Administration of liposomal agents and blood clearance capacity of the mononuclear phagocyte system

As liposomes are cleared from the circulation to a substantial extent by the phagocytic cells of the mononuclear phagocyte system (MPS), there is a question whether administration of liposome-based therapeutic agents interferes with clearance of infectious organisms by the MPS from blood. In the present study, at first the effect of administration of three types of empty liposomes (devoid of drug), differing in blood residence time, on carbon clearance and bacterial clearance from blood was studied with mice. Classical liposomes (LIP A) and placebo liposomes with lipid composition as in AmBisome (LIP B) or as in Doxil (LIP C) were used. Liposomes were administered intravenously as a single dose. Second, the effect of multiple-dose administration of AmBisome on bacterial blood clearance was studied with rats. AmBisome was administered with two different dosage schedules. The blood clearance capacity of the MPS was monitored at different time points after the last liposome injection. It was shown that the carbon blood clearance capacity of the MPS was impaired only at a high lipid dose of empty classical liposomes. The bacterial blood clearance capacity was never impaired, not even after prolonged treatment with AmBisome administered in a clinically relevant regimen

Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis

Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine–aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets

Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.</p

An experimental Staphylococcus aureus carriage and decolonization model in rhesus macaques (Macaca mulatta)

Our human model of nasal colonization and eradication of S. aureus is limited by safety issues. As rhesus macaques are closely related to humans and natural hosts for S. aureus, we developed an experimental decolonization and inoculation protocol in these animals. Animals were screened for nasal carriage of S. aureus and 20 carriers were selected. Decolonization was attempted using nasal mupirocin (10 animals) or mupirocin plus trimethoprim/ sulfadiazine intramuscularly (10 animals) both once daily for 5 days, and checked by followup cultures for 10 weeks. Intranasal inoculation was performed with S. aureus strain 8325–4 in culture-negative animals. 11/20 animals, of which 5 received mupirocin and 6 the combination treatment, became culture-negative for S. aureus for 10 weeks and these 11 animals were subsequently inoculated. Swabs were taken once a week for 5 weeks to test for the presence of the inoculated strain. In 3 animals, strain 8325–4 was cultured from the nose 1 week after inoculation, indicating short-term survival of this strain only, a finding similar to that previously found in our human model. These data demonstrate that rhesus macaques may constitute a relevant animal model to perform S. aureus eradication and inoculation studies with relatively limited invasive handling of the animals

Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BμG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688

Early regular egg exposure in infants with eczema: a randomized controlled trial

BackgroundObservational studies suggest that early regular ingestion of allergenic foods might reduce the risk of food allergy.ObjectiveWe sought to determine whether early regular oral egg exposure will reduce subsequent IgE-mediated egg allergy in infants with moderate-to-severe eczema.MethodsIn a double-blind, randomized controlled trial infants were allocated to 1 teaspoon of pasteurized raw whole egg powder (n = 49) or rice powder (n = 37) daily from 4 to 8 months of age. Cooked egg was introduced to both groups after an observed feed at 8 months. The primary outcome was IgE-mediated egg allergy at 12 months, as defined based on the results of an observed pasteurized raw egg challenge and skin prick tests.ResultsA high proportion (31% [15/49]) of infants randomized to receive egg had an allergic reaction to the egg powder and did not continue powder ingestion. At 4 months of age, before any known egg ingestion, 36% (24/67) of infants already had egg-specific IgE levels of greater than 0.35 kilounits of antibody (kUA)/L. At 12 months, a lower (but not significant) proportion of infants in the egg group (33%) were given a diagnosis of IgE-mediated egg allergy compared with the control group (51%; relative risk, 0.65; 95% CI, 0.38-1.11; P = .11). Egg-specific IgG4 levels were significantly (P ConclusionInduction of immune tolerance pathways and reduction in egg allergy incidence can be achieved by early regular oral egg exposure in infants with eczema. Caution needs to be taken when these high-risk infants are first exposed to egg because many have sensitization already by 4 months of age.Debra J. Palmer, Jessica Metcalfe, Maria Makrides, Michael S. Gold, Patrick Quinn, Christina E. West, Richard Loh, and Susan L. Prescot

Genetic and metabolic links between the murine microbiome and memory.

BackgroundRecent evidence has linked the gut microbiome to host behavior via the gut-brain axis [1-3]; however, the underlying mechanisms remain unexplored. Here, we determined the links between host genetics, the gut microbiome and memory using the genetically defined Collaborative Cross (CC) mouse cohort, complemented with microbiome and metabolomic analyses in conventional and germ-free (GF) mice.ResultsA genome-wide association analysis (GWAS) identified 715 of 76,080 single-nucleotide polymorphisms (SNPs) that were significantly associated with short-term memory using the passive avoidance model. The identified SNPs were enriched in genes known to be involved in learning and memory functions. By 16S rRNA gene sequencing of the gut microbial community in the same CC cohort, we identified specific microorganisms that were significantly correlated with longer latencies in our retention test, including a positive correlation with Lactobacillus. Inoculation of GF mice with individual species of Lactobacillus (L. reuteri F275, L. plantarum BDGP2 or L. brevis BDGP6) resulted in significantly improved memory compared to uninoculated or E. coli DH10B inoculated controls. Untargeted metabolomics analysis revealed significantly higher levels of several metabolites, including lactate, in the stools of Lactobacillus-colonized mice, when compared to GF control mice. Moreover, we demonstrate that dietary lactate treatment alone boosted memory in conventional mice. Mechanistically, we show that both inoculation with Lactobacillus or lactate treatment significantly increased the levels of the neurotransmitter, gamma-aminobutyric acid (GABA), in the hippocampus of the mice.ConclusionTogether, this study provides new evidence for a link between Lactobacillus and memory and our results open possible new avenues for treating memory impairment disorders using specific gut microbial inoculants and/or metabolites. Video Abstract