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.

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.

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.

Surface attachment of Salmonella typhimurium to intestinal epithelia imprints the subepithelial matrix with gradients chemotactic for neutrophils.

During intestinal disease induced by Salmonella typhimurium transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. Among the events stimulated by these interactions is the release of chemotaxins that guide PMN through the subepithelial matrix and subsequently through the epithelium itself (McCormick, B.A., S.P. Colgan, C. Delp-Archer, S.I. Miller, and J.L. Madara. 1993. J. Cell Biol. 123:895-907). Given the substantial volume flow that normally characterizes matrix compartments underlying transporting epithelia, it is unclear how such transmatrix signaling is sustained. Here we show that when underlying matrices are isolated from biophysically confluent polarized monolayers of the human intestinal epithelial cell line T84, they fail to support substantial transmatrix migration of PMN unless an exogenous chemotactic gradient is imposed. In contrast, such matrices isolated from confluent monolayers apically colonized with S. typhimurium support spontaneous transmatrix migration of PMN. Such chemotactic imprinting of underlying matrices is resistant to volume wash and is paralleled by secretion of the known matrix-binding chemokine IL-8. Chemotactic imprinting of the matrix underlying S. typhimurium-colonized monolayers is dependent on epithelial protein synthesis, is directional implying the existence of a gradient, and is neutralized by antibodies either to IL-8 or to the IL-8 receptor on PMN. An avirulent S. typhimurium strain, PhoPc, which attaches to epithelial cells as efficiently as wild-type S. typhimurium, fails to induce basolateral secretion of IL-8 and likewise fails to imprint matrices. Together, these observations show that the epithelial surface can respond to the presence of a luminal pathogen and subsequently imprint the subepithelial matrix with retained IL-8 gradients sufficient to resist washout effects of the volume flow that normally traverses this compartment. Such data further support the notion that the primary role for basolateral secretion of IL-8 by the intestinal and likely other epithelia is recruitment of PMN through the matrix to the subepithelial space, rather than directing the final movement of PMN across the epithelium.

Elevated superoxide dismutase in black alcoholics.

Superoxide dismutase concentrations in lysates of erythrocytes from black alcoholics were higher than those of white alcoholics and of nonalcoholics of both races. Higher concentrations of enzyme protein, as determined by competition radioimmunoassay, correspond to proportionately higher enzyme activity. Elevated superoxide dismutase levels were not related to any other clinical, historical, or demographic variables. Increased superoxide dismutase levels may delay or prevent some of the pathological sequelae of alcoholism and may be a useful biological marker for alcohol abuse.

Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ.

Bacterial pathogenesis requires proteins that sense host microenvironments and respond by regulating virulence gene transcription. For Salmonellae, one such regulatory system is PhoP-PhoQ, which regulates genes required for intracellular survival and resistance to cationic peptides. Analysis by mass spectrometry revealed that Salmonella typhimurium PhoP-PhoQ regulated structural modifications of lipid A, the host signaling portion of lipopolysaccharide (LPS), by the addition of aminoarabinose and 2-hydroxymyristate. Structurally modified lipid A altered LPS-mediated expression of the adhesion molecule E-selectin by endothelial cells and tumor necrosis factor-alpha expression by adherent monocytes. Thus, altered responses to environmentally induced lipid A structural modifications may represent a mechanism for bacteria to gain advantage within host tissues.

Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa.

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Synthesis of and evaluation of lipid A modification by 4-substituted 4-deoxy arabinose analogs as potential inhibitors of bacterial polymyxin resistance.

Three sets of novel 4-deoxy-l-arabinose analogs were synthesized and evaluated as potential inhibitors of the bacterial resistance mechanism in which lipid A, on the outer membrane, is modified with 4-amino-4-deoxy-l-arabinose (l-Ara4N). One compound diminished the transfer of l-Ara4N onto lipid A. These results suggest that small molecules might be designed that would effect the same reversal of bacterial resistance observed in genetic knockouts.

trans splicing in Leishmania enriettii and identification of ribonucleoprotein complexes containing the spliced leader and U2 equivalent RNAs.

