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A H Limper

Publications and source records attributed to A H Limper.

At least 19 recordsLinked to original sources
PubMed2005

Angiotensin II antagonism fails to ameliorate bleomycin-induced pulmonary fibrosis in mice.

Based on current evidence, transforming growth factor (TGF)-beta plays a central pathogenic role in the development of pulmonary fibrosis. There is growing evidence that angiotensin II can serve as a stimulus for TGF-beta-mediated lung fibrosis. However, the role of angiotensin II in the pathobiology of pulmonary fibrosis in vivo remains unclear and the therapeutic potential for targeting angiotensin II in a bleomycin-induced pulmonary fibrosis model is not well known. Therefore, the aim of this study was to test whether the angiotensin II antagonist, losartan, attenuated the development of bleomycin-induced pulmonary fibrosis in two distinct murine strains, C57/BL6 and Sv129. This was determined by histopathology and quantification of collagen content by hydroxyproline assay. Despite demonstrable angiotensin II antagonism in vivo and a reduction in measures of acute lung injury, losartan therapy, at a dose shown to reduce renal and cardiac fibrosis in mice, failed to significantly ameliorate bleomycin-induced pulmonary fibrosis. In conclusion, these data suggest that the pulmonary fibrotic disease process in vivo is not solely dependent on angiotensin II activity and the potential for angiotensin II receptor blockers as a therapeutic strategy in patients with pulmonary fibrosis may be limited.

Angiotensin II Type 1 Receptor Blockers↗
PubMed2001

Differential regulation of growth and checkpoint control mediated by a Cdc25 mitotic phosphatase from Pneumocystis carinii.

Pneumocystis carinii is an opportunistic fungal pathogen phylogenetically related to the fission yeast Schizosaccharomyces pombe. P. carinii causes severe pneumonia in immunocompromised patients with AIDS and malignancies. Although the life cycle of P. carinii remains poorly characterized, morphologic studies of infected lung tissue indicate that P. carinii alternates between numerous small trophic forms and fewer large cystic forms. To understand further the molecular mechanisms that regulate progression of the cell cycle of P. carinii, we have sought to identify and characterize genes in P. carinii that are important regulators of eukaryotic cell cycle progression. In this study, we have isolated a cDNA from P. carinii that exhibits significant homology, but unique functional characteristics, to the mitotic phosphatase Cdc25 found in S. pombe. P. carinii Cdc25 was shown to rescue growth of the temperature-sensitive S. pombe cdc25-22 strain and thus provides an additional tool to investigate the unique P. carinii life cycle. Although P. carinii Cdc25 could also restore the DNA damage checkpoint in cdc25-22 cells, it was unable to restore fully the DNA replication checkpoint. The dissociation of checkpoint control at the level of Cdc25 indicates that Cdc25 may be under distinct regulatory control in mediating checkpoint signaling.

Amino Acid Sequence↗
PubMed2001

Characterization of Pneumocystis carinii PHR1, a pH-regulated gene important for cell wall Integrity.

Pneumocystis carinii remains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region coding GSC-1, a gene mediating beta-glucan cell wall synthesis in P. carinii. The predicted amino acid sequence of this new gene, termed P. carinii PHR1, exhibited 38% homology to Saccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology to Candida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking beta-1,3- and beta-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression of P. carinii PHR1 was examined. As in C. albicans, P. carinii PHR1 expression was repressed under acidic conditions but induced at neutral and more alkaline pH. PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties with P. carinii culture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression of P. carinii genes in related fungi to address functional correlates of P. carinii-encoded proteins. Therefore, the potential role of P. carinii PHR1 in cell wall integrity was examined by assessing its ability to rescue an S. cerevisiae gas1 mutant with absent endogenous Phr1p-like activity. Interestingly, P. carinii PHR1 DNA successfully restored proliferation of S. cerevisiae gas1 mutants under lethal conditions of cell wall stress. These results indicate that P. carinii PHR1 encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.

Amino Acid Sequence↗
PubMed2001

Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: comparison of LightCycler PCR, in situ hybridization, direct fluorescence antigen detection, and culture.

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.

