Host-Imposed Copper Poisoning Impacts Fungal Micronutrient Acquisition during Systemic Candida albicans Infections

This work was supported by the European Research Council (http://erc.europa.eu/: STRIFE Advanced Grant ERC-2009-AdG-249793). A.J.P.B. was also supported by the UK Biotechnology and Biological Research Council (www.bbsrc.ac.uk: Research Grants BB/F00513X/1, BB/K017365/1), the UK Medical Research Council (www.mrc.ac.uk: Programme Grant MR/M026663/1; Centre Grant MR/ N006364/1), and the Wellcome Trust (www.wellcome.ac.uk: Strategic Award 097377)Peer reviewedPublisher PD

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Modulation of lytic molecules restrain serial killing in γδ T lymphocytes

Abstract γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies

The <i>C</i>. <i>albicans CRP1</i> copper exporting ATPase is required for copper tolerance <i>in vitro</i> and plays a role in fungus-host interaction <i>in vivo</i>.

<p>(A) The deletion of <i>CRP1</i> severely impairs the ability of <i>C</i>. <i>albicans</i> to grow in the presence of copper. <i>C</i>. <i>albicans</i> cells were grown at 30°C overnight in YPD medium. Cultures were diluted at OD600 = 0.001 into fresh medium containing the appropriate CuSO₄ concentrations, and growth was monitored after 21 h, at 30°C. The values represent differences between the final and initial OD600 of cultures (+/- SD from two technical replicates), and are representative of three separate experiments performed. (B) Nevertheless, <i>CRP1</i> deletion does not impair the interaction of <i>C</i>. <i>albicans</i> with murine renal epithelial cells <i>in vitro</i>. The <i>CRP1</i> mutant elicits a similar degree of damage to the renal epithelial cell monolayer as the parental strain SC5314, as assessed by measurements of LDH release. At least three independent <i>C</i>. <i>albicans</i> inocula were used in at least two independent co-incubation experiments, in duplicate. (C) Similarly, there is no difference in the release of KC by the renal cells when incubated with the <i>crp1</i> mutant versus the SC5314 isogenic control.</p

High affinity copper acquisition influences fungal pathogenicity factors.

<p><i>C</i>. <i>albicans ctr1</i> cells are susceptible to copper deprivation imposed using the copper chelator BCS (95 μM) (A). The <i>C</i>. <i>albicans ctr1</i> mutant is also sensitive to iron chelation by BPS (B). The <i>C</i>. <i>albicans ctr1</i> strain is highly sensitive to H₂O₂ (C). Both of these defects are reversed by copper supplementation. Further, the <i>C</i>. <i>albicans ctr1</i> mutant displays growth defects on various carbon sources, a phenotype that is suppressed by copper supplementation, but not by haem or Fe³⁺ addition (D). Images were taken after 72 h incubation at 30°C. Number of cells spotted in every subset is given at the bottom of the panel. (E) The transcript levels for key metabolic genes are perturbed in <i>ctr1</i> cells, in the absence (top) and presence (bottom) of copper. These data represent averages of three biological replicates with two technical replicates. Numerical data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#pone.0158683.s014" target="_blank">S10 Table</a>. (F) Changes in <i>C</i>. <i>albicans</i> transcript levels during renal colonisation. These data represent duplicate measurements from at least four biological replicates from the kidneys of animals at early (24 h, replicates E1–E4, 4 animals) or late (96 h, replicates L1–L5, 5 animals) infection stage. Colour scale is as in (E). Fungal transcript abundances were normalised to the <i>ACT1</i> mRNA. Numerical data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#pone.0158683.s015" target="_blank">S11 Table</a>.</p

Systemic candidiasis perturbs copper homeostatic functions in the liver and spleen.

<p>Progressive <i>C</i>. <i>albicans</i> infection affects the expression of copper homeostatic functions in the liver at transcript (A) and protein (B) levels. As the infection progresses, the abundance of hepatic transcripts for the metal storage protein metallothionein MT1 and the copper-containing ferroxidase ceruloplasmin (CP) increase, while the abundance of the copper importer <i>Ctr1</i> transcript decreases (A). In contrast, hepatic ceruloplasmin protein levels do not increase with progressive <i>C</i>. <i>albicans</i> infection, while CTR1 protein levels diminish (B), as assessed by immunohistochemistry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#sec002" target="_blank">Materials and Methods</a>). All transcript data were acquired in duplicate from four biological replicates: healthy mice (replicates H1–H4), animals at early (24 h, replicates E1–E4) or late (96 h, replicates L1–L4) infection stages, following injection with saline (controls, H) or <i>C</i>. <i>albicans</i> SC5314. Transcript abundances were normalised against the <i>GAPDH</i> mRNA. Fold differences in expression are given when <i>p</i>≤0.05. Numerical data are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#pone.0158683.s008" target="_blank">S4 Table</a>. (C) In the spleen, the red pulp macrophage population remains relatively constant, as revealed by detection of the red pulp macrophage antigen F4/80. The haem oxygenase HO-1, CTR1 importer and trans-Golgi ATPase ATP7B all decrease in the red pulp over the course of infection. Images are representative of four biological replicates. Brown colour indicates positive reaction and blue colour no reaction. Size bars: 5 mm (B) or 200 μm (C).</p

