Search PubMed⌕ Search

Biomedical subjects

S I Miller

Publications and source records attributed to S I Miller.

At least 73 records · Page 4Linked to original sources
PubMed1995

Factors predicting patients' satisfaction with managed mental health care.

Patients' satisfaction with a managed mental health care program was examined using a mail survey that included questions about patients' perceptions of various elements of service delivery and of their functioning after treatment. Completed surveys were returned by 292 patients who received outpatient psychotherapy, a 30 percent return rate. Patients who reported better psychological functioning and who rated the managed care staff as helpful and their therapist as skillful and conveniently located were more likely to be satisfied with the services they received.

Adult↗
PubMed1994

Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes.

Light microscopic studies of phagocytosis showed that Salmonella typhimurium entered mouse macrophages enclosed in spacious phagosomes (SP). Viewed by time-lapse video microscopy, bone marrow-derived macrophages exposed to S. typhimurium displayed generalized plasma membrane ruffling and macropinocytosis. Phagosomes containing Salmonella were morphologically indistinguishable from macropinosomes. SP formation was observed after several methods of bacterial opsonization, although bacteria opsonized with specific IgG appeared initially in small phagosomes that later enlarged. In contrast to macropinosomes induced by growth factors, which shrink completely within 15 min, SP persisted in the cytoplasm, enlarging often by fusion with macropinosomes or other SP. A Salmonella strain containing a constitutive mutation in the phoP virulence regulatory locus (PhoPc) induced significantly fewer SP. Similar to Yersinia enterocolitica, PhoPc bacteria entered macrophages in close-fitting phagosomes, consistent with that expected for conventional receptor-mediated phagocytosis. These results suggest that formation of SP contributes to Salmonella survival and virulence.

Animals↗
PubMed1994

Further characterization of the PhoP regulon: identification of new PhoP-activated virulence loci.

Salmonella typhimurium survival within macrophages is an essential virulence property necessary to enteric fever pathogenesis. This survival requires coordinate transcriptional activation of virulence genes within acidified macrophage phagosomes. Virulence gene transcription is regulated by a two-component system comprising the PhoP (transcriptional activator) and PhoQ (sensor-kinase) proteins. Thirteen new PhoP-activated loci (designated pagD to pagP) encoding membrane or secreted proteins have been identified by use of the transposon TnphoA. Three of these loci have a chromosomal location that was linked to the previously identified pagC locus. Strains with TnphoA insertions in pagD, pagJ, pagK, and pagM were significantly attenuated for mouse virulence (50% lethal dose greater than 1,000 times that of wild-type bacteria). No strains with pag::TnphoA insertions were found to have altered sensitivity to the cationic antimicrobial peptide NP-1 defensin. PhoP and PhoQ are pleotropic regulators of membrane or secreted proteins, suggesting that the ability to effect a global change in the expression of these proteins is required for S. typhimurium survival within acidified macrophage phagosomes.

Animals↗
PubMed1993

Acute bacterial meningitis in adults. A review of 493 episodes.

BACKGROUND AND METHODS: To characterize acute bacterial meningitis in adults, we reviewed the charts of all persons 16 years of age or older in whom acute bacterial meningitis was diagnosed at Massachusetts General Hospital from 1962 through 1988. We included patients who were admitted after initial treatment at other hospitals. RESULTS: During the 27-year period, 445 adults were treated for 493 episodes of acute bacterial meningitis, of which 197 (40 percent) were nosocomial. Gram-negative bacilli (other than Haemophilus influenzae) caused 33 percent of the nosocomial episodes but only 3 percent of the community-acquired episodes. In the 296 episodes of community-acquired meningitis, the most common pathogens were Streptococcus pneumoniae (37 percent), Neisseria meningitidis (13 percent), and Listeria monocytogenes (10 percent); these organisms accounted for only 8 percent of the nosocomial episodes. Only 19 of the 493 episodes of meningitis (4 percent) were due to H. influenzae. Nine percent of all patients had recurrent meningitis; many had a cerebrospinal fluid leak. Seizures occurred in 23 percent of patients with community-acquired meningitis, and 28 percent had focal central nervous system findings. Risk factors for death among those with single episodes of community-acquired meningitis included older age (> or = 60 years), obtunded mental state on admission, and seizures within the first 24 hours. Among those with single episodes, the in-hospital mortality rate was 25 percent for community-acquired and 35 percent for nosocomial meningitis. The overall case fatality rate was 25 percent and did not vary significantly over the 27 years. CONCLUSIONS: In our large urban hospital, a major proportion of cases of acute bacterial meningitis in adults were nosocomial. Recurrent episodes of meningitis were frequent. The overall mortality rate remained high.

Acute Disease↗
PubMed1993

The PhoP virulence regulon and live oral Salmonella vaccines.