Antigens, Bacterial↗
PubMed2001

Carbohydrate recognition domain of surfactant protein D mediates interactions with Pneumocystis carinii glycoprotein A.

Pneumocystis carinii continues to cause severe pneumonia in immunocompromised patients. Surfactant protein D (SP-D), a lung collectin, markedly accumulates during P. carinii pneumonia and binds to glycoprotein A (gpA) on the surface of P. carinii, thereby enhancing interactions with alveolar macrophages. Herein, we report the structural basis of the interaction of SP-D with gpA. We demonstrate that natural SP-D binds to purified gpA in the presence of 2 mM calcium in a saturable, concentration-dependent manner, which is abolished by 10 mM ethylenediaminetetraacetic acid. Increasing concentrations of calcium under otherwise cation-free conditions significantly enhance SP-D binding to gpA, whereas manganese and magnesium cations have minimal effect. Maximal SP-D binding occurs at pH 7.4, with significant inhibition at pH 4. SP-D binding to gpA is also competitively inhibited by maltose>glucose>mannose>N-acetyl-glucosamine. Comparison of the binding of various natural and recombinant forms of SP-D to gpA reveals that the number of carbohydrate recognition domains (CRDs) in a given SP-D form determines the relative extent of binding to gpA. Maximal binding is observed with natural SP-D (dodecamers and higher order SP-D complexes) followed by recombinant dodecamers. In contrast, recombinant full-length trimers exhibit substantially less binding, which is similar to that observed with a recombinant truncated molecule consisting of the CRD and neck regions, and containing trimers of this portion of the molecule. Taken together, these findings strongly indicate that the CRD of SP-D mediates interaction with P. carinii gpA through its attached oligosaccharides and that the extent of SP-D binding to P. carinii is greatest with dodecamers and higher order forms of SP-D.

Acetylglucosamine↗
PubMed2001

Vitronectin and fibronectin function as glucan binding proteins augmenting macrophage responses to Pneumocystis carinii.

beta-glucans represent major structural components of fungal cell walls. We recently reported that Pneumocystis carinii beta-glucans stimulate alveolar macrophages to release proinflammatory cytokines. Macrophage activation by beta-glucan is augmented by serum, implying the presence of circulating factors that interact with beta-glucans and enhance their ability to stimulate macrophages. Using beta-glucan-enriched cell wall fractions from P. carinii and Saccharomyces cerevisiae, two prominent proteins were precipitated from serum and demonstrated to be vitronectin (VN) and fibronectin (FN) by immune analysis. Preincubation of beta-glucan with VN or FN enhanced macrophage activation in response to this cell wall component. Because VN and FN accumulate in the lungs during P. carinii pneumonia, we further investigated hepatic and pulmonary expression of VN and FN messenger RNA during infection. P. carinii pneumonia in rodents is associated with increased hepatic expression of VN and FN as well as increased local expression of FN in the lung. Because interleukin (IL)-6 represents the major regulator of VN and FN expression during inflammatory conditions, we measured macrophage IL-6 release in response to stimulation with P. carinii beta-glucan. Stimulation of macrophages with P. carinii beta-glucan induced significant release of IL-6. Elevated concentrations of IL-6 were noted in the blood of infected animals compared with uninfected control animals. These studies indicate that VN and FN bind to beta-glucan components of P. carinii and augment macrophage inflammatory responses. P. carinii cell wall beta-glucan stimulates secretion of IL-6 by macrophages, thereby enhancing hepatic synthesis of both VN and FN, and lung synthesis of FN during pneumonia.

Animals↗
PubMed2000

Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition.

Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of beta-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of beta-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5'-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733-560, an antagonist of Gsc-1-type beta-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of beta-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal beta-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5'-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate that P. carinii contains a unique catalytic subunit of beta-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of beta-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.

Anti-Bacterial Agents↗
PubMed2000

Isolated Pneumocystis carinii cell wall glucan provokes lower respiratory tract inflammatory responses.

