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A R Hauser

Publications and source records attributed to A R Hauser.

18 recordsLinked to original sources
PubMed1999

Pseudomonas aeruginosa induces type-III-secretion-mediated apoptosis of macrophages and epithelial cells.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on macrophages, we infected J774A.1 cells and primary bone-marrow-derived murine macrophages with the P. aeruginosa strain PA103 in vitro. PA103 caused type-III-secretion-dependent killing of macrophages within 2 h of infection. Only a portion of the killing required the putative cytotoxin ExoU. By three criteria, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays, cytoplasmic nucleosome assays, and Hoechst staining, the ExoU-independent but type-III-secretion-dependent killing exhibited features of apoptosis. Extracellular bacteria were capable of inducing apoptosis, and some laboratory and clinical isolates of P. aeruginosa induced significantly higher levels of this form of cell death than others. Interestingly, HeLa cells but not Madin-Darby canine kidney cells were susceptible to type-III-secretion-mediated apoptosis under the conditions of these assays. These findings are consistent with a model in which the P. aeruginosa type III secretion system transports at least two factors that kill macrophages: ExoU, which causes necrosis, and a second, as yet unidentified, effector protein, which induces apoptosis. Such killing may contribute to the ability of this organism to persist and disseminate within infected patients.

Animals↗
PubMed1999

Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia.

Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. The pilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant. In a mouse model of acute pneumonia, the pilT and pilU mutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

Adenosine Triphosphatases↗
PubMed1998

PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence.

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of hospital-acquired pneumonia. We identified a 73kDa protein, designated Pseudomonas exoprotein A (PepA), that was secreted by P. aeruginosa strain PA103. PepA was necessary for in vitro killing of epithelial cells as well as virulence in a mouse model of acute pneumonia. Several properties of PepA suggested that it was secreted by a type III system. Secretion occurred without cleavage of a signal peptide and in low-calcium environments in the presence of a divalent cation chelator, as is the case for characterized P. aeruginosa type III secreted proteins. Secretion of PepA was absent from isogenic mutants with defective type III pathways. Finally, amino-terminal peptide sequence analysis indicated that the amino-terminal five residues of PepA were identical to those of ExoS and ExoT, two type III secreted proteins of P. aeruginosa. After secretion, PepA underwent cleavage at two sites, each with the sequence A-X-K-S, suggesting that the cleavage may be caused by a protease. The gene encoding PepA, designated pepA, was cloned and sequenced, and comparisons with the genetic database using BLAST alignments indicated that the nucleotide sequence of pepA and the inferred protein sequence of PepA had no homology to known sequences. A nucleotide sequence identical to the consensus element for binding of ExsA, a transcriptional activator of P. aeruginosa type III secretion genes, was located 84 bp 5' of the translational start codon. Analysis of transposon insertion mutants indicated that the carboxy terminus was required for cytotoxicity. Examination of respiratory clinical isolates demonstrated that pepA was a variable trait and probably acquired by horizontal transmission. Consistent with this hypothesis was the identification of a putative insertion element 94 bp 5' of the PepA translational start site. Analysis of G + C content of the PepA coding sequence and the adjacent insertion element suggested that they were acquired together from a different species. In summary, PepA is a secreted protein of P. aeruginosa that is necessary for epithelial cell cytotoxicity in vitro and virulence in a mouse model of pneumonia.

Amino Acid Sequence↗
PubMed1998

Defects in type III secretion correlate with internalization of Pseudomonas aeruginosa by epithelial cells.

Previous characterization of Pseudomonas aeruginosa clinical isolates has demonstrated an inverse correlation between cytotoxicity and internalization by epithelial cells. To further investigate this relationship, we tested PA103, a cytotoxic P. aeruginosa strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby canine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bacterial uptake by epithelial cells is not absolute. Six of the noncytotoxic mutants, however, demonstrated measurable levels of internalization by standard aminoglycoside exclusion assays even though internalization of wild-type strain PA103 was not detectable. All six had evidence of protein secretion defects involving two proteins, a 40-kDa protein and a 32-kDa protein. These proteins, designated PepB (for Pseudomonas exoprotein B) and PepD, respectively, each had characteristics of type III transported proteins. In addition, nucleotide sequencing studies demonstrated that PepB and PepD are homologs of YopB and YopD, respectively, type III secreted proteins of Yersinia spp. necessary for the translocation of effector molecules into the cytoplasmic compartment of eukaryotic cells. Thus, while many mutations in PA103 result in loss of cytotoxicity without an appreciable increase in internalization, defects in transport of type III secretion proteins PepB and PepD correlate with both loss of cytotoxicity and gain of internalization. These results are consistent with type III secretion of an inhibitor of internalization that requires PepB and PepD for translocation into the host cell.

