythroid progenitor cells, re-emerging after DLI treatment of an MDS patient relapsing after allo-SCT, can provide a normal peripheral red blood cell count.

A 54-year-old RhD-negative male with del(20q)-positive myelodysplastic syndrome was transplanted with bone marrow from an HLA-identical RhD-positive sibling donor. Cytogenetic relapse was detected 21 months after stem cell transplantation (SCT), with reappearance of the original del(20q)-positive clone and reversion to recipient RhD-negative blood group. The patient received sequential donor lymphocyte infusions (DLIs), resulting in mild graft-versus-host disease and pure red cell aplasia. At 2 years post DLI, the patient remains in a stable condition, despite a dominance of recipient-derived erythro- and granulopoiesis originating in del(20q)-carrying progenitor cells. We conclude that reappearance of autologous erythropoiesis, upon relapse after allogeneic SCT, may be predictive of erythropenia after DLI and that re-emerging autologous del(20q)-positive erythropoiesis post DLI can provide a normal peripheral red blood cell count. Furthermore, in patients relapsing after blood-group-mismatched transplantation, a possible reversion to recipient blood group should be considered prior to blood transfusion or DLI.

Characterisation of the biosynthesis and processing of the neutrophil granule membrane protein CD63 in myeloid cells.

The biosynthesis and processing of the neutrophil granule membrane protein CD63, present in azurophil granules, was investigated in four myeloid cell lines. The amount of CD63 synthesised differed, so did the amount of protein processed to high molecular weight forms, with the demonstration of a more prominent synthesis of CD63 in K562 cells. Newly synthesised CD63 was initially detected as two precursor forms with molecular weight of 32 and 35 kDa, respectively. These two initial forms were processed further to yield high molecular weight forms of CD63 with a mean molecular weight of 50 kDa. Treatment with endoglycosidase H or N-glycosidase F revealed a protein core, free from asparagine-linked carbohydrates, with a molecular weight of 23 kDa. Newly synthesised CD63 was susceptible to digestion with endoglycosidase H, and the protein was not completely resistant to endoglycosidase H until after 4 h of chase, indicating that transport through the medial and trans-Golgi complex with conversion of high-mannose carbohydrates to complex oligosaccharide side chains had occurred. This finding indicates a relatively long processing time for CD63 compared to that of soluble azurophil granule proteins. By digestion with O-glycanase, the existence of O-linked oligosaccharides on CD63 could not be demonstrated. Biosynthetic labelling of cells in the presence of brefeldin A showed the importance of a functional Golgi apparatus for processing of the protein to its high molecular weight forms.

Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/azurocidin.

Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproazurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human azurocidin.

Processing and targeting of granule proteins in human neutrophils.

Neutrophils contain an assembly of granules destined for regulated secretion, each granule type with distinct constituents formed before terminal differentiation. The earliest granules are designated azurophil (primary), followed in time by specific (secondary), and gelatinase granules as well as secretory vesicles. Transcription factors regulate the genes for the granule proteins to ensure that expression of the gene products to be stored in different organelles is separated in time. Similar to lysosomal enzymes, many granule proteins, in particular those of the heterogeneous azurophil granules, are trimmed by proteolytic processing into mature proteins. Rodent myeloid cell lines have been utilized for research on the processing and targeting of human granule proteins after transfection of cDNA. Results from extensive work on the hematopoietic serine proteases of azurophil granules, employing in vitro mutagenesis, indicate that both an immature and a mature conformation are compatible with targeting for storage in granules. On the other hand, the amino-terminal propeptide of myeloperoxidase facilitates both the export from the endoplasmic reticulum and targeting for storage in granules. Similarly, targeting of defensins rely on an intact propeptide. The proteolytic processing into mature granule protein is most commonly a post-sorting event. Mis-sorting of specific granule proteins into azurophil or lysosome-like granules can result in premature activation and degradation, but represents a potential for manipulating the composition and function of neutrophil granules.

On the role of the proform-conformation for processing and intracellular sorting of human cathepsin G.

