The 67P/Churyumov-Gerasimenko observation campaign in support of the Rosetta mission.

We present a summary of the campaign of remote observations that supported the European Space Agency's Rosetta mission. Telescopes across the globe (and in space) followed comet 67P/Churyumov-Gerasimenko from before Rosetta's arrival until nearly the end of the mission in September 2016. These provided essential data for mission planning, large-scale context information for the coma and tails beyond the spacecraft and a way to directly compare 67P with other comets. The observations revealed 67P to be a relatively 'well-behaved' comet, typical of Jupiter family comets and with activity patterns that repeat from orbit to orbit. Comparison between this large collection of telescopic observations and the in situ results from Rosetta will allow us to better understand comet coma chemistry and structure. This work is just beginning as the mission ends-in this paper, we present a summary of the ground-based observations and early results, and point to many questions that will be addressed in future studies.This article is part of the themed issue 'Cometary science after Rosetta'.

Assessing public health policy approaches to level-up the gradient in health inequalities: the Gradient Evaluation Framework.

OBJECTIVES: This paper seeks to introduce and analyse the development of the Gradient Evaluation Framework (GEF) to facilitate evaluation of policy actions for their current or future use in terms of their 'gradient friendliness'. In particular, this means their potential to level-up the gradient in health inequalities by addressing the social determinants of health and thereby reducing decision-makers' chances of error when developing such policy actions. STUDY DESIGN: A qualitative developmental study to produce a policy-based evaluation framework. METHODS: The scientific basis of GEF was developed using a comprehensive consensus-building process. This process followed an initial narrative review, based on realist review principles, which highlighted the need for production of a dedicated evaluation framework. The consensus-building process included expert workshops, a pretesting phase, and external peer review, together with support from the Gradient project Scientific Advisory Group and all Gradient project partners, including its Project Steering Committee. RESULTS: GEF is presented as a flexible policy tool resulting from a consensus-building process involving experts from 13 European countries. The theoretical foundations which underpin GEF are discussed, together with a range of practical challenges. The importance of systematic evaluation at each stage of the policy development and implementation cycle is highlighted, as well as the socio-political context in which policy actions are located. CONCLUSIONS: GEF offers potentially a major contribution to the public health field in the form of a practical, policy-relevant and common frame of reference for the evaluation of public health interventions that aim to level-up the social gradient in health inequalities. Further research, including the need for practical field testing of GEF and the exploration of alternative presentational formats, is recommended.

Alloanergization of human T cells results in expansion of alloantigen-specific CD8(+) CD28(-) suppressor cells.

Allostimulation with concurrent costimulatory blockade induces alloantigen-specific hyporesponsiveness in responder T cells ("alloanergization"). Alloanergized responder cells also acquire alloantigen-specific suppressive activity, suggesting this strategy induces active immune tolerance. While this acquired suppressive activity is mediated primarily by CD4(+) FOXP3(+) cells, other cells, most notably CD8(+) suppressor cells, have also been shown to ameliorate human alloresponses. To determine whether alloanergization expands CD8(+) cells with allosuppressive phenotype and function, we used mixed lymphocyte cultures in which costimulatory blockade was provided by belatacept, an FDA-approved, second-generation CTLA-4-immunoglobulin fusion protein that blocks CD28-mediated costimulation, as an in vitro model of HLA-mismatched transplantation. This strategy resulted in an eightfold expansion of CD8(+) CD28(-) T cells which potently and specifically suppressed alloresponses of both CD4(+) and CD8(+) T cells without reducing the frequency of a range of functional pathogen-specific T cells. This CD8-mediated allosuppression primarily required cell-cell contact. In addition, we observed expansion of CD8(+) CD28(-) T cells in vivo in patients undergoing alloanergized HLA-mismatched bone marrow transplantation. Use of costimulatory blockade-mediated alloanergization to expand allospecific CD8(+) CD28(-) suppressor cells merits exploration as an approach to inducing or supporting immune tolerance to alloantigens after allogeneic transplantation.

