Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a).

Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.

Deficient serum bactericidal activity against Escherichia coli in patients with cirrhosis of the liver.

The serum bactericidal activity (SBA) of cirrhotic patients was compared with that of normal individuals using the release of (51)Cr from radiolabeled Escherichia coli as the assay method. 80% (22/27) of patients were found to have deficient SBA against at least one of three smooth, serum-sensitive test strains of E. coli. Cirrhotic patients were found to have normal levels of serum lysozyme. Although some patients were mildly hypocomplementemic, this abnormality did not correlate with the presence of a bactericidal defect. Bactericidal antibody in normal and cirrhotics' sera was limited to the immunoglobulin (Ig)M class. Purified IgM from patients with deficient SBA against E. coli 0111 had lower concentrations of bactericidal antibody for that E. coli than did IgM from normal sera; the calculated bactericidal activity of total serum IgM was also lower. The bactericidal defect in cirrhotic serum could be completely corrected by either human antiserum to the homologous strain of E. coli or by purified, normal human IgM. However, because higher concentrations of IgM were required to restore normal SBA to a cirrhotic's serum than to agammaglobulinemic serum, there may be an inhibitor of bactericidal antibody in addition to a deficiency of bactericidal IgM antibody to E. coli in the serum of patients with cirrhosis. The bactericidal activity of the alternative complement pathway was also assessed. Sera from cirrhotic patients had no deficit in SBA attributable to the alternative complement pathway. In fact, in some, the activity of the alternative complement pathway was supernormal, compensating in part for the deficit in IgM-mediated SBA.

Diverse virulence traits underlying different clinical outcomes of Salmonella infection.

Salmonella strains have evolved to infect a wide variety of reptiles, birds, and mammals resulting in many different syndromes ranging from colonization and chronic carriage to acute fatal disease. Adaptation to a large number of different evolutionary niches has undoubtedly driven the high degree of phenotypic and genotypic diversity in Salmonella strains. Differences in LPS and flagellar structure generate the antigenic variation that is reflected in the more than 2,000 known serotypes. Moreover, variations of LPS structure affect the virulence of the strain. The differential expression of various fimbriae by Salmonella is likely to be due to the wide variety of mucosal surfaces that are encountered by various strains, and the host immune response may select for a different expression pattern. As with these surface structures, a variety of other important virulence determinants show a variable distribution in Salmonella strains and also serve to delineate the divergence of the Salmonella lineage from E. coli. The acquisition of the SPI-1 region may have represented the defining genetic event in the separation of the Salmonella and E. coli lineages. The SPI-1 cell invasion function allowed Salmonella to establish a separate niche in epithelial cells. The mgtC locus on SPI-3 is also present in all lineages and facilitates the adaptation of the bacteria to the low Mg2+, low pH environment of the endosome that results from SPI-1-mediated invasion. Subsequent acquisition of SPI-2 allowed Salmonella to manipulate the sorting of the endosome or phagosome, altering the intracellular environment and facilitating bacterial growth within infected cells. The ability to disseminate from the bowel and establish extraintestinal niches is promoted by the spv locus. Since Salmonella proliferates within macrophages and must avoid phagocytosis by neutrophils to establish a systemic infection, the spv genes appear to promote the macrophage phase of the disease process. Here the polymorphism of the spv locus is clearly demonstrated, since the serovars that cause most cases of nontyphoid bacteremia contain the spv genes. The absence of the spv genes from S. typhi is particularly puzzling and is a strong indication that the pathogenesis of typhoid fever is fundamentally different from that of bacteremia due to nontyphoid Salmonella. There is currently no genetic explanation for the phenotype of host adaptation or for the finding that only a few serovars cause the majority of human infections. Based on recent findings that multiple individual virulence genes have a variable distribution in Salmonella, it is unlikely that a single locus will be found to be responsible for these complex biological traits. Instead, a complicated combination of genes are likely to contribute to the overall virulence phenotype.

Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck.