The serine protease cathepsin G is synthesized during the promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an amino-terminal propeptide from the catalytically inactive proform, the active protease acquires a mature conformation and is stored in azurophil granules. To investigate the importance of the proform-conformation for targeting to granules, a cDNA encoding a double-mutant form of human preprocathepsin G lacking functional catalytic site and amino-terminal prodipeptide (CatG/Gly201/triangle upGly19Glu20) was constructed, because we were not able to stably express a mutant lacking only the propeptide. Transfection of the cDNA to the rat basophilic leukemia RBL-1 and the murine myeloblast-like 32D cl3 cell lines resulted in stable, protein-expressing clones. In contrast to wild-type proenzyme, CatG/Gly201/triangle upGly19Glu20 adopted a mature conformation cotranslationally, as judged by the early acquisition of affinity to the serine protease inhibitor aprotinin, appearing before the carboxyl-terminal processing and also in the presence of the Golgi-disrupting agent brefeldin A. The presence of a mature amino-terminus was confirmed by amino-terminal radiosequencing. As with wild-type proenzyme, CatG/Gly201/triangle upGly19Glu20 was proteolytically processed carboxyl-terminally and glycosylated with asparagine-linked carbohydrates that were converted into complex forms. Furthermore, it was targeted to granules, as determined by subcellular fractionation. Our results show that the initial proform-conformation is not critical for intracellular sorting of human cathepsin G. Moreover, we demonstrate that double-mutant cathepsin G can achieve a mature conformation before carboxyl-terminal processing of the proform.

Biosynthesis, processing and sorting of neutrophil proteins: insight into neutrophil granule development.

Neutrophil granulocytes are specialized phagocytic cells that carry a collection of granules for regulated secretion, each with distinct constituents. The granules can be classified as azurophil (primary), developed first, followed in time by specific (secondary) granules gelatinase granules, and secretory vesicles. Stage- and tissue-specific transcription factors govern the successive expression of genes for granule proteins to allow storage of the gene products in these organelle categories whose packaging is separated in time. Many of the granule proteins, in particular those of the heterogeneous lysosome-like azurophil granules, are subject to extensive post-translational proteolytic processing into mature proteins, most commonly as a post-sorting event. A selective aggregation of proteins destined for storage in granules, as discussed in this review, would facilitate their retention and eliminate a need for distinct sorting motifs on each granule protein. Aggregation of granule proteins, that are often cationic, would be assisted by the anionic serglycin proteoglycans present in neutrophils. The antibacterial granule proteins can serve as models for antibiotics and some of them possess a potentially useful therapeutic ability to bind and neutralize endotoxin. Because aberrant expression of transcription factors regulating the synthesis of granule proteins is often found in leukemia, the clarification of mechanisms regulating the timed expression of granule proteins will shed light on the maturation block in myeloid leukemias.

Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines.

Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegener's granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre-proproteinase 3 cloned from U-937 cells was transfected to the rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D c13. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. After pulse labeling for 30 min two proforms of PR3 (32 and 35 kDa), differing in carbohydrate content but with protein cores of identical size, were demonstrated. Chase of the label resulted in a processed 32-kDa form clearly visible in RBL, but only faintly in 32D cells, probably indicating delayed intracellular transfer in the latter cell line. Partial digestion with N-glycosidase F showed that both potential N-glycosylation sites on PR3 were occupied and conversion of the oligosaccharide side chains into complex forms was demonstrated by acquisition of resistance to endoglycosidase H. Translocation of PR3 to granules was shown by subcellular fractionation and immunocytochemistry. Enzymatic activation of PR3 was suggested by affinity to diisopropylfluorophosphate and removal of an amino-terminal propeptide. Cells transfected with PR3 showed positive immunofluorescence for ANCA-containing sera from patients with Wegener's granulomatosis. Our results show that human PR3 transfected to RBL or 32D cells is synthesized as a 29-kDa protein core glycosylated on two distinct sites. Oligosaccharide trimming and proteolytic processing occur and the protein is targeted for granular storage in a form antigenic for ANCA.