Low dose antithymocyte globulin for the treatment of older patients with aplastic anaemia.

We report 14 older patients with aplastic anaemia (AA) who were treated with 'low dose' antithymocyte globulin (ATG). The aims of the study were to assess the efficacy and safety of reduced dose ATG in patients over the age of 60 years. Median age was 71 years (range 62-74 years). At the study endpoint (response to treatment at 6 months) 12 patients were evaluable. All patients received lymphoglobuline (horse ATG; Genzyme) at a dose of 0.5vials/10kg/day for 5 days (5mg/kg/day, equivalent to one-third of the standard dose). There were no deaths attributed to ATG. Two patients died during follow-up, from sepsis and anaphylaxis following platelet transfusion, respectively. Only one of the 12 evaluable patients responded to treatment and remains transfusion independent at 14 months after ATG. These results suggest that this lower dose of ATG, though well tolerated, had low efficacy in the treatment of AA.

New advances in acute graft-versus-host disease prophylaxis.

Immunocompetent donor T cells in Allogeneic Haematopoietic Stem Cell grafts mediate acute Graft versus Host Disease (GvHD), still a major cause of recipient morbidity and mortality post transplant. Despite the advent of high resolution HLA-typing and matching at HLA loci, acute GvHD remains a significant problem, even in HLA matched siblings, due primarily to minor histocompatability antigen mismatches. Treatment of GvHD remains ineffective and highly immunosuppressive and the challenge to find effective methods of prevention continues. Non selective removal of donor T cells from the graft has been proven to be effective in preventing GvHD but the beneficial effects of donor T cells, namely effective immune reconstitution and anti tumour activity, are lost. This review considers mechanisms by which acute GvHD may be prevented in the context of the current model of GvHD immunopathogenesis, with a special emphasis on the recent techniques of selective removal or destruction of donor allogeneic T cells that have been described.

Starvation survival, growth and recovery of Enterococcus faecalis in human serum.

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose-, phosphate- or amino acid-limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient-limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 106 colony-forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.

Autoimmune cytopenias in the 22q11.2 deletion syndrome.

We describe two cases of recurrent autoimmune cytopenias, which were subsequently diagnosed with a 22q11.2 deletion/DiGeorge syndrome. The cases are of particular interest as both possessed limited clinical features of this syndrome, and the investigation of haematological abnormalities led to the establishment of a definitive genetic diagnosis.

Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide.

AIM: This study sought to clarify the mechanisms that enable E. faecalis to survive the high pH of calcium hydroxide. METHODOLOGY: E. faecalis strain JH2-2 was exposed to sublethal concentrations of calcium hydroxide, with and without various pretreatments. Blocking agents were added to determine the role of stress-induced protein synthesis and the cell wall-associated proton pump. RESULTS: E. faecalis was resistant to calcium hydroxide at a pH of 11.1, but not pH 11.5. Pre-treatment with calcium hydroxide pH 10.3 induced no tolerance to further exposure at pH 11.5. No difference in cell survival was observed when protein synthesis was blocked during stress induction, however, addition of a proton pump inhibitor resulted in a dramatic reduction of cell viability of E. faecalis in calcium hydroxide. CONCLUSIONS: Survival of E. faecalis in calcium hydroxide appears to be unrelated to stress induced protein synthesis, but a functioning proton pump is critical for survival of E. faecalis at high pH.

HST and VLT investigations of the fragments of comet C/1999 S4 (LINEAR).