The 5' ends of Leishmania mRNAs contain an identical 35-nucleotide sequence termed the spliced leader (SL) or 5' mini-exon. The SL sequence is at the 5' end of an 85-nucleotide primary transcript that contains a consensus eucaryotic 5' intron-exon splice junction immediately 3' to the SL. The SL is added to protein-coding genes immediately 3' to a consensus eucaryotic 3' intron-exon splice junction. Our previous work demonstrated possible intermediates in discontinuous mRNA processing that contain the 50 nucleotides of the SL primary transcript 3' to the SL, the SL intron sequence (SLIS). These RNAs have a 5' terminus at the splice junction of the SL and the SLIS. We examined a Leishmania nuclear extract for these RNAs in ribonucleoprotein (RNP) particles. Density centrifugation analysis showed that the SL RNA is predominantly in RNP complexes at 60S, while the SLIS-containing RNAs are in complexes at 40S. We also demonstrated that the SLIS can be released from polyadenylated RNA by incubation with a HeLa cell extract containing debranching enzymatic activity. These data suggested that Leishmania enriettii mRNAs are assembled by bimolecular or trans splicing as has been recently demonstrated for Trypanosoma brucei. Furthermore, we determined the partial sequence of the Leishmania U2 equivalent RNA and demonstrated that it cosediments with the SL RNA at 60S in a nuclear extract. These RNP particles may be analogous to so-called spliceosomes that have been demonstrated in other systems.

Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo.

Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.

A multidimensional problem-oriented review and evaluation system.

The need for evaluation of psychiatric services is becoming more and more apparent as third-party coverage becomes more available. A variety of expensive and complex data and computer systems have been developed. Small institutions and programs, however, cannot afford them. The system described in this report uses diagnosis and a standardized problem list in order to define the treatment outcome of a variety of therapies delivered in an interdisciplinary setting. The system, which has been developed and is operating at low cost, satisfies a variety of needs.

Predicting readmission to the psychiatric hospital in a managed care environment: implications for quality indicators.

OBJECTIVE: This study examined predictors of hospital readmission to determine whether readmissions can serve as a quality indicator for an inpatient psychiatric service. METHOD: A series of 255 patients consecutively admitted to any of seven psychiatric hospitals in a regional managed care program were followed to determine whether they were readmitted within 6 months of discharge. Case managers assessed patients with the use of a reliable outcome management/decision support system designed for acute psychiatric services. RESULTS: Patients with greater impairment in self-care, more severe symptoms, and more persistent illnesses were more likely to be readmitted than other patients. Suicidal patients were less likely to be readmitted. There was no evidence to suggest that poor hospital outcome or premature discharge was associated with readmission either within 30 days or within 6 months. CONCLUSIONS: Although patients at risk for hospital admission can be identified, it does not appear that the success of the hospital intervention per se influences the likelihood of readmission. Use of readmission rates as quality indicators for hospital care providers is not recommended.

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.

Salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity.

Resistance to innate immunity is essential for salmonellae pathogenesis. The salmonellae PhoP/PhoQ regulators sense host environments to promote remodeling of the bacterial envelope. This remodeling includes enzymes that modify lipopolysaccharide (LPS). Modified LPS promotes bacterial survival by increasing resistance to cationic antimicrobial peptides and by altered host recognition of LPS.

New insights on molecular pathways utilized by salmonella species in cell binding.

Salmonella infections are a principal source of gastroenteritis and enteric fever in a variety of animals, including humans. An essential step in the development of Salmonella pathogenesis is the entry of bacteria into non-phagocytic cells, including those that line the intestinal epithelium. As a consequence of specific cues from the host intestinal micro-environment, Salmonella entry into the intestinal epithelium is the product of a multistep process that culminates in host cell membrane ruffling, and subsequent bacterial uptake. The events that trigger the internalization event appear to require an array of bacterial secreted proteins, exemplified by the formation of bacterial surface appendages (invasomes) which are important for the induction of host-cell signal transduction pathways that lead to membrane ruffling. In addition, during intestinal disease states induced by Salmonella typhimurium, transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial membrane. Current evidence indicates that the intestinal epithelium plays a key role in orchestrating the inflammatory response to surface attached S. typhimurium. In this review, we explore current insights on the molecular pathways utilized by Salmonella spp. in cell binding that are important not only in the processes of Salmonella internalization but also in the generation of signals which lead to active states of intestinal inflammation.

Hypoglycemia as a manifestation of sepsis.

Hypoglycemia has rarely been described as a clinical sign of severe bacterial sepsis. We recently encountered nine patients in whom hypoglycemia (mean serum glucose of 22 mg/dl) was associated with overwhelming sepsis. Clinical disease in these patients included pneumonia and cellulitis; in three patients, no focus of infection was apparent. Altered mental status, metabolic acidosis, leukopenia, abnormal clotting studies and bacteremia were common features in these cases. In four patients, no cause for hypoglycemia other than sepsis was present. In five patients, another possible metabolic cause for hypoglycemia was present (alcoholism in four and chronic renal insufficiency in one) although none had been observed to be hypoglycemic on previous hospitalizations. Streptococcus pneumoniae (three cases) and Hemophilus influenzae, type b, (two cases) were the most common pathogens, and the over-all mortality was 67 per cent. The mechanism(s) for hypoglycemia with sepsis is not well defined. Depleted glycogen stores, impaired gluconeogenesis and increased peripheral glucose utilization may all be contributing factors. Incubation of bacteria in fresh blood at room temperature does not increase the normal rate of breakdown of glucose suggesting that the hypoglycemia occurs in vivo. Hypoglycemia is an important sign of overwhelming sepsis that may be more common than has previously been recognized.

