Bacteriophage populations mirror those of bacterial pathogens at sites of infection.

Bacteriophages, viruses that parasitize bacteria, are known to be abundant at sites of bacterial colonization, but the relationship between phages and bacteria at sites of infection is unclear. Bacteriophages are highly specific to their bacterial host species, and so we hypothesize that phage populations would mirror those of bacterial pathogens within infected tissues. To test this, here we study publicly available cell-free DNA (cfDNA) generated using next-generation sequencing of infected bodily fluids, including urine, joint fluid, peritoneal fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, and abscess fluid, as well as uninfected control samples. These were analyzed using a computational pipeline for identifying bacteriophage sequences in cfDNA. We find that bacteriophage sequences are present in both infected and uninfected bodily fluids and represent a variety of bacteriophage morphologies and bacterial hosts. Additionally, phages from Escherichia coli, Streptococcus, and Staphylococcus aureus are overrepresented both in terms of proportion and diversity in fluids infected with these same pathogens. These data indicate that phages reflect the relative abundance of their bacterial hosts at sites of infection. Bacteriophage sequences may help inform future investigative and diagnostic approaches that utilize cell-free DNA to study the microbiome within infected tissues. IMPORTANCE Bacteriophages are an active area of investigation in microbiome research, but most studies have focused on phage populations at sites of bacterial colonization. Little is known about bacteriophage ecology at sites of active infection. To address this gap in knowledge, we utilized a publicly available data set to study bacteriophage populations in cell-free DNA collected from sites of infection. We find that phages reflect the relative abundance of their bacterial hosts at sites of infection. These studies may lead to future investigative and diagnostic approaches that incorporate phages as well as bacterial cell-free DNA.

The Extra-Islet Pancreas Supports Autoimmunity in Human Type 1 Diabetes.

In autoimmune Type 1 diabetes (T1D), immune cells progressively infiltrate and destroy the islets of Langerhans - islands of endocrine tissue dispersed throughout the pancreas. However, it is unclear how this process, called 'insulitis', develops and progresses within this organ. Here, using highly multiplexed CO-Detection by indEXing (CODEX) tissue imaging and cadaveric pancreas samples from pre-T1D, T1D, and non-T1D donors, we examine pseudotemporal-spatial patterns of insulitis and exocrine inflammation within large pancreatic tissue sections. We identify four sub-states of insulitis characterized by CD8 + T cells at different stages of activation. We further find that exocrine compartments of pancreatic lobules affected by insulitis have distinct cellularity, suggesting that extra-islet factors may make particular lobules permissive to disease. Finally, we identify "staging areas" - immature tertiary lymphoid structures away from islets where CD8 + T cells appear to assemble before they navigate to islets. Together, these data implicate the extra-islet pancreas in autoimmune insulitis, greatly expanding the boundaries of T1D pathogenesis.

Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media.

Chronic suppurative otitis media (CSOM) is a widespread, debilitating problem with poorly understood immunology. Here, we assess the host response to middle ear infection over the course of a month post-infection in a mouse model of CSOM and in human subjects with the disease. Using multiparameter flow cytometry and a binomial generalized linear machine learning model, we identified Ly6G, a surface marker of mature neutrophils, as the most informative factor of host response driving disease in the CSOM mouse model. Consistent with this, neutrophils were the most abundant cell type in infected mice and Ly6G expression tracked with the course of infection. Moreover, neutrophil-specific immunomodulatory treatment using the neutrophil elastase inhibitor GW 311616A significantly reduces bacterial burden relative to ofloxacin-only treated animals in this model. The levels of dsDNA in middle ear effusion samples are elevated in both humans and mice with CSOM and decreased during treatment, suggesting that dsDNA may serve as a molecular biomarker of treatment response. Together these data strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection in CSOM and suggest that immunomodulatory strategies may benefit drug-tolerant infections for chronic biofilm-mediated disease.

Quantitative assessment of bacterial growth phase utilizing flow cytometry.

Flow cytometry is currently underutilized for bacterial phenotyping and standard microbiological techniques do not provide phenotypic information about the state of the bacterial disease. Pseudomonas aeruginosa is a human pathogen of increased importance in public health due to both the ability to cause chronic diseases and the prevalence of functionally different subsets that can be difficult to treat and diagnose. In the present study, we used flow cytometry to analyze the growth phase of P. aeruginosa. A simple method for single cell quantitative detection of bacterial biofilm and planktonic cells was established with a combination of membrane permeable (SYTO 60) and impermeable (TOTO-1) dyes plus the addition of polystyrene counting beads. The specificity of the dye combination for biofilm detection was determined by comparison with impaired biofilm forming strains of P. aeruginosa LasI/RhlI-/- and ∆PfPhage. Results suggest that flow cytometric bacterial phenotyping serves as an expandable platform that may be useful for enumeration of population level variation in P. aeruginosa studies.

The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.

Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan (HA) synthesis in mouse models of cancer, autoimmunity and a variety of other inflammatory disorders where HA has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after 1 week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a 1-week loading period on the drug is required for most protocols. At steady state, more than 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulphated metabolite, 4-methylumbelliferyl sulphate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0·65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice.

Tissue integrity signals communicated by high-molecular weight hyaluronan and the resolution of inflammation.

The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior. Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory. We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a novel form of pattern recognition that recognizes intact tissues and communicates "tissue integrity signals" that promote resolution of local immune responses.

Evaluation of in vivo T cell kinetics: use of heavy isotope labelling in type 1 diabetes.

CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.

Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes.

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

Reconstructing the complex evolutionary history of hepatitis B virus.

A detailed analysis of the evolutionary history of hepatitis B virus (HBV) was undertaken using 39 mammalian hepadnaviruses for which complete genome sequences were available, including representatives of all six human genotypes, as well as a large sample of small S gene sequences. Phylogenetic trees of these data were ambiguous, supporting no single place of origin for HBV, and depended heavily on the underlying model of DNA substitution. In some instances genotype F, predominant in the Americas, was the first to diverge, suggesting that the virus arose in the New World. In other trees, however, sequences from genotype B, prevalent in East Asia, were the most divergent. An attempt was also made to determine the rate of nucleotide substitution in the C open reading frame and then to date the origin of HBV. However, no relationship between time and number of substitutions was found in two independent data sets, indicating that a reliable molecular clock does not exist for these data. Both the pattern and the rate of nucleotide substitution are therefore complex phenomena in HBV and hinder any attempt to reconstruct the past spread of this virus.

Recombination between sequences of hepatitis B virus from different genotypes.

A comparison of 25 hepatitis B virus (HBV) isolates for which complete genome sequences are available revealed two that occupied different positions in phylogenetic trees reconstructed from different open reading frames. Further analysis indicated that this incongruence was the result of recombination between viruses of different genomic and antigenic types. Both putative recombinants originated from geographic regions where multiple genotypes are known to cocirculate. A search of the sequence databases showed evidence of similar intergenotypic recombinants. These observations indicate that recombination between divergent strains may represent an important source of genetic variation in HBV.