At least 16 fragments were detected in images of comet C/1999 S4 (LINEAR) taken on 5 August 2000 with the Hubble Space Telescope (HST) and on 6 August with the Very Large Telescope (VLT). Photometric analysis of the fragments indicates that the largest ones have effective spherical diameters of about 100 meters, which implies that the total mass in the observed fragments was about 2 x 10(9) kilograms. The comet's dust tail, which was the most prominent optical feature in August, was produced during a major fragmentation event, whose activity peaked on UT 22.8 +/- 0.2 July 2000. The mass of small particles (diameters less than about 230 micrometers) in the tail was about 4 x 10(8) kilograms, which is comparable to the mass contained in a large fragment and to the total mass lost from water sublimation after 21 July 2000 (about 3 x 10(8) kilograms). HST spectroscopic observations during 5 and 6 July 2000 demonstrate that the nucleus contained little carbon monoxide ice (ratio of carbon monoxide to water is less than or equal to 0.4%), which suggests that this volatile species did not play a role in the fragmentation of C/1999 S4 (LINEAR).

Polymorphisms in pilin glycosylation Locus of Neisseria meningitidis expressing class II pili.

We have located a locus, pgl, in Neisseria meningitidis strain NMB required for the glycosylation of class II pili. Between five and eight open reading frames (ORFs) (pglF, pglB, pglC, pglB2, orf2, orf3, orf8, and avtA) were present in the pgl clusters of different meningococcal isolates. The Class I pilus-expressing strains Neisseria gonorrhoeae MS11 and N. meningitidis MC58 each contain a pgl cluster in which orf2 and orf3 have been deleted. Strain NMB and other meningococcal isolates which express class II type IV pili contained pgl clusters in which pglB had been replaced by pglB2 and an additional novel ORF, orf8, had been inserted between pglB2 and pglC. Insertional inactivation of the eight ORFs of the pgl cluster of strain NMB showed that pglF, pglB2, pglC, and pglD, but not orf2, orf3, orf8, and avtA, were necessary for pilin glycosylation. Pilin glycosylation was not essential for resistance to normal human serum, as pglF and pglD mutants retained wild-type levels of serum resistance. Although pglB2 and pglC mutants were significantly sensitive to normal human serum under the experimental conditions used, subsequent examination of the encapsulation phenotypes revealed that pglB2 and pglC mutants expressed almost 50% less capsule than wild-type NMB. A mutation in orf3, which did not affect pilin glycosylation, also resulted in a 10% reduction in capsule expression and a moderately serum sensitive phenotype. On the basis of these results we suggest that pilin glycosylation may proceed via a lipid-linked oligosaccharide intermediate and that blockages in this pathway may interfere with capsular transport or assembly.

Enterococcal vanB resistance locus in anaerobic bacteria in human faeces.

While developing a rapid method to detect carriers of vancomycin-resistant enterococci (VRE), we found the vanB gene by PCR in 13 of 50 human faecal specimens that did not contain culturable VRE. Passaging under antibiotic selection allowed us to isolate two species of anaerobic bacteria that were vanB PCR positive, vancomycin resistant, and teicoplanin sensitive. Sequence analysis of the 16S rRNA genes showed that one isolate resembled Eggerthella lenta (98% identity), and the other Clostridium innocuum (92% identity). Southern hybridisation and nucleotide sequencing showed a vanB locus homologous to that in VRE. We propose that vanB resistance in enterococci might arise from gene transfer in the human bowel.

A randomized controlled trial of interleukin-1 receptor antagonist in a rabbit model of ascending infection in pregnancy.

OBJECTIVE: To determine whether treatment with interleukin-1 receptor antagonist (IL1-ra) would affect amniotic fluid concentrations of tumor necrosis factor alpha (TNF-alpha) and prostaglandins or clinical or microbiological outcomes in a model of ascending bacterial infection in pregnancy. METHODS: Timed pregnant New Zealand white rabbits at 70% of gestation underwent endoscopic inoculation of the cervices with 10(6) - 10(7) cfu Escherichia coli. Animals were randomly assigned in a blinded manner to a 5-h intravenous infusion of human IL1-ra (10 mg/kg) or placebo beginning 1-2 h after inoculation. Blood was drawn from the does for assay of serum IL1-ra concentration before inoculation, at mid-infusion, after the infusion ended and at necropsy. At necropsy, temperature and cultures were taken, and aspirated amniotic fluid was pooled for assays of TNF-aalpha, prostaglandin E2 (PGE2) and ILI-ra. RESULTS: Serum IL1-ra concentrations rose to a mean of 2 microg/ml at mid-infusion and fell markedly after the infusion to concentrations barely detectable at necropsy. Between the two groups, there were no significant differences in the rates of fever or positive cultures or in amniotic fluid concentrations of PGE2 or TNF-alpha. One unique finding was the demonstration that administration of human IL1-ra to the does resulted in measurable concentrations of human IL1-ra in the amniotic fluid. CONCLUSIONS: Treatment with an intravenous infusion of human IL1-ra after cervical inoculation with E. coli did not affect clinical or microbiological outcomes or amniotic fluid concentrations of TNF-alpha or PGE2. This experiment providesthefirstdemonstration of passage of human IL1-ra from the maternal bloodstream to the amniotic fluid.