Amino Acid Sequence↗
PubMed1998

Another toxic shock syndrome. Streptococcal infection is even more dangerous than the staphylococcal form.

In the battery of diseases caused by group A streptococci, streptococcal toxic shock syndrome is among the most severe. Its nonspecific presentation and rapid progression require that the treating physician be both knowledgeable and vigilant. Certain signs and symptoms suggest that caution is in order: Pain out of proportion to physical findings may herald deep tissue involvement in a patient who at first glance appears to have cellulitis. Streptococcal infection accompanied by hypotension, confusion, or unexplained acute renal insufficiency is a clue to streptococcal toxic shock syndrome. Treatment consists of antibiotic and supportive care, with aggressive surgical debridement of soft-tissue foci of infection when necessary. Anecdotal evidence suggests that intravenous immunoglobulin may have a place in neutralizing the secreted streptococcal toxins that are thought to mediate features of the disease. At present, even with aggressive therapy, the mortality rate of streptococcal toxic shock syndrome can exceed 50%. Understanding of its pathogenesis is progressing. However, until effective interventions are developed, early detection appears to be the best weapon.

Humans↗
PubMed1997

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury.

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa-induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pill, based on their resistance to phage PO4 and/or loss of twitching motility (twt-). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process.

Amino Acid Sequence↗
PubMed1992

Clonal basis for resurgence of serious Streptococcus pyogenes disease in the 1980s.

During the 1980s there was a resurgence of serious Streptococcus pyogenes infections with complications, including rheumatic fever, sepsis, severe soft-tissue invasion, and toxic-shock-like syndrome (TSLS). We have investigated the suggested association between expression of a scarlet fever toxin, SPE A, and systemic toxicity, and the possibility that a new highly virulent clone of S pyogenes has emerged and spread world wide. We studied serotype M1 strains, the serotype most commonly associated with serious complications. 19 isolates from patients with sepsis, with or without TSLS, and 48 from patients with uncomplicated pharyngitis or superficial skin infection were subjected to restriction-enzyme digestion and electrophoresis; 56 isolates (19 serious, 37 uncomplicated disease) were then examined by hybridisation to an speA gene probe. 17 (90%) of the 19 serious-disease isolates had a characteristic ("invasive", I) restriction-fragment profile and were positive for the speA gene. Significantly lower proportions of the isolates from patients with uncomplicated disease had the I profile (21/48 [44%]; p = 0.0035) and speA (20/37 [54%]; p less than 0.001). These findings suggest that the strains from patients with serious disease are a unique clone, which became the predominant cause of severe streptococcal infections in the United States and elsewhere in the late 1980s.

DNA, Bacterial↗
PubMed1991

Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression.

Genetic diversity and relationships among 108 isolates of the bacterium Streptococcus pyogenes recently recovered from patients in the United States with toxic-shock-like syndrome or other invasive diseases were estimated by multilocus enzyme electrophoresis. Thirty-three electrophoretic types (ETs), representing distinctive multilocus clonal genotypes, were identified, but nearly half the disease episodes, including more than two-thirds of the cases of toxic-shock-like syndrome, were caused by strains of two related clones (ET 1 and ET 2). These two clones were also represented by recent pathogenic European isolates. A previous report of a relatively high frequency of expression of exotoxin A among isolates recovered from toxic-shock-like syndrome patients in the United States was confirmed; and the demonstration of this association both within clones and among distantly related clones supports the hypothesis that exotoxin A is a causal factor in pathogenesis of this disease. Near identity of the nucleotide sequences of the exotoxin A structural gene of six isolates of five ETs in diverse phylogenetic lineages was interpreted as evidence that the gene has been horizontally distributed among clones, presumably by bacteriophage-mediated transfer.

Bacterial Proteins↗
PubMed1991

Molecular analysis of pyrogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome.

Toxic shock-like syndrome (TSLS) is characterized by hypotension or shock, fever, multiorgan system involvement, and a concurrent group A streptococcal infection. We analyzed 34 streptococcal strains isolated from patients with clinically well-documented TSLS for their pyrogenic toxin profiles and M-protein types. Although strains of nine different M types were represented in the sample, 74% of the isolates were of either M type 1 or 3. It was determined that 53% produced streptococcal pyrogenic exotoxin type A under in vitro growth conditions and that 85% contained the gene encoding this toxin. These values are in contrast to the published value of 15% for the incidence of this gene in a sample of general group A streptococcal isolates. As has been found with all group A streptococci examined to date, regardless of disease association, 100% of TSLS-associated isolates contained the gene encoding pyrogenic exotoxin type B. This toxin was detectably produced by 59% of isolates. The gene encoding pyrogenic toxin type C was found in only 21% of isolates. We conclude that the pyrogenic exotoxin type A gene is associated with group A streptococcal strains isolated from patients with TSLS and may play a causative role in this illness. However, other factors are also likely to be important, since not all strains from patients with TSLS contained the A toxin gene.