We find that pADEO16, a recombinant cosmid carrying the rck gene of the Salmonella typhimurium virulence plasmid, when cloned into either rough or smooth Escherichia coli and Salmonella strains, confers high level resistance to the bactericidal activity of pooled normal human serum. The rck gene encodes a 17-kD outer membrane protein that is homologous to a family of virulence-associated outer membrane proteins, including pagC and Ail. Complement depletion, C3 and C5 binding, and membrane-bound C3 cleavage products are similar in strains with and without rck. Although a large difference in C9 binding was not seen, trypsin cleaved 55.7% of bound 125I-C9 counts from rough S. typhimurium with pADEO16, whereas only 26.4% were released from S. typhimurium with K2011, containing a mutation in rck. The majority of C9 extracted from rck strain membranes sediments at a lower molecular weight than in strains without rck, suggesting less C9 polymerization. Furthermore, SDS-PAGE analysis of gradient peak fractions indicated that the slower sedimenting C9-containing complexes in rck strains did not contain polymerized C9 typical of the tubular membrane attack complex. These results indicate that complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes.

A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion.

Pathogenic bacteria that penetrate the intestinal epithelial barrier stimulate an inflammatory response in the adjacent intestinal mucosa. The present studies asked whether colon epithelial cells can provide signals that are important for the initiation and amplification of an acute mucosal inflammatory response. Infection of monolayers of human colon epithelial cell lines (T84, HT29, Caco-2) with invasive strains of bacteria (Salmonella dublin, Shigella dysenteriae, Yersinia enterocolitica, Listeria monocytogenes, enteroinvasive Escherichia coli) resulted in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, IL-8, monocyte chemotactic protein-1, GM-CSF, and TNF alpha, as assessed by mRNA levels and cytokine secretion. Expression of the same cytokines was upregulated after TNF alpha or IL-1 stimulation of these cells. In contrast, cytokine gene expression was not altered after infection of colon epithelial cells with noninvasive bacteria or the noninvasive protozoan parasite, G. lamblia. Notably, none of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, IL-12p40, IFN-gamma, or significant levels of IL-1 or IL-10 in response to the identical stimuli. The coordinate expression of IL-8, MCP-1, GM-CSF and TNF alpha appears to be a general property of human colon epithelial cells since an identical array of cytokines, as well as IL-6, also was expressed by freshly isolated human colon epithelial cells. Since the cytokines expressed in response to bacterial invasion or other proinflammatory agonists have a well documented role in chemotaxis and activation of inflammatory cells, colon epithelial cells appear to be programmed to provide a set of signals for the activation of the mucosal inflammatory response in the earliest phases after microbial invasion.

Physical and genetic mapping of the Salmonella dublin virulence plasmid pSDL2. Relationship to plasmids from other Salmonella strains.

Plasmids of approximately 80 kb in size are found in nearly all clinical isolates of Salmonella dublin and are believed to be essential for virulence. We have shown previously that the 80-kb plasmid pSDL2 is required for the S. dublin Lane strain to establish a lethal systemic infection in BALB/c mice after oral or intraperitoneal inoculation. We now present a physical and genetic characterization of pSDL2. We have established a complete restriction endonuclease cleavage map of pSDL2 for five enzymes: Xba I, Bam HI, Xho I, Sal I, and Hind III. The region specifying autonomous replication has been localized to a 10.5-kb region of the Sal I A fragment by subcloning on the vector pBR322. Using transposon insertion mutagenesis with Tn5-oriT, a region encoding the virulence phenotype has been mapped within a 6.4-kb portion of the Sal I B fragment. Deletions generated by partial Eco RI restriction digestion demonstrate that at least 50 kb of the plasmid DNA are not required for replication or virulence functions, confirming the map location of these phenotypes. Plasmids of different sizes and restriction patterns were found in mouse virulent strains of S. dublin Vi+, S. enteritidis, and S. choleraesuis. By Southern hybridization, these putative virulence plasmids share a common 4-kb Eco RI fragment with the virulence region of pSDL2, and the plasmids from S. dublin Vi+ and S. enteritidis were shown to express mouse virulence comparable to pSDL2.

Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin h synthase-2 expression and prostaglandin E2 and F2alpha production.

Increased intestinal fluid secretion is a protective host response after enteric infection with invasive bacteria that is initiated within hours after infection, and is mediated by prostaglandin H synthase (PGHS) products in animal models of infection. Intestinal epithelial cells are the first host cells to become infected with invasive bacteria, which enter and pass through these cells to initiate mucosal, and ultimately systemic, infection. The present studies characterized the role of intestinal epithelial cells in the host secretory response after infection with invasive bacteria. Infection of cultured human intestinal epithelial cell lines with invasive bacteria, but not noninvasive bacteria, is shown to induce the expression of one of the rate-limiting enzymes for prostaglandin formation, PGHS-2, and the production of PGE2 and PGF2alpha. Furthermore, increased PGHS-2 expression was observed in intestinal epithelial cells in vivo after infection with invasive bacteria, using a human intestinal xenograft model in SCID mice. In support of the physiologic importance of epithelial PGHS-2 expression, supernatants from bacteria-infected intestinal epithelial cells were shown to increase chloride secretion in an in vitro model using polarized epithelial cells, and this activity was accounted for by PGE2. These studies define a novel autocrine/paracrine function of mediators produced by intestinal epithelial cells in the rapid induction of increased fluid secretion in response to intestinal infection with invasive bacteria.

Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis.

Chlamydia species infect epithelial cells at mucosal surfaces, and are major causes of sexually transmitted diseases. Infection is characterized by inflammation which is exacerbated upon reinfection, ultimately leading to tissue damage and scarring. Although central for the development of disease manifestations, little is known about the mechanisms that initiate and sustain the inflammatory response to Chlamydia. Infection of cervical and colonic epithelial cells with Chlamydia trachomatis and Chlamydia psittaci is shown in the present studies to upregulate mRNA expression and secretion of the proinflammatory cytokines IL-8, GRO alpha, GM-CSF, and IL-6. In contrast to the rapid, but transient, cytokine induction following infection with other invasive bacteria, the epithelial cytokine response to Chlamydia was delayed until 20-24 h after infection, persisted throughout the chlamydial growth cycle (2-4 d), and required bacterial protein synthesis. Moreover, epithelial cell lines and primary endocervical epithelial cells released IL-1alpha after Chlamydia infection, and increased secretion of the proinflammatory cytokines could be inhibited by anti-IL-1alpha. This suggests that IL-1alpha, released following lysis of infected epithelial cells, may amplify the inflammatory response by stimulating additional cytokine production by noninfected neighboring cells. These findings suggest a novel pathophysiologic concept wherein the acute host response to Chlamydia at mucosal surfaces is primarily initiated and sustained by epithelial cells, the first and major targets of chlamydial infection.

Intestinal epithelial cells as watchdogs for the natural immune system.

Intestinal epithelial cells secrete a spectrum of chemoattractant and proinflammatory cytokines after invasion by bacteria. We suggest the novel concept that epithelial cells not only act as a mechanical barrier to invasive bacteria, but that they also signal the presence of invasive pathogens to the mucosal immune and inflammatory cells.

Growth-phase regulation of plasmid virulence genes in Salmonella.

Virulence genes in the genus Salmonella are regulated by growth phase and by environmental signals, which allows a sequential program of expression during infection. Conditions that promote the expression of loci required in systemic infection, including the plasmid-encoded spv genes, are the opposite of the factors that induce genes involved in the invasion of epithelial cells in the gastrointestinal tract.

High-fat, high-cholesterol diet increases the incidence of gastritis in LDL receptor-negative mice.

Transgenic and knockout mice are widely used as models for atherogenesis studies. While developing a Helicobacter infection model in LDL receptor-negative (LDLR(-/-)) mice, we noticed that mice fed a high-fat, high-cholesterol diet often contracted gastritis independent of infection. To further investigate this finding, we studied 27 male and 18 female LDLR(-/-) mice fed high-fat, 1% or 1.25% cholesterol diets for 3 to 4 months. The extent of atherosclerosis was morphometrically analyzed in the whole aorta, and the degree of gastric inflammation was scored histologically in hematoxylin-eosin-stained stomach sections. The autoantibody titers to epitopes of oxidized LDL were also measured. Mice fed high-fat, high-cholesterol diets had a significantly higher incidence of gastritis than mice fed normal chow, 62% versus 5%, respectively (P<0.0001). This effect was specific for LDLR(-/-) mice, because no difference in gastritis was found in wild-type mice fed either diet. Animals with gastritis showed slightly more atherosclerosis than animals without gastritis: 16.3+/-6.4% versus 12.8+/-3.4% in males and 9.4+/-3.5% versus 6.5+/-3.3% in females. Cholesterol-fed mice also had significantly higher IgG autoantibody titers against modified LDL than normal chow-fed animals, but no difference was seen between the gastritis and nongastritis groups. We conclude that the standard high-fat, high-cholesterol diet commonly used in many murine models to induce atherosclerosis increased the incidence of gastritis significantly in LDLR(-/-) mice.

Polymorphonuclear leukocytes and innate immunity to Salmonella infections in mice.

