Biomedical subjects
Publications and source records attributed to Kristian Riesbeck.
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Eosinophils are a characteristic component of the inflammatory response seen in several diseases, including allergic asthma and chronic obstructive pulmonary disease. After activation, eosinophil-derived products may exert proinflammatory effects and cause considerable tissue damage. In the present study, we investigated innate interactions between the respiratory tract pathogen nontypeable Haemophilus influenzae (NTHi) and human eosinophils. Bacterial binding to eosinophils was dependent on (1-3)-beta-D-glucan receptors, as deduced from blocking experiments using the soluble glucan derivatives laminarin and scleroglucan. In addition, expression of the beta-glucan receptor dectin-1 was shown in eosinophils by reverse transcriptase-polymerase chain reaction. Activation of the beta-glucan receptors by bacteria elicited a time- and dose-dependent respiratory burst in eosinophils. NTHi caused increased expression of the proinflammatory chemokine interleukin-8 as measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Incubation of eosinophils in the presence of NTHi for 4.5 h revealed upregulation of 245 different genes as detected by microarray. Signal transduction-related transcripts were most strongly upregulated, followed by cytokine mRNAs. Our findings suggest that NTHi can induce an innate inflammatory response in eosinophils that is mainly mediated via beta-glucan receptors. This points to possible pathophysiologic mechanisms involving innate recognition of NTHi by eosinophils during infection of the airways, thus promoting inflammation in chronic pulmonary disease.
The Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is a 200-kDa outer membrane protein displaying a unique and specific affinity for human IgD. MID is found in the majority of M. catarrhalis strains. In the present paper, we show that MID-expressing M. catarrhalis strains agglutinate human erythrocytes and bind to type II alveolar epithelial cells. In contrast, M. catarrhalis isolates with low MID expression levels and two mutants deficient in MID, but with readily detectable UspA1 expression, do not agglutinate erythrocytes and have a 50% lower adhesive capacity. To examine the adhesive part of MID, the protein was dissected into nine fragments covering the entire molecule. The truncated MID proteins were expressed in Escherichia coli, purified, and used for raising polyclonal antibodies in rabbits. Interestingly, by using recombinant fragments, we show that the hemagglutinating and adhesive part of MID is localized within the 150-amino-acid fragment MID(764-913). In addition, antibodies against full-length MID, MID(764-913), or a 30-amino-acid consensus sequence (MID(775-804)) inhibited adhesion to alveolar epithelial cells. Antibodies against UspA1, an outer membrane protein expressed in essentially all M. catarrhalis strains, also inhibited adhesion, suggesting that both MID and UspA1 are needed for optimal attachment to epithelial cells. Taken together, in addition to MID-dependent IgD binding, we have demonstrated that the outer membrane protein MID is a novel adhesin that would be a suitable target for a future vaccine against M. catarrhalis.
The prevalence of the Moraxella catarrhalis immunoglobulin D (IgD)-binding outer membrane protein MID and its gene was determined in 91 clinical isolates and in 7 culture collection strains. Eighty-four percent of the clinical Moraxella strains expressed MID-dependent IgD binding. The mid gene was detected in all strains as revealed by homology of the signal peptide sequence and a conserved area in the 3' end of the gene. When MID proteins from five different strains were compared, an identity of 65.3 to 85.0% and a similarity of 71.2 to 89.1% were detected. Gene analyses showed several amino acid repeat motifs in the open reading frames, and MID could be called a putative autotransport protein. Interestingly, homopolymeric [polyguanine [poly(G)]] tracts were detected at the 5' ends within the open reading frames. By flow cytometry, using human IgD and fluorescein isothiocyanate-conjugated anti-IgD polyclonal antibodies, most strains showed two peaks: one high- and one low-intensity peak. All isolates expressing high levels of MID had 1, 2, or 3 triplets of G's in their poly(G) tracts, while strains not expressing MID had 4, 7, 8, or 10 G's in their poly(G) tracts or point mutations causing a putative preterminated translation. Northern blot analysis revealed that the mid gene was regulated at the transcriptional level. Experiments with nonclumping variants of M. catarrhalis proved that bacteria lost their MID expression by removing a G in their poly(G) tracts. Moraxella strains isolated from the nasopharynx or from blood and sputum specimens expressed MID at approximately the same frequency. In addition, no variation was observed between strains of different geographical origins (Australia, Europe, Japan, or the United States). MID and the mid gene were found solely in M. catarrhalis, whereas related Neisseria and Moraxella species did not express MID. Taken together, MID appears to be a conserved protein that can be found in essentially all M. catarrhalis strains. Furthermore, MID is governed by poly(G) tracts when bacteria undergo phase variation.
Prevotella bivia is mainly associated with endometritis. The case of a patient with paronychia in a thumb due to P. bivia resulting in osteitis and amputation is reported. The species was not acknowledged in the first bacterial culture 2 weeks before surgery.