Macrophage-induced lung inflammation contributes substantially to respiratory failure during Pneumocystis carinii pneumonia. We isolated a P. carinii cell wall fraction rich in glucan carbohydrate, which potently induces TNF-alpha and macrophage-inflammatory protein-2 generation from alveolar macrophages. Instillation of this purified P. carinii carbohydrate cell wall fraction into healthy rodents is accompanied by substantial increases in whole lung TNF-alpha generation and is associated with neutrophilic infiltration of the lungs. Digestion of the P. carinii cell wall isolate with zymolyase, a preparation containing predominantly beta-1,3 glucanase, substantially reduces the ability of this P. carinii cell wall fraction to activate alveolar macrophages, thus suggesting that beta-glucan components of the P. carinii cell wall largely mediate TNF-alpha release. Furthermore, the soluble carbohydrate beta-glucan receptor antagonists laminariheptaose and laminarin also substantially reduce the ability of the P. carinii cell wall isolate to stimulate macrophage-inflammatory activation. In contrast, soluble alpha-mannan, a preparation that antagonizes macrophage mannose receptors, had minimal effect on TNF-alpha release induced by the P. carinii cell wall fraction. P. carinii beta-glucan-induced TNF-alpha release from alveolar macrophages was also inhibited by both dexamethasone and pentoxifylline, two pharmacological agents with potential activity in controlling P. carinii-induced lung inflammation. These data demonstrate that P. carinii beta-glucan cell wall components can directly stimulate alveolar macrophages to release proinflammatory cytokines mainly through interaction with cognate beta-glucan receptors on the phagocyte.

Animals↗
PubMed2000

Pneumocystis carinii uses a functional cdc13 B-type cyclin complex during its life cycle.

Pneumocystis carinii causes severe pneumonia in immunocompromised patients. Recent studies indicate that P. carinii uses a Cdc2 cyclin-dependent kinase to control its proliferation. To further study the regulation of the life cycle of P. carinii, we characterized the P. carinii B-type cyclin termed Cdc13, whose binding to Cdc2 is necessary for kinase activity. Antibodies to B-type cyclins (Cdc13) specifically immunoprecipitated Cdc2/ Cdc13 complexes with associated kinase activity from P. carinii extracts. To clone P. carinii cdc13, degenerate polymerase chain reaction was undertaken using primers generated from amino-acid motifs conserved in fungal Cdc13 proteins. This amplicon was used to obtain full-length genomic and complementary DNA (cDNA) clones. A specific synthetic peptide antibody generated to P. carinii Cdc13 further demonstrated differential Cdc2/Cdc13 activity over the life cycle of P. carinii, with greater activity in cysts compared with trophic forms of the organism. Finally, P. carinii cdc13 cDNA was used to rescue mutant Schizosaccharomyces pombe strains containing temperature-sensitive deficiencies of endogenous Cdc13 activity, thus verifying function of the P. carinii Cdc13 protein. Therefore, P. carinii contains a Cdc13 cyclin, which is variably active over its life cycle and which promotes fungal proliferation.

Amino Acid Sequence↗
PubMed2000

Mechanisms of defence in the lung: lessons from Pneumocystis carinii pneumonia.

Pneumocystis carinii continues to represent an important complication of individuals with compromised immunity. P. carinii interacts with immune and non-immune cells in the lung and mediates lung injury through a variety of mechanisms. CD4+ T lymphocytes are the cornerstone in defence against P. carinii. Recent studies indicate that alveolar macrophages provide essential functions that significantly enhance clearance of P. carinii infection. P. carinii also attaches to alveolar epithelial cells, causing inhibition of epithelial growth and replication. In addition to cellular interactions, P. carinii organisms bind to a variety of host adhesive proteins present in the lower respiratory tract. Binding of these proteins to P. carinii modulates host cell recognition and immune responses to the parasite. During the course of P. carinii pneumonia, several inflammatory mediators are produced in the lung. Although necessary for control of infection, exuberant inflammatory responses also predispose the host to the development of acute lung injury. Thus, host defences against P. carinii depend on complex interactions between immune and non-immune cells as well as several mediators that facilitate host recognition and eventual elimination of infection. Understanding these complex processes may enable development of novel therapeutic approaches for management of this important infection.

CD4 Antigens↗