Neutropenia makes normal mice more susceptible to infection with spv (+) but not spv (-) Salmonella dublin. This shows the important role of polymorphonuclear leukocytes in resistance to Salmonella that can grow in host macrophages. Polymorphonuclear leukocytes, part of the innate immune system, kill Salmonella in a complement-dependent manner, and work in concert with macrophages.

Human infection with Salmonella dublin.

Twenty-seven cases of human infection with Salmonella dublin were identified over a 12-year period at the University of California at San Diego-affiliated hospitals. Important epidemiologic risk factors were the ingestion of unpasteurized dairy products or treatment with nutritional therapy that included raw calf-liver extracts. Nearly all patients had underlying chronic diseases. Like Salmonella choleraesuis, S. dublin infections were associated with a high incidence of bacteremia (91%), metastatic sites of infection (30%), and mortality (26%) relative to other non-typhoidal Salmonellae. This pattern of disease expression may be related to a plasmid-encoded virulence factor common to both of these organisms.

Age-related changes in isolated rat hepatocytes. Comparison of size, morphology, binucleation, and protein content.

Hepatocytes isolated from 6-, 12-, 18-, and 30-month-old female Fischer F344 rats were examined by scanning electron microscopy. No significant change in cell size with age was observed. However, the surface morphology of the cells isolated from the older animals exhibited a significant increase in surface folds. This feature did not exceed 10% of the cell population until 12 months of age and continued to increase to 31% of the cells in 30-month-old rats. From 6 to 12 months of age, there was a significant increase in protein content of the hepatocytes. No further increase in protein content occurred during senescence. An increase in percentage of binuclear cells occurred after 24 months of age. Because ploidy and binucleation increase with increasing age, it appears that the nuclear/cytoplasmic ratio changes as a function of age.

Fluconazole in the treatment of chronic pulmonary and nonmeningeal disseminated coccidioidomycosis. NIAID Mycoses Study Group.

PURPOSE: To determine the efficacy and safety of fluconazole as treatment for coccidioidomycosis. PATIENTS AND METHODS: This was a multicenter, open-label, single-arm study. Of 78 patients enrolled, 22 had soft-tissue, 42 had chronic pulmonary, and 14 had skeletal coccidioidomycosis. Forty-nine had at least one concomitant disease, 7 of whom had HIV infection. Patients were given oral fluconazole 200 mg/d. Nonresponders were increased to 400 mg/d. Treatment courses were long: a mean of 323 +/- 230 days at 200 mg and 433 +/- 178 days at 400 mg. Predefined assessment of disease-related abnormalities was performed at the time of enrollment and repeated at least every 4 months. A satisfactory response was defined as any reduction of baseline abnormality by month 4 and at least 51% reduction by month 8. RESULTS: Among 75 evaluable patients, a satisfactory response was observed in 12 (86%) of the 14 patients with skeletal, 22 (55%) of the 40 patients with chronic pulmonary, and 16 (76%) of the 21 patients with soft-tissue disease. Five patients (7%) required modification of treatment due to toxicity. Forty-one patients who responded were followed off drug. Fifteen (37%) of them experienced reactivation of infection. CONCLUSION: Fluconazole 200 or 400 mg/d is well tolerated and a moderately effective treatment for chronic pulmonary or nonmeningeal disseminated coccidioidomycosis. The relapse rate following therapy is high. Treatment trials with higher doses appear warranted. The relative efficacy of fluconazole versus other azoles or amphotericin B remains unknown.

Fluconazole in the treatment of persistent coccidioidomycosis.

Fluconazole is one of the new antifungal triazoles undergoing clinical trials. We used fluconazole at a dose of 50 or 100 mg/day in an open trial for the treatment of patients with persistent coccidioidomycosis. Fourteen patients were enrolled and treated for a mean of 13 +/- 7 months. Two failed to respond. Of the 12 who responded, one reactivated while being treated, and one died of myocardial infarction after successful treatment of his fungal infection; six had relapses from nine days to 15 months after treatment was stopped. Only four patients are asymptomatic at a mean of 14 +/- 3 months after cessation of treatment. Fluconazole is well tolerated at this dose. In view of its low toxicity, the partial clinical efficacy observed, and the high recurrence rate of chronic coccidioidal infection, it would be justified to try higher doses.

Plantar compartmental infection in the diabetic foot. The role of computed tomography.