The PhoP virulence regulon is essential to Salmonella typhimurium mouse typhoid fever pathogenesis and survival within macrophages. This virulence regulon is composed of the PhoP (transcriptional regulator) and PhoQ (environmental sensor) proteins and the genetic loci they positively (pags for PhoP activated genes) and negatively (prgs for PhoP repressed genes) regulate. Three regulated loci pagC, pagD, and prgH, when singly mutated, affect the virulence of S. typhimurium for mice. Strains with phoP locus mutations are effective as live vaccines in mice, and strains with a constitutive regulatory mutation, a point mutation in PhoQ, can protect mice against typhoid fever when only very few organisms are administered. The addition of various PhoP regulon mutations further attenuates aroA mutants of S. typhimurium, suggesting that these mutations would be useful in further attenuating vaccine strains with metabolic pathway mutations. The phoP, phoQ, pagC, and pagD genes are highly conserved between S. typhimurium and S. typhi and may be valuable as mutations in live vaccines for human typhoid fever. A plasmid suicide vector that allows deletion of the pagC gene and stable insertion of heterologous antigen genes within the deleted pagC locus has been constructed and used successfully in S. typhimurium and S. typhi.

Administration, Oral↗
PubMed1993

Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.

Bacterial Adhesion↗
PubMed1993

A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells.

The Salmonella typhimurium transcriptional regulators, PhoP/PhoQ, induce phoP-activated gene (pag) expression to promote virulence and intracellular survival within macrophages. This response to the macrophage intracellular environment is simulated by phoP/phoQ constitutive mutations (phenotype PhoPc) that increase the expression of pag genes and repress the synthesis of approximately 20 proteins encoded by phoP-repressed genes (prg genes) (S. I. Miller and J. J. Mekalanos, J. Bacteriol. 172:2485-2490, 1990). PhoPc bacteria are attenuated for mouse virulence, suggesting that prg genes are virulence genes. We now report the identification of five unlinked prg loci by use of the transposon TnphoA. In general, medium conditions (i.e., starvation) that activate pag expression repress prg expression. However, variable effects on the PhoP regulon were observed when bacteria were grown under different oxygen tensions (pag and prg genes) or exposed to low pH (prg genes), suggesting heterogenous control of the regulon. One prg locus, prgH, was demonstrated to contribute to mouse virulence by both the oral and the intraperitoneal routes. prgH was located at 59 min on the Salmonella chromosome, a region where other genes essential to invasion of epithelial cells are clustered. The prgH locus was highly linked to one invasion locus, hil (C.A. Lee, B.D. Jones, and S. Falkow, Proc. Natl. Acad. Sci. USA 89:1847-1851, 1992), although transcription of prgH was opposite that of the Tn5B50-encoded promoters that result in a hyperinvasive or hil phenotype. Both PrgH and PhoPc mutant S. typhimurium were found to be defective in induction of endocytosis by Madin-Darby canine kidney (MDCK) epithelial cells. The invasion defect of PrgH but not that of PhoPc mutant bacteria was complemented by plasmids containing prgH (hil) DNA. Therefore, two virulence properties of Salmonella species, induction of endocytosis by epithelial cells and survival within macrophages, are oppositely modulated by the PhoP/PhoQ virulence regulators.

Animals↗
PubMed1992

Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.

Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.

Ammonium Chloride↗
PubMed1992

Purification and primary structure of murine cryptdin-1, a Paneth cell defensin.

We have purified and determined the amino acid sequence of cryptdin-1, a murine Paneth cell defensin. The peptide corresponds to a previously characterized mRNA that accumulates to high abundance during postnatal ontogeny of the small bowel. Acid-extracted intestinal protein was fractionated by cation-exchange chromatography and fractions were assayed for antimicrobial activity. One peak of anti-Salmonella activity contained a putative defensin, based on its predicted electrophoretic migration in acid-urea PAGE. The peptide was purified to homogeneity by RP-HPLC and sequenced. These studies demonstrate defensin expression in non-myeloid tissue. The N-terminal extension of cryptdin-1 is a unique structural feature of this novel epithelial defensin.

Amino Acid Sequence↗
PubMed1992

Enteric defensins: antibiotic peptide components of intestinal host defense.

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.

Amino Acid Sequence↗
PubMed1992

An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae.

Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells. It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail. This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage. Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC. A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64. In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion. Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion. The observation that pagC did not confer an invasive phenotype to E. coli further suggests that PagC is not an invasion factor. A cloned pagC gene complemented the macrophage survival defect of S. typhimurium pagC1 mutants, but the cloned ail gene did not. Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function. Interestingly, S. enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S. enteritidis carrying the pagC1 allele that results only in a macrophage survival defect.

Alkaline Phosphatase↗
PubMed1991

PhoP/PhoQ: macrophage-specific modulators of Salmonella virulence?