Antigens, Bacterial↗
PubMed1990

Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor.

The streptococcal pyrogenic exotoxin (SPE) type B-encoding structural gene, speB, was subcloned from a 4.5-kilobase streptococcal DNA insert onto a 2.4-kilobase insert, which was then sequenced. Studies indicated that a 1,194-base-pair open reading frame encoded a 398-amino-acid protein. Removal of the putative signal peptide resulted in a mature protein with 371 residues (molecular weight, 40,314), which was subsequently proteolyzed to yield a 253-residue breakdown product (molecular weight, 27,588). This processing was confirmed by amino-terminal sequencing of both the 40,314-molecular-weight protein and the breakdown product. Monte Carlo analysis indicated that SPE B was relatively dissimilar to other members of the pyrogenic toxin family that also includes SPEs A and C, toxic shock syndrome toxin 1, and the staphylococcal enterotoxins. Comparison with the published amino acid sequence of streptococcal proteinase precursor as well as DNA hybridization experiments indicated that SPE B is a variant of this protein even though the particular gene sequenced did not encode a proteolytically active molecule.

Amino Acid Sequence↗
PubMed1989

Type IV collagen-mediated melanoma cell adhesion and migration: involvement of multiple, distinct domains of the collagen molecule.

Tumor cell metastasis involves a complex series of interdependent events, including repeated invasion of basement membranes. Studies from several laboratories have implicated tumor cell adhesion and migration in response to laminin as a major contributing factor in tumor cell invasion. The current studies address the direct role of type IV collagen in promoting tumor cell adhesion, spreading, and migration. The observations of type IV collagen-mediated cellular behavior are contrasted with cellular behavior on type I collagen. The highly metastatic K1735 M4 melanoma cell line adhered, spread, and migrated in response to type IV collagen in a concentration-dependent manner. Functional assays using well-defined proteolytic fragments of type IV collagen demonstrated that melanoma cells interact with multiple domains of this protein. Highly metastatic melanoma cells adhered, spread, and exhibited motile behavior in response to 0.2 to 200 nM concentrations of a purified pepsin-generated, triple helix-rich domain of type IV collagen. In contrast, cells adhered and spread but were essentially nonmotile in response to a purified major noncollagenous domain of the protein. In addition, de novo protein synthesis was required for cell adhesion to the major noncollagenous domain, whereas adhesion to the helical domain was less dependent upon de novo protein synthesis. Arg-Gly-Asp (RGD)-related peptides were used to study the adhesion and spreading of melanoma cells on type IV collagen. The results demonstrated that a serine containing RGD-related peptide (GRGDSP) has virtually no effect on melanoma cell adhesion on type IV collagen-coated substrata, whereas this peptide inhibited melanoma cell adhesion to fibronectin-coated substrata in a concentration-dependent manner. In contrast, when threonine was substituted for serine (GRGDTP), cell adhesion to type IV collagen was significantly (45%) inhibited. The threonine-containing peptide virtually eliminated cell adhesion on substrata coated with type I collagen. These data demonstrate that adhesion, spreading, and migration of melanoma cells on type IV collagen have a complex molecular basis which is partially dependent on RGD-related sequences.

Amino Acid Sequence↗
PubMed1988

Cloning of the gene, speB, for streptococcal pyrogenic exotoxin type B in Escherichia coli.

The structural gene encoding streptococcal pyrogenic exotoxin type B, designated speB, was cloned in Escherichia coli and localized onto a 4.5-kilobase BamHI-BglII DNA fragment. Streptococcal pyrogenic exotoxin type B, partially purified from E. coli clones, was immunologically related to streptococcus-derived toxin. Also, toxin derived from either E. coli or Streptococcus pyogenes had similar lymphocyte mitogenic activity and molecular weight (29,300) and displayed comparable microheterogeneity when evaluated by isoelectric focusing.

Bacterial Proteins↗
PubMed1985

Determination of cimetidine and metabolites in plasma by reversed-phase high-performance liquid chromatographic radial compression technique.

A high-performance liquid chromatographic method for the detection of cimetidine and its metabolites in plasma was developed which has a short analysis time and good resolution. The total analysis time was approximately 11 min. The standard curves were linear over the concentrations used for all compounds and the sensitivity limits were good. The coefficient of variation for within-day and between-day analysis was less than 4.2% for all compounds with the exception of guanyl urea cimetidine, which was approximately 10%. Currently, this assay is being used in a pharmacokinetic study of plasma and gastric aspirate samples obtained from a critically ill pediatric population.

Biotransformation↗
PubMed1985

Pharmacokinetics and pharmacodynamics of cimetidine and metabolites in critically ill children.