Histologic inflammation in the maternal and fetal compartments in a rabbit model of acute intra-amniotic infection.

OBJECTIVE: This study was undertaken to determine the course of acute inflammation in the maternal and fetal compartments during experimentally induced ascending intra-amniotic infection. STUDY DESIGN: Forty pregnant rabbits at 70% gestation were inoculated endocervically with 10(5) colony-forming units of Escherichia coli. Does were killed at 0, 4, 8, 16, 24, and 30 hours after inoculation. At necropsy, blood, peritoneal fluid, amniotic fluid, and uterine tissue were cultured. Fetal brain, lung, heart, gut, and kidney were collected for histologic examination. Necrosis, infiltrates, congestion, and edema were each assessed semiquantitatively, and mean composite histologic-inflammation scores were compared with analysis of variance. Inflammation, mitotic activity, and apoptosis were evaluated in the fetal brain, and groups were compared with analysis of variance. RESULTS: Twenty-six animals were evaluated after 14 were excluded (lack of fever or positive culture results). A significant increase in histologic inflammation score was seen in the uterus (P<.001), placenta(P = .011), and fetal lung (P = .001) but not in other fetal tissues. These changes were seen earlier in the uterus and placenta and later in the fetal lung. Mitotic activity in the fetal brain decreased significantly by 8 hours after cervical inoculation. There was no inflammation in the fetal brain, and apoptosis in the fetal brain did not increase with time. CONCLUSIONS: Histologic inflammation occurs early in both the uterus and the placenta and later in the fetal lung in the rabbit model of acute intra-amniotic infection. This contrasts with the previously reported chronic model of intra-amniotic infection in the rabbit.

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence.

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.

Identification of Mycobacterium ulcerans in the environment from regions in Southeast Australia in which it is endemic with sequence capture-PCR.

We recently described the use of PCR to identify the environmental source of Mycobacterium ulcerans during an outbreak of ulcerative disease that occurred in a localized region of southeast Australia. The PCR used was based on amplification of the M. ulcerans-specific insertion sequence, IS2404. In this study we developed a new test that is a substantial improvement over the original PCR method in terms of sensitivity, reliability, and ease of use. In the new method magnetic bead sequence capture-PCR is used to detect two M. ulcerans sequences (IS2404 and IS2606) and total mycobacterial 16S ribosomal DNA. We used sequence capture-PCR to test water and plant material collected over a 12-month period during 1998 and 1999 from sites near the centers of two distinct foci of M. ulcerans infections. A golf course irrigation system in one area and a small shallow lake in another area repeatedly were PCR positive for M. ulcerans. Nearby sites and sites unrelated to the endemic areas were negative. Based on the PCR data, a most-probable-number method was used to estimate the concentration of M. ulcerans cells in positive samples from both regions. This procedure resulted in average concentrations of 0.5 cell per 100 ml of water and 40 cells per 100 g of detritus. Loss of the PCR signal coincided with a decrease in ulcerative disease in each area. These results provide further evidence that M. ulcerans may be transmitted from a point environmental source and demonstrate the utility of magnetic bead sequence capture-PCR for identification of nonculturable microbial pathogens in the environment.