Human cathepsin G lacking functional glycosylation site is proteolytically processed and targeted for storage in granules after transfection to the rat basophilic/mast cell line RBL or the murine myeloid cell line 32D.

The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines. The proform was proteolytically processed into a stable form with an apparent molecular mass of 27.5 kDa, indicating removal of the carboxyl-terminal prodomain. The mutant cathepsin G was enzymatically activated as determined by acquisition of affinity to aprotinin, a serine protease inhibitor. As for wild type cathepsin G, small amounts of the unprocessed form of the mutated enzyme were released from the cells, while the major part was transferred to a granular compartment as demonstrated by subcellular fractionation. Thus, neither processing leading to enzymatic activation nor granular sorting was obviously affected by the lack of oligosaccharides on the mutant cathepsin G. Our results therefore indicate that glycosylation is not essential for these processes. In addition to the previously utilized cell line RBL, we propose the 32D cell line as a suitable cellular model for transgenic expression of human neutrophil serine proteases.

Carboxyl-terminal prodomain-deleted human leukocyte elastase and cathepsin G are efficiently targeted to granules and enzymatically activated in the rat basophilic/mast cell line RBL.

The hematopoietic neutral serine proteases leukocyte elastase and cathepsin G are synthesized as inactive precursors, but become activated by removal of an amino-terminal dipeptide and are stored in granules. Moreover, the pro forms of elastase and cathepsin G show carboxyl-terminal prodomains of 20 and 11 amino acids, respectively, which are not present in the mature enzymes. To investigate mechanisms of processing, activation, and granular targeting, we have utilized transgenic expression of myeloid serine proteases in the rat basophilic/mast cell line RBL-1 (Gullberg, U., Lindmark, A., Nilsson, E., Persson, A.-M., and Olsson, I. (1994) J. Biol. Chem. 269, 25219-25225). Leukocyte elastase was stably expressed in RBL-1 cells, and the translation products were characterized by biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Processing of a main pro form of 34 kDa into mature 31- and 29-kDa forms was demonstrated. Translocation of mature forms to granule-containing fractions was shown by subcellular fractionation experiments. The processed forms were enzymatically active, judging by the occurrence of amino-terminal processing demonstrated by radiosequence analysis, the acquisition of affinity for the protease inhibitor aprotinin, and the appearance of elastase activity in transfected RBL cells. To investigate the function of the carboxyl-terminal prodomains, deletion mutants of leukocyte elastase and cathepsin G lacking the carboxyl-terminal extension were constructed and transfected into RBL cells. Our results show that as full-length proteins, the deletion mutants were converted to active enzymes and transferred to granules with kinetics similar to that of wild-type enzymes. We conclude that human leukocyte elastase and cathepsin G are converted into enzymatically active forms when expressed in RBL cells and targeted for storage in granules; the carboxyl-terminal prodomains are necessary neither for enzymatic activation nor for targeting to granules in RBL cells.

Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL.

The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.

Processing and intracellular transport of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937-modulation by brefeldin A, ammonium chloride, and monensin.

The effects of brefeldin A, monensin, and the weak base NH4Cl on the biosynthesis and processing of cathepsin G and neutrophil elastase of myeloid cells were investigated. Monoblast-like U-937 cells were biosynthetically labeled with [35S]methionine, followed by subcellular fractionation, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Brefeldin A inhibited proteolytic processing, intracellular transport, and secretion. The effects were reversible inasmuch as removal of brefeldin A resulted in a normal pattern of processing and transfer to high-density organelles, corresponding to lysosomes, and restitution of constitutive secretion of precursor forms. Both cathepsin G and neutrophil elastase acquired resistance to endoglycosidase-H, suggesting that conversion to complex oligosaccharide side chains also occurs in the presence of brefeldin A. Monensin and NH4Cl inhibited final proteolytic processing, indicating either that acidification is necessary for directing cathepsin G and neutrophil elastase to lysosomal-like organelles or that the protease(s) responsible for processing requires an acid pH. We conclude that pH-dependent proteolytic processing of cathepsin G and neutrophil elastase occurs in post-Golgi structures and that transfer to lysosomes or an immediately prelysosomal compartment is mandatory for complete processing.