Systemic candidiasis perturbs copper homeostatic functions of the kidney and is accompanied by renal metal redistribution.

<p>(A) As the infection develops, renal metabolism shifts towards increased copper acquisition. Immunohistochemistry revealed an increase in CTR1 copper importer, ceruloplasmin (CP), and the trans-Golgi copper transporting ATPases ATP7A and ATP7B, during infection. The location of the renal medulla is indicated inside the dotted area of the top left panel. Arrows mark <i>C</i>. <i>albicans</i> lesions. Brown colour indicates positive reaction and blue colour no reaction. (B) Fluorescent detection of ATP7B protein with Alexa Fluor 647 antibody conjugate showed a progressive increase in signal (false-coloured red, over the blue DAPI signal) in both the medulla and cortex, with no apparent changes in subcellular distribution of the protein (Size bars: 20 μm). (C) As the <i>C</i>. <i>albicans</i> infection develops, there is a transient redistribution of copper in renal tissue as shown by ⁶³Cu measurements using LA-ICP MS. ¹³C levels remain relatively unchanged throughout, whereas there is a transient increase in tissue ⁶³Cu early in the infection (far right). Histology of the corresponding tissue sections is shown (‘pas_h’, middle right). The sequential transverse kidney sections shown in (A-C) are representative of at least two technical replicates, with at least three biological replicates. (D) The shifts in copper distribution are reinforced by changes in the expression of genes encoding copper-associated functions. These transcript abundance data were acquired in duplicate from at least three biological replicates. Transcript abundances were normalised to the <i>GAPDH</i> mRNA. Fold differences in expression are given when <i>p</i>≤0.05. Numerical data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#pone.0158683.s009" target="_blank">S5 Table</a>.</p

Rumination in Migraine: Mediating Effects of Brooding and Reflection between Migraine and Psychological Distress

OBJECTIVE: The relationship between migraine and psychological distress has been consistently reported in cross-sectional and longitudinal studies. We hypothesized that a stable tendency to perseverative thoughts such as rumination would mediate the relationship between migraine and psychological distress. Design and Main Outcomes Measures: Self-report questionnaires measuring depressive rumination, current psychological distress and migraine symptoms in two independent European population cohorts, recruited from Budapest (N=1139) and Manchester (N=2004), were used. Structural regression analysis within structural equation modelling was applied to test the mediational role of brooding and reflection, the components of rumination, between migraine and psychological distress. Sex, age and lifetime depression were controlled for in the analysis. RESULTS: Migraine predicted higher brooding and reflection scores, and brooding proved to be a mediator between migraine and psychological distress in both samples, while reflection mediated the relationship significantly only in the Budapest sample. CONCLUSIONS: Elevated psychological distress in migraine is partially attributed to ruminative response style. Further studies are needed to expand our findings to clinical samples and to examine how rumination links to the adjustment to migraine

Table_1_Trickle infection with Heligmosomoides polygyrus results in decreased worm burdens but increased intestinal inflammation and scarring.xlsx

IntroductionIntestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides polygyrus, the parasites are cleared more quickly. Whether this more effective host response has any negative consequences for the host is not known.ResultsUsing a trickle model of infection, we found that worm clearance was associated with known resistance-related host responses: increased granuloma and tuft cell numbers, increased levels of granuloma IgG and decreased intestinal transit time, as well as higher serum IgE levels. However, we found that the improved worm clearance was also associated with an inflammatory phenotype in and around the granuloma, increased smooth muscle hypertrophy/hyperplasia, and elevated levels of Adamts gene expression.DiscussionTo our knowledge, we are the first to identify the involvement of this protein family of matrix metalloproteinases (MMPs) in host responses to helminth infections. Our results highlight the delicate balance between parasite clearance and host tissue damage, which both contribute to host pathology. When continually exposed to parasitic worms, improved clearance comes at a cost.</p