Fluoroquinolones have immunomodulatory properties and interfere with cytokine production. The aim of this study was to characterize the extent of the superinduced mRNA levels in activated human lymphocytes incubated with ciprofloxacin (5 and 80 micro g/ml) using a cytokine gene expression microarray from R and D Systems (Abingdon, UK). Several gene transcripts (n=104) were up-regulated in cells treated with ciprofloxacin at 80 micro g/ml, whereas 98 transcripts were down-regulated out of 847 total genes included on the microarray. The increased mRNAs were distributed between major gene programs, including interleukins (36.5%), signal-transduction molecules (13.5%), adhesion molecules (10.6%), tumor necrosis factor and transforming growth factor-beta superfamilies (10.6%), cell-cycle regulators (9.6%), and apoptosis-related molecules (8.7%). To determine the specificity of the microarray, a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), which contained a panel of 12 different cytokine mRNAs, was used. Eleven out of the 12 gene transcripts were up-regulated in the specific RT-PCR, whereas only eight were found to be increased in the microarray. A microarray from Clontech (Hampshire, UK), containing 588 different genes, was also included. Results obtained with this broad-coverage expression array slightly differed compared with the other microarray. We conclude that the fluoroquinolone ciprofloxacin at high concentrations interferes with several gene programs, which is in accordance with a mammalian stress response. From a technical point of view, a discrepancy may exist between data obtained by different microarrays and more specific methods such as quantitative RT-PCR.
Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID(962-1200)). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID(985-1142)) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID(962-1200) was efficiently attracted to a standard IgD serum and to purified myeloma IgD(kappa) and IgD(lambda) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID(962-1200) using transposons suggested that alpha-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID(962-1200) was a tetramer. Interestingly, tetrameric MID(962-1200) attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID(962-1200) is crucial for optimal IgD-binding capacity.
Moraxella IgD binding protein (MID) is a novel bacterial outer membrane protein with IgD-binding properties. MID was purified from the respiratory pathogen Moraxella catarrhalis and is here shown to have B cell stimulatory properties. Purified MID in the range of 0.01-0.1 microg/ml was optimal to induce a proliferative response in human PBL. MID coupled to Sepharose and formalin-fixed M. catarrhalis preparations induced similar proliferative responses in PBL cultures. MID or MID-Sepharose stimulated purified human peripheral B cells as measured by proliferation. In contrast, MID or MID-Sepharose did not activate T cells. Preincubation of purified B cells with anti-IgD Abs inhibited MID-Sepharose-induced B cell proliferation. The addition of IL-4 specifically induced IL-6 production in MID-Sepharose-activated B cells. IgM secretion was detected in B cell cultures stimulated with MID or MID-Sepharose and IL-2 for 10 days. Secretion of IgG and IgA was efficiently induced in cultures from purified B cells stimulated with the combination of MID or MID-Sepharose and IL-4, IL-10, and soluble CD40 ligand, suggesting that Th2-derived cytokines were required for optimal plasma cell generation. Taken together, MID has properties that make it an important tool to study IgD-targeted activation of B cells.
CTLA4Ig and anti-LFA-1 are members of a new generation of immunomodulatory drugs which inhibit important signaling pathways in T cell activation. Both substances target molecules which have pivitol functions in the activation of CD4+ and CD8+ T cells and have been theorized to have an interdependent relationship. These drugs have been used independently in various treatment regimens and have shown great promise in prolonging the survival of allografts. In order to test whether these substances have synergistic or potentiating effects when combined, we performed mixed lymphocyte reactions, skin transplantation and vascularised heterotopic heart transplantation in the Balb/c (H-2(d)) to C3H/HeJ (H-2(k)) strain combination. When anti-LFA-1 and CTLA4Ig were combined at low doses, there was a substantial inhibition of lymphocyte proliferation. When each drug was used as a mono-therapy in skin graft recipients, there was no significant effect on median graft survival (anti-LFA-1, 15 days; CTLA4Ig, 16 days) when compared to untreated controls (13 days), whereas a combination of anti-LFA-1 and CTLA4Ig extended graft survival significantly to 32 days. Untreated vascularised heart grafts rejected at a median of 8 days, CTLA4Ig-treated mice rejected at a median time of 79 days and anti-LFA-1-treated mice rejected at 43 days (n = 9). When CTLA4Ig and anti-LFA-1 were combined, all animals had functioning heart grafts at 100 days after transplantation. Histological analysis of combined-therapy hearts showed no signs or only minor changes associated with chronic rejection. In conclusion, these results indicate a synergistic effect of combining anti-LFA-1 with CTLA4Ig in inhibiting lymphocyte proliferation and prolonging the survival of fully MHC-mismatched allografts.
Non-typeable Haemophilus influenzae, which is a cause of disease in the upper and lower respiratory tract, can survive intracellularly in human epithelial cells and macrophages. We studied the in vitro activity of five antibiotics against intracellular non-typeable H. influenzae in human type II alveolar epithelial cells. The eukaryotic cells were loaded with bacteria, and extracellular bacteria were killed by gentamicin. After the cells were washed, antibiotics were added at concentrations of 0.12-64 mg/L for 18 h before the numbers of viable intracellular bacteria were determined. Of the antibiotics tested, ciprofloxacin and quinupristin/dalfopristin were the most potent agents, followed by clarithromycin and telithromycin. Ampicillin was not active against intracellularly localized, non-typeable H. influenzae.
The susceptibility pattern of Mycobacterium marinum was determined. Quinupristin-dalfopristin and telithromycin were less active than clarithromycin. Linezolid showed good antimicrobial activity at clinically achievable concentrations. Gatifloxacin, levofloxacin, and moxifloxacin displayed activities similar to those of ciprofloxacin. Gemifloxacin was less active. The Etest method showed variable agreement with the reference method.
Salmonella enterica serotype Brandenburg is one of the more uncommon serotypes isolated from patients with gastroenteritis. Few cases of extraintestinal infections with serotype Brandenburg have been documented. The first case of a serotype Brandenburg-dependent thigh abscess originating from an atherosclerotic pseudoaneurysm of the femoral artery is reported.