Persistent Staphylococcus aureus isolates from two independent cases of bacteremia display increased bacterial fitness and novel immune evasion phenotypes.

Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.

Influence of temperature on complement-dependent immune damage to liposomes.

Maximal release of trapped liposomal glucose, in the presence of saturating amounts of liposomal antigen (galactocerebroside), antiserum (anti-galactocerebroside), and complement, was dependent on temperature. At lower temperatures (20--25 degrees C), maximal glucose release was inversely related to liposomal phospholipid fatty acyl chain length (dimyristoyl phosphatidylcholine > dipalmitoyl phosphatidylcholine > distearoyl phosphatidylcholine > sphingomyelin). At higher temperatures (32--35 degrees C) a limiting plateau of glucose release, at approx. 60%, was reached, or approached, by all preparations. Sphingomyelin liposomes still released less glucose than those prepared from other phospholipids, even at 35 degrees C. The titers of antiserum and complement (ABL50/ml and CL50/ml) were dependent on temperature, and differences based on liposomal phospholipid fatty acyl chain length were observed. Analysis of antiserum and complement-dependence on temperature, and on phospholipid type, revealed that although antibody binding to galactocerebroside undoubtedly was subject to steric hindrance due to interference by surrounding phospholipids at 20--25 degrees C, steric hindrance did not play a major role in blocking antibody binding above 32 degrees C.

Binding of choleragen and anti-ganglioside antibodies to gangliosides incorporated into preformed liposomes.

Exogenously added gangliosides were taken up and incorporated into liposomes just as they are incorporated into cells. Ganglioside GM1 was rapidly taken up by liposomes containing dimyristoyl- or dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate. When incubated with a wide range of GM1 concentrations for 18 h, the liposomes incorporated about 10% of the added ganglioside. The rate of GM1 uptake by preformed liposomes was both time- and temperature-dependent. The liposomes also incorporated other gangliosides to a similar extent. The GM1 taken up by preformed liposomes was predominantly located on the outer surface of the liposomes and did not appear to be internalized into the inner half of the lipid bilayer. Liposomes containing GM1 added after liposome formation bound as many anti-GM1 antibodies and as much choleragen as liposomes having GM1 added during the formation of the lipid bilayers. Thus, preformed liposomes sensitized by incubation with GM1 are a good model system for studying the interactions of antibodies and toxins with membrane-associated gangliosides.

Influence of vesicle size on complement-dependent immune damage to liposomes.

Complement-dependent antibody-mediated damage to multilamellar lipid vesicles (MLVs) normally results in a maximum release of 50-60% of trapped aqueous marker. The most widely accepted explanation for this is that only the outermost lamellae of MLVs are attacked by complement. To test this hypothesis, complement damage to two different types of large unilamellar vesicles (LUVs), large unilamellar vesicles prepared by the reverse-phase evaporation procedure (REVs) and large unilamellar vesicles prepared by extrusion techniques (LUVETs), were determined. In the presence of excess antibody and complement the LUVs released a maximum of only approx. 25 to 40% of trapped aqueous marker, instead of close to 100% that would be expected. Since small unilamellar vesicles apparently differ from LUVs in that they can release 100% of trapped aqueous marker it appeared that the size of the vesicles was an important factor. Because of these observations the influence of MLV size on marker release was examined. Three populations of MLVs of different sizes were separated by a fluorescence activated cell sorter. Assays of the separated MLV populations showed that the degree of complement-dependent marker release was inversely related to MLV size. No detectable glucose was taken up by MLVs when glucose was present only outside the liposomes during complement lysis. Our results can all be explained by the closing, or loss, of complement channels. We conclude that complement channels are only transiently open in liposomes, and that loss of channel patency may be due to either channel closing or to loss of channels.

Liposome spin immunoassay: a new sensitive method for detecting lipid substances in aqueous media.

A new sensitive immunoassay procedure is described for quantitative detection of glycolipids and other lipids in aqueous media. As with other immunoassays specific antiserum is first reacted with the free lipid hapten. The amount of antibody activity remaining is measured by assaying the release, in the presence of complement, of spin label marker from liposomes containing the same lipid hapten. Using this method, 2.6 pmol of aqueous Forssman hapten was detected, and the sensitivity could be increased further.