Chromosome location of four genes in Leishmania.

Chromosome-sized DNA molecules from Leishmania isolates (L. mexicana amazonensis, L. mexicana mexicana, L. chagasi, L. major, L. donovani, and L. braziliensis) were separated by orthogonal field alternation gel electrophoresis. The chromosome locations of four genes were mapped. The alpha-tubulin and rRNA genes each mapped to a single chromosome size class. The beta-tubulin and the 5'-spliced-leader-sequence genes were found on more than one chromosome size class and showed variation of hybridization profiles across species.

Transcriptional mapping of Leishmania enrietti tubulin mRNAs.

We have mapped the mRNAs for alpha- and beta-tubulins of Leishmania enriettii promastigotes and amastigotes and have demonstrated that each RNA contains a 35 nucleotide sequence on its 5' end which is not encoded contiguously with the rest of the message. Sequencing of the 5' end of the alpha- and beta-tubulin mRNAs revealed that this 35 nucleotide leader sequence is identical in both messages and that it is homologous to the spliced leader found on the 5' end of mRNAs in the related parasite Trypanosoma brucei. Additionally, we have sequenced regions of the alpha- and beta-tubulin genomic clones upstream from the mRNA encoding regions and have shown that the leader sequence is not encoded within these regions of DNA. Hence, Leishmania tubulin mRNAs may be synthesized by a discontinuous transcription process that links together the transcription products of two separate gene families, as suggested previously by others for the assembly of T. brucei mRNAs. Homologies between the Leishmania alpha- and beta-tubulin genes themselves and between these genes and the T. brucei alpha- and beta-tubulin genes have revealed sequences which may be important in synthesis or processing of Leishmania tubulin mRNAs.

Significant determinants of susceptibility to alcohol teratogenicity.

Typically, the rate of abusive drinking during pregnancy considerably exceeds the rates of fetal alcohol syndrome (FAS) and alcohol-related birth defects, suggesting that other factors may modify the impact of alcohol on the developing organism. Data in the literature supporting this susceptibility hypothesis are sparse. In this paper, two studies in different samples, using different analytic strategies to examine susceptibility to different adverse outcomes are presented. Among 176 pregnancies in which lowered birth weight for gestational age was detected as an effect attributable to frequent beer drinking, 27 infants weighted less than 2,700 grams and 149 weighed more. Using discriminant analysis to contrast these groups, lowered birth weight for gestational age was associated with black race and lower maternal weight and weight gain. The effects of these factors were additive with that of persistent alcohol exposure; no interactions were detected, but pregnancies with risks in addition to alcohol were more likely to yield growth-retarded infants. In a second study, pregnancies resulting in 25 FAS cases were contrasted with 50 controls. A four-factor model accounted for nearly two-thirds of the explainable variance in the occurrence of FAS. Adjusted for frequency of maternal drinking, chronic alcohol problems and parity, there was a sevenfold increase in risk for FAS among black infants. The findings from both studies are consistent with the susceptibility hypothesis and have potentially important implications for public health and clinical approaches to prevention, as well as for future research.

The treatment of dangerous patients in managed care. Psychiatric hospital utilization and outcome.

The legal criteria for civil commitment dictates that individuals must be mentally ill, and either a danger to themselves, a danger to others, or substantially impaired in their ability to provide for their basic needs. These criteria, which have been adopted as medical necessity criteria by managed care programs, may result in a change in the clinical mix of the psychiatric inpatient population. The present study assesses the incidence of dangerousness among psychiatric inpatients and compares dangerous and nondangerous patients in terms of characteristics and treatment outcomes. The results indicate that for a large regional managed care program, 30% of psychiatric inpatients have a history of dangerousness in the past year. Patients who are rated as dangerous to others during admission have higher rates of complications for treatment and psychiatric disorders such as residential and vocational instability, family disruption, and higher premorbid dysfunction. They are also more likely to engage in disruptive and aggressive behavior during their hospital stays. Despite the higher incidence of acute and long-term dysfunction for dangerous patients, their hospitalization length of stay was comparable to that of patients not rated as dangerous.