In vitro activity of DMG-Mino and DMG-DM Dot, two new glycylcyclines, against anaerobic bacteria.

The in vitro activity of DMG-Mino and DMG-DM Dot against 350 anaerobic bacterial strains including anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, other Bacteroides species and fusobacteria was determined by the agar dilution method. Their activity was compared with that of minocycline, doxycycline, piperacillin, cefoxitin, imipenem, clindamycin and metronidazole. DMG-Mino and DMG-DM Dot and imipenem were the most active agents tested. DMG-Mino and DMG-DM Dot had in vitro activity superior to that of minocycline and doxycycline.

In vitro activity of the new quinolone BAY y 3118 against anaerobic bacteria.

The in vitro activity of BAY y 3118 against anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, other Bacteroides spp. and fusobacteria was determined by an agar dilution method. This activity was compared with that of ciprofloxacin, ofloxacin, piperacillin, cefoxitin, imipenem, clindamycin and metronidazole. BAY y 3118, imipenem, clindamycin and metronidazole were the most active agents tested. The in vitro activity of BAY y 3118 against anaerobic bacteria was superior to that of ciprofloxacin and ofloxacin.

In vitro activity of L-627 against anaerobic bacteria.

The in vitro activity of L-627 against 370 anaerobic bacterial strains including anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, other Bacteroides spp. and fusobacteria was determined by the agar dilution method. This activity was compared with that of piperacillin, cefoxitin, imipenem, meropenem, clindamycin, metronidazole and chloramphenicol. L-627, imipenem, meropenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested. L-627 had in vitro activity similar to that of the other carbapenems tested.

Susceptibility of anaerobic bacteria to tosufloxacin.

The in vitro activity of tosufloxacin against anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, Bacteroides spp. and fusobacteria was determined by the agar dilution method. This activity was compared with that of ciprofloxacin, piperacillin, cefoxitin, imipenem, clindamycin, metronidazole and chloramphenicol. Tosufloxacin, imipenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested. Tosufloxacin has an antibacterial activity that warrants investigation in clinical trials.

Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937.

The processing of the neutral proteases cathepsin G and neutrophil elastase, normally synthesized in myeloid precursor cells and stored in azurophil granules, were investigated by biosynthetic labeling with 14C-leucine of the monoblastic cell line U-937. The proteases were precipitated with specific antibodies and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. The transfer to lysosomes of newly synthesized proteases was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenates in a Percoll gradient. The presence of a closely spaced polypeptide band-doublet at intermediate gradient density suggested cleavage of the specific aminoterminal pro dipeptide extension before storage in lysosomes. The molecular heterogeneity observed for cathepsin G and neutrophil elastase seemed to be due to modifications occurring after sorting into lysosomes, most likely because of C-terminal processing. Modifications of the secreted enzymes were not detectable by SDS-PAGE. In contrast to other lysosomal enzymes, no phosphorylation was demonstrated. Newly synthesized cathepsin G and neutrophil elastase rapidly became resistant to endoglycosidase H, indicating transport through the medial and trans cisternae of the Golgi complex and conversion to "complex" oligosaccharide side chains. This conversion was inhibited by an agent swainsonine, but translocation from the Golgi complex and secretion were unaffected. The processing described may play a role in activation of the proteases.

Susceptibility of anaerobic bacteria to ALP 201.

The activity of ALP 201 against 350 strains of anaerobic bacteria was determined by an agar dilution method. Its activity was compared with those of piperacillin, cefoxitin, imipenem, clindamycin, metronidazole, and chloramphenicol. ALP 201 and imipenem were the most active agents tested. Based on these results, ALP 201 appears to be a promising antimicrobial agent for anaerobic infections and warrants further clinical investigations.