This review discusses the role of computed tomography (CT) in the evaluation of extent of plantar soft tissue infection in the diabetic foot. CT abnormalities are correlated with conventional radiography, results of preoperative aspiration cultures, intraoperative assessment, and bone, gallium, and 111In-leukocyte scan findings. Plantar soft tissue disease respects compartmental boundaries in general, with transcompartmental spread possible along musculotendinous units that normally transgress the intervening fascial septae. CT correlates well with the extent of infection as determined by other modalities, but cannot precisely predict its proximal boundary due to gradual transition between unequivocally abnormal and normal tissue. CT may be useful in establishing an appropriate level for contemplated amputation and can detect extension of superficial diabetic foot infections at an earlier stage than existing clinical methods, potentially resulting in less extensive surgical procedures.

Nosocomial infections in HIV-infected patients: preliminary results from a multicenter surveillance system (1989-1995).

OBJECTIVE: To describe the characteristics of and trends in nosocomial infection among human immunodeficiency virus (HIV)-infected patients. DESIGN: Multicenter prospective cohort study. SETTING/PATIENTS: HIV-infected patients were enrolled at time of first inpatient admission at five Veterans' Administration Medical Centers (VAMCs). RESULTS: As of March 1995, 2,541 patients with 6,625 inpatient admissions had been monitored in the five VAMCs. A total of 530 nosocomial infections were detected using standard Centers for Disease Control and Prevention definitions. Overall distribution by infection site was 31% for primary bloodstream infections (BSIs), 28% for urinary tract infections, 15% for pneumonia, and 26% for all other sites. Of BSIs, 63% were central line-associated bloodstream infections (CLABs). The rate of CLABs per 1,000 central line days was 6.5 (range, 2.3-8.3) for all patients from participating hospitals, similar to the median CLAB rate of 6.0 for patients in medical intensive-care units (ICUs) of National Nosocomial Infections Surveillance (NNIS) System hospitals from January 1990 through September 1994. For ICU-specific CLABs, the rate from hospitals reporting at least one ICU CLAB was 12.7 (range, 12.1-13.1), comparable to the 90th percentile of NNIS hospital medical ICUs (13.1). Staphylococcus aureus, associated with 35% of BSIs, was the most common nosocomial BSI pathogen. Our data demonstrated the following: 13 (10%) of 134 patients with CD4 counts > or = 200 cells/mm3 had a CLAB, compared with 61 (6%) of 1,011 patients with CD4 counts < 200 cells/mm3, P = .08; the per-day risk of CLABs did not change with increased duration of catheterization (P = .4); and the per-day risk of a temporary (ie, short-term) CLAB was greater than that of a permanent CLAB (P < .001). CONCLUSIONS: The data suggest that HIV-infected patients were at higher risk of acquiring a BSI than were patients in the NNIS population; patients with CD4 counts > or = 200 cell/mm3 and temporary central lines were at increased risk for BSI, perhaps reflecting widespread prophylaxis with trimethoprim-sulfamethoxazole among patients with CD4 counts < 200 cells/mm3, and, in contrast to most studies, S aureus, not coagulase-negative Staphylococcus, was the most common BSI pathogen.

Infections and infection risk in residents of long-term care facilities: a review of the literature, 1970-1984.

We reviewed the English-language peer-reviewed journals and the Centers for Disease Control's Morbidity and Mortality Weekly Reports between 1970 and 1984 presenting information about infections and infection risk in residents of long-term care facilities. More than 50 articles met review criteria. Approximately one third of the articles were reports of outbreaks, primarily of respiratory and gastrointestinal infections. Seven articles reported rates for several infection sites, but most rates were not directly comparable to one another because numerators and/or denominators were different. Many of the studies have been done in Veterans Administration hospitals with largely male populations, which may limit their applicability to freestanding long-term care facilities with largely female clients. This review establishes the need for high-quality observational studies of infections in long-term care facilities. Such studies are needed before intervention studies can be done to measure the effect of manipulation of risk factors on infection outcome.

Non-pulmonary Rhodococcus equi infections in patients with acquired immune deficiency syndrome (AIDS).

Rhodococcus equi, formerly known as Corynebacterium equi, was isolated repeatedly from the blood of two patients with the acquired immune deficiency syndrome (AIDS). Neither of the patients had pneumonia while they were bacteraemic, whereas pneumonia has been present in all previously reported cases of human infection with R equi. One of our patients had diarrhoea and the organism was isolated from a stool culture; the other patient had a large granulomatous soft tissue mass in his pelvis caused by R equi. Both isolates were resistant to penicillin and one produced a beta-lactamase. Both patients were treated with vancomycin but only one recovered.