The regulation of gene expression by the two-component regulatory system PhoP/PhoQ is necessary for Salmonella typhimurium survival within macrophages, defensin resistance, acid resistance, and murine typhoid fever pathogenesis. Salmonella experience multiple environments during mammalian infection and survival requires tightly regulated gene expression. After phagocytosis by macrophages, signal transduction by PhoQ results in the transcription of phoP-activated genes (pags) encoding proteins essential to bacterial survival and virulence. One such gene, pagC, encodes an envelope protein with amino acid similarity to an epithelial cell invasion protein of Yersina enterocolitica, Ail, and a bacteriophage lambda outer membrane protein, Lom. The PhoP and PhoQ proteins can also repress the synthesis of proteins, encoded by phoP repressed genes (prgs), when pags are maximally expressed. If prgs encode receptors for toxic compounds, prg repression may protect the cell within macrophages when pag expression is most necessary. At least one prg locus, prgH, is required for full S. typhimurium mouse virulence. Within the macrophage, different environments may stimulate a switch from pag to prg expression that is necessary to Salmonella survival. prg expression may also be necessary for surviving nonmacrophage environments. Study of the PhoP regulon should lead to the discovery of new virulence factors, increase knowledge of how gene regulation is essential to bacterial virulence, and perhaps lead to the development of better vaccines for typhoid fever.

Animals↗
PubMed1991

A Salmonella typhimurium virulence protein is similar to a Yersinia enterocolitica invasion protein and a bacteriophage lambda outer membrane protein.

The phoP-phoQ-regulated pagC locus is essential for full virulence and survival within macrophages of Salmonella typhimurium. The protein product, DNA sequence, and transcript of pagC were determined. The pagC locus encodes a single 188-amino-acid membrane protein that is similar to the ail-encoded eucaryotic cell invasion protein of Yersinia enterocolitica and the lom-encoded protein of bacteriophage lambda. The similarity of PagC and Ail to Lom leads us to hypothesize that Lom is a virulence protein and that bacteriophage gene transfer and lysogeny could have led to the development of proteins essential to survival within macrophages and eucaryotic cell invasion.

Adhesins, Bacterial↗
PubMed1990

Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium.

The defensin sensitivities of Salmonella typhimurium strains with mutations in the phoP/phoQ two-component virulence regulon were tested by using purified defensins NP-1 and NP-2. Strains with mutations in either gene of the regulatory pair (phoP [transcriptional activator] or phoQ [membrane sensor kinase]) had increased sensitivities to defensin. The predicted periplasmic domain of the PhoQ protein contained a markedly anionic domain that could interact with cationic proteins and that could be responsible for resistance to defensin. Because insertion mutations in phoP are polar on phoQ, we constructed strains that expressed the PhoQ protein in the absence of PhoP to test whether resistance to defensin requires only the phoQ gene product. We found that resistance to defensin requires the function of both components of this regulatory system, because strains expressing PhoQ without PhoP were still markedly sensitive to defensins. This implied that a pag (phoP-activated gene) product is responsible for defensin resistance. We also tested for the ability of defensins NP-1, NP-5, and HNP-1 to activate pag expression and found that these peptides have no effect. Defensin resistance is not the only virulence characteristic controlled by the PhoP-PhoQ regulon because mutations in pagC, as well as ones in the phoP locus that resulted in constitutive pag activation (phenotype PhoPc), had no effect on defensin resistance, even though they rendered the organism avirulent and deficient in survival within macrophages. The virulence defect conferred by mutations in the phoP-phoQ two-component regulatory system is not completely explained by alterations in resistance to cationic proteins and involves the control of other proteins necessary for S. typhimurium survival within macrophages.

Animals↗
PubMed1990

Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.

The phoP genetic locus is a two-component regulatory system (phoP-phoQ) that controls the expression of genes essential for Salmonella typhimurium virulence and survival within macrophages. Strains with a phoP constitutive mutation (phenotype PhoPC) showed up to 10-fold greater expression of phoP-activated genes (pag loci) than did strains with a wild-type phoP locus (phenotype PhoP+). While the phoP constitutive mutation resulted in increased expression of pag loci, it also dramatically reduced the expression of other protein species. Comparison of the protein content of PhoP+ and PhoPC strains by two-dimensional protein gel electrophoresis demonstrated that at least 40 separate protein species were changed in expression as a result of this mutation. The PhoPC S. typhimurium were found to be attenuated for virulence and survival within macrophages. This finding suggests that a balanced PhoP-PhoQ regulatory response, which allows expression of phoP-repressed as well as -activated genes, is required for full virulence of S. typhimurium. We have further shown that small numbers of PhoPC bacteria can be used as a live attenuated vaccine to protect against mouse typhoid. As few as 15 PhoPC bacteria protected mice against challenge with 10(5) 50% lethal doses of wild-type organisms, suggesting that important protective antigens are regulated by the PhoP-PhoQ virulence regulon.

Animals↗
PubMed1990

Use of commercially available rapid chloramphenicol acetyltransferase test to detect resistance in Salmonella species.

Chloramphenicol resistance among Salmonella spp. has important public health and clinical implications, especially in areas of the world where these strains are endemic. The availability of rapid and sensitive screening methods for detection of antibiotic resistance is important. Therefore, we tested 33 strains of Salmonella for chloramphenicol acetyltransferase (CAT) activity using two rapid techniques. Evaluation of a 1-h tube method and a 30-min commercial disk procedure demonstrated that they are as accurate as standardized susceptibility techniques. Both the 1-h tube and 30-min disk methods detected CAT enzymatic activity produced by one CAT gene copy per cell.

Chloramphenicol O-Acetyltransferase↗