The dilute phospholipid APTT: a sensitive assay for verification of lupus anticoagulants.

A simple sensitive method for verification of lupus anticoagulants utilizing dilution of phospholipid in the activated partial thromboplastin time (APTT) system is described. Patient plasma, mixed with an equal volume of normal plasma, is activated with micronized silica. To this mixture are added different dilutions of Thrombofax and then calcium chloride. Clotting times are plotted linearly against the logarithm of the phospholipid dilutions and slopes are calculated by regression analysis. In this assay the mean negative slope of 19 plasmas that contained anti-phospholipid activity was five times greater than those of normal plasma or those obtained from patients having single or multiple coagulation factor deficiencies such as those induced by warfarin. The assay can be modified to test heparinized plasmas. Thus, it is a sensitive means by which to verify the presence of lupus anticoagulants in patients who have congenital or acquired factor deficiencies or who are receiving anticoagulant therapy.

Liposomes containing lipid A: a potent nontoxic adjuvant for a human malaria sporozoite vaccine.

Liposomes containing lipid A have been developed as adjuvants for inducing humoral immunity to synthetic antigens containing repeat sequence epitopes from the circumsporozoite protein of Plasmodium falciparum. Preclinical studies demonstrated that liposomes containing lipid A and encapsulated antigen could overcome immunosuppression observed with antigen alone. When liposomes containing lipid A were adsorbed with aluminum hydroxide (alum), further stimulation of humoral immunity against encapsulated antigen was observed in animals. In the presence of huge doses of liposomal lipid A pyrogenicity was not observed and adjuvant activity was enhanced. A phase I human clinical trial has been initiated utilizing a vaccine containing a synthetic recombinant antigen and monophosphoryl lipid A in liposomes and nonliposomal alum as a further adjuvant. Preliminary results confirm that the vaccine lacks significant acute toxicity in humans and causes very strong specific humoral immunity against the appropriate epitopes of the target antigen.

Murine IgG subclass antibodies to antigens incorporated in liposomes containing lipid A.

The IgG subclass responses to antigens incorporated in liposomes containing lipid A were investigated using a synthetic malarial antigen (SPf66) and cholera toxin (CT). The antigen-specific IgG subclass response was determined in BALB/c mice immunized with either: (a) SPf66 encapsulated in liposomes containing lipid A, (b) CT bound to the surface of liposomes containing lipid A, or (c) both encapsulated SPf66 and surface-bound CT in the same liposomes. In each case the antibodies to SPf66, CT and lipid A demonstrated an IgG2a predominance. Liposomes containing lipid A not only increased the magnitude of the antibody response to liposomal antigens but elicited predominantly IgG2a subclass antibodies as well.

Epidemiological study of complete and partial hydatidiform mole in Abu Dhabi: influence age and ethnic group.

An unmatched case control study of molar pregnancy was carried out at this hospital between 1978 and 1987 to investigate the influence of maternal age and ethnic group on the incidence of complete and partial hydatidiform mole. The age specific incidence of complete mole was minimal between the ages of 30 and 34 years (relative risk 1), showed a minor peak in teenagers (relative risk 3.1, 95% confidence interval 6.5-1.4), and a major peak in those of 35 years and over. Between 35 and 39 years the relative risk was 2.5 (95% CI 6.2-1.0) and at 40 years or more the relative risk was 9.8 (95% CI 28.9-3.3). No age group showed a significantly increased risk of partial mole. The women of Abu Dhabi had increased risks of both forms of molar pregnancy relative to women in Nottingham, England (relative risk 1): the risk of complete mole was increased threefold (95% CI 4.2-2.2) and that of partial mole twofold (95% CI 4.0-1.2). The increased risk of complete mole was greatest in Gulf Arabs (mainly Omanis and Yemenis) who had a sixfold increase in crude relative risk (95% CI 10.7-3.5). The increased risks of complete mole associated with maternal ethnic group remained after adjustment for maternal age distribution.

Novel adjuvant strategies for experimental malaria and AIDS vaccines.