Cimetidine and antacids are the mainstays of therapy for the prophylaxis of stress-induced ulceration in critically ill children. Previous cimetidine dosing recommendations have been empiric because of a lack of knowledge about cimetidine disposition kinetics in children. Thirty children, mean age 9 +/- 3.2 years, were admitted to the study with the following primary diagnoses: closed head injury (23 patients), sepsis (four), gunshot wound (two), and bleeding gastric ulceration (one). The mean dose of cimetidine was 26 mg/kg/day, administered intravenously over 15 minutes in four divided doses. Cimetidine disposition was best described by a biphasic elimination curve with t1/2 values for cimetidine, cimetidine sulfoxide, and hydroxymethyl cimetidine of 1.39, 2.6, and 4.7 hours, respectively. Cimetidine plasma concentrations were maintained at greater than or equal to 0.5 microgram/ml for a significantly longer time in patients who received greater than or equal to 20 mg/kg/day. Most patients had a plasma cimetidine concentration below 0.5 to 1.0 microgram/ml 4 hours after infusion. The mean apparent volume of distribution and total body clearance for cimetidine were 1.23 L/kg and 10.4 ml/min/kg, respectively. A significant correlation was found between age and either apparent volume of distribution (r = 0.76, P less than 0.001) or total body clearance (r = 0.75, P less than 0.001). No significant correlation between cimetidine concentrations in either plasma or gastric juice and gastric pH could be determined. However, seven of nine patients who received only cimetidine had a gastric pH of greater than or equal to 4 at 2 hours after infusion when the plasma cimetidine concentration was greater than or equal to 1.0 or the gastric juice concentration was greater than or equal to 2.0 microgram/ml. The mean gastric pH was 2.2 at 6 hours, when plasma and gastric juice concentrations of cimetidine were greater than or equal to 1.0 microgram/ml. On the basis of our data, a cimetidine dosage of 20 to 30 mg/kg/day administered in six divided doses should provide for average steady-state plasma cimetidine concentrations of 1.3 to 2.0 micrograms/ml.

Adolescent↗
PubMed1984

Estramustine phosphate sodium.

Estramustine phosphate is approved by the Food and Drug Administration for oral use in the palliative treatment of patients with metastatic and/or progressive carcinoma of the prostate. Estramustine is a conjugate of 17 beta-estradiol and the carbamate of nitrogen mustard. Although its therapeutic efficacy has been demonstrated, it is not clear to what extent each constituent contributes to estramustine's effectiveness. Estramustine phosphate therapy achieves objective response rates of 60-90 percent in advanced stage D prostatic cancer patients with no prior hormonal therapy. These results are consistent with those obtained with conventional hormonal therapy in similar patient populations. Therapeutic efficacy does not appear to increase when estramustine is used concurrently with other cytotoxic chemotherapeutic agents. An objective response rate of 20-30 percent can be anticipated in patients refractory to conventional hormonal therapy. It is in this group, the estrogen-resistant patients, that estramustine shows the most promise. Adverse effects of estramustine are similar to those of diethylstilbestrol. Gastrointestinal and cardiovascular side effects appear to be the most important and may be severe enough to require discontinuation of therapy.

Animals↗
PubMed1983

In vitro effect of cimetidine concentration and pH on guaiac-slide tests.

The in vitro effect of cimetidine concentration and pH on two brands of guaiac-slide tests was studied. Samples of simulated gastric fluid at pH 1.2, 2.8, and 4.0 were prepared. Cimetidine was added to each of these solutions, and subsequent dilutions of the three samples were made to produce cimetidine concentrations of 10, 5, 2.5, 2.0, 1.5, 1.25, and 0.625 mg/ml. Samples of each pH-concentration combination were placed on two test areas of two Hemoccult and two Fe-Cult slides and tested in accordance with manufacturers' instructions. To determine more specifically the effect of pH, seven aliquots of a cimetidine solution of 1.5 mg/ml in distilled water were prepared (initial pH 8.8). Hydrochloric acid was added to six aliquots to adjust the pH to 7.3, 6.5, 4.6, 3.4, 2.6, and 1.5. A sample of each aliquot was applied to two test areas of four Hemoccult and four Fe-Cult slides. In general, at a given concentration, fewer positive results were obtained with samples prepared from the solution lowest in pH. All results were negative at cimetidine concentrations less than 1.5 mg/ml. When the cimetidine concentration was constant at 1.5 mg/ml, pH values greater than 4.6 for Hemoccult and greater than 7.3 for Fe-Cult were associated with positive results. Both tests appeared to become less sensitive to the effect of cimetidine at lower pH values. Positive Hemoccult and Fe-Cult guaiac-slide test reactions occur in vitro at cimetidine concentrations greater than or equal to 1.5 mg/ml. Because it is doubtful that in vivo concentrations of cimetidine in gastric fluid exceed 1.5 mg/ml, the clinical importance of this interaction is probably minor.

Cimetidine↗