Adjuvant research has improved the ability of biotechnology to generate novel vaccines. Numerous strategies for enhancing the immunogenicity of synthetic peptides and proteins have been identified. This overview focuses on adjuvant development and vaccine delivery systems that provide new tools for amplifying the effectiveness of ongoing malaria and AIDS vaccine development programs. In addition, some of the complex challenges and issues that have become associated with the delivery of modern vaccines in man are outlined. As adjuvant research continues to open new opportunities in vaccine development, there is renewed expectation that further generations of safe and potent vaccines will be possible against a broad spectrum of infectious agents and cancer.

Liposomal subunit vaccines: effects of lipid A and aluminum hydroxide on immunogenicity.

Protein and peptide antigens frequently are only slightly immunogenic when utilized alone in vaccines. Formulation of these antigens in a carrier vehicle, particularly when an adjuvant is included, often results in markedly enhanced immune responses. Encapsulation of peptide and protein antigens in liposomes generally results in a relatively slight enhancement of antibody production compared with that observed with the antigen alone. However, when lipid A is included in the liposomes, immunogenicity is markedly increased compared both with antigen alone and with antigen encapsulated in liposomes lacking lipid A. The enhancement of the immune response caused by lipid A is dependent on the liposomal lipid A dose. Aluminum salts, such as aluminum hydroxide and aluminum phosphate, act as adjuvants for some antigens and are used in a variety of human vaccines. When liposomes containing encapsulated protein or peptide antigens were adsorbed with aluminum hydroxide, an enhancement of the antibody response was observed with some antigens, whereas with other antigens the presence of aluminum hydroxide either had no effect or resulted in a diminished antibody response. Immunogenicity of protein and peptide antigens can be enhanced by formulation in liposomes containing lipid A and, depending on the antigen, can be enhanced further by adsorption of the liposomal antigen formulation with aluminum salts.

Predicting hospital mortality in patients with acute myocardial infarction.

BACKGROUND Patients who have a myocardial infarction are a heterogeneous group. If those at risk for early mortality could be readily identified, it would provide a more solid basis for management decisions. Although past research has explored factors associated with mortality, findings are inconsistent. Variables have also been combined into prognostic indices, but these tools have yet to be evaluated adequately. OBJECTIVES To determine factors predictive of hospital mortality in patients with acute myocardial infarction, and to examine the usefulness of two severity-of-illness indices. METHODS The medical records of 392 patients diagnosed with acute myocardial infarction who had undergone coronary angiography during 1989 at a university medical center were reviewed. RESULTS Overall mortality was 9.4% (n = 37). Logistic regression analysis demonstrated that history of myocardial infarction, cardiogenic shock, age, left ventricular ejection fraction, and the number of occluded coronary vessels were significantly associated with hospital mortality in patients with acute myocardial infarction. The two severity-of-illness indices were significant predictors of mortality, although sensitivity, specificity, and predictive values varied. A formula for determining the probability of mortality, based on logistic regression analysis, is also presented. CONCLUSIONS Five factors were found to predict hospital mortality. The two severity-of-illness indices were moderately useful in predicting mortality. Unlike previous indices that did not incorporate currently available diagnostic data, the new formula included data from coronary angiography and nuclear scans. Although this formula requires validation on independent samples of patients with myocardial infarction, the findings of this study advance clinicians' ability to predict patient outcome.

Amyloidosis in Hodgkin's disease: a Scottish survey.

Hodgkin's disease is a rare cause of secondary amyloidosis. In Scotland in the period 1961 to 1974 four patients in whom these 2 conditions were associated have been identified. In one of these the presence of amyloidosis was an unexpected finding at a staging laparotomy for Hodgkin's disease. The clinical and pathological features are summarised. The development of amyloid disease is usually suspected by the finding of proteinuria, which is rarely present in uncomplicated Hodgkin's disease. A distinction is made between a nephrotic syndrome due to glomerulopathy, which is an early complication of Hodgkin's disease and improves with treatment of the primary condition, and the nephrotic syndrome due to amyloidosis which occurs late in the course of the illness and is irreversible and rapidly progressive.