Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

Unconventional life history in a migratory shorebird: desegregating reproduction and migration.

Conventional life-history theory predicts that energy-demanding events such as reproduction and migration must be temporally segregated to avoid resource limitation. Here, we provide, to our knowledge, the first direct evidence of 'itinerant breeding' in a migratory bird, an incredibly rare breeding strategy (less than 0.1% of extant bird species) that involves the temporal overlap of migratory and reproductive periods of the annual cycle. Based on GPS-tracking of over 200 female American woodcock, most female woodcock (greater than 80%) nested more than once (some up to six times) with short re-nest intervals, and females moved northwards on average 800 km between first and second nests, and then smaller distances (ca 200+ km) between subsequent nesting attempts. Reliance on ephemeral habitat for breeding, ground-nesting and key aspects of life history that reduce both the costs of reproduction and migration probably explain the prevalence of this rare phenotype in woodcock and why itinerant breeding so rarely occurs in other bird species.

Conduction block in immune-mediated neuropathy: paranodopathy versus axonopathy.

BACKGROUND AND PURPOSE: Conduction block is a pathognomonic feature of immune-mediated neuropathies. The aim of this study was to advance understanding of pathophysiology and conduction block in chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN). METHODS: A multimodal approach was used, incorporating clinical phenotyping, neurophysiology, immunohistochemistry and structural assessments. RESULTS: Of 49 CIDP and 14 MMN patients, 25% and 79% had median nerve forearm block, respectively. Clinical scores were similar in CIDP patients with and without block. CIDP patients with median nerve block demonstrated markedly elevated thresholds and greater threshold changes in threshold electrotonus, whilst those without did not differ from healthy controls in electrotonus parameters. In contrast, MMN patients exhibited marked increases in superexcitability. Nerve size was similar in both CIDP groups at the site of axonal excitability. However, CIDP patients with block demonstrated more frequent paranodal serum binding to teased rat nerve fibres. In keeping with these findings, mathematical modelling of nerve excitability recordings in CIDP patients with block support the role of paranodal dysfunction and enhanced leakage of current between the node and internode. In contrast, changes in MMN probably resulted from a reduction in ion channel density along axons. CONCLUSIONS: The underlying pathologies in CIDP and MMN are distinct. Conduction block in CIDP is associated with paranodal dysfunction which may be antibody-mediated in a subset of patients. In contrast, MMN is characterized by channel dysfunction downstream from the site of block.

Oxidative stress and lung pathology following geogenic dust exposure.

This study was designed to evaluate markers of systemic oxidative stress and lung histopathology following subacute exposure to geogenic dust with varying heavy metal content collected from a natural setting prone to wind erosion and used heavily for off-road vehicle recreation. Adult female B6C3F1 mice were exposed to several concentrations of dust collected from seven different types of surfaces at the Nellis Dunes Recreation Area in Clark County, Nevada, designated here as CBN 1-7. Dust representing each of the seven surface types, with an average median diameter of 4.2 µm, was selected and administered via oropharyngeal aspiration to mice at concentrations from 0.01 to 100 mg of dust kg(-1) of body weight. Exposures were given four times spaced a week apart over a 28 day period to mimic a month of weekend exposures. Lung pathology was evaluated while plasma markers of oxidative stress included levels of reactive oxygen and nitrogen species, superoxide dismutase, total antioxidant capacity and total glutathione. Overall, results of these assays to evaluate markers of oxidative stress indicate that no single CBN surface type was able to consistently induce markers of systemic oxidative stress at a particular dose or in a dose-response manner. All surface types were able to induce some level of lung inflammation, typically at the highest exposure levels. These data suggest that dust from the Nellis Dunes Recreation Area may present a potential health risk, but additional studies are necessary to characterize the full extent of health risks to humans. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic variants and effect modifiers of QT interval prolongation in patients with sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is a common inherited blood disorder among African Americans (AA), with premature mortality which has been associated with prolongation of the heart rate-corrected QT interval (QTc), a known risk factor for sudden cardiac death. Although numerous genetic variants have been identified as contributors to QT interval prolongation in the general population, their impact on SCD patients remains unclear. This study used an unweighted polygenic risk score (PRS) to validate the previously identified associations between SNPs and QTc interval in SCD patients, and to explore possible interactions with other factors that prolong QTc interval in AA individuals with SCD. METHODS: In SCD patients, candidate genetic variants associated with the QTc interval were genotyped. To identify any risk SNPs that may be correlated with QTc interval prolongation, linear regression was employed, and an unweighted PRS was subsequently constructed. The effect of PRS on the QTc interval was evaluated using linear regression, while stratification analysis was used to assess the influence of serum alanine transaminase (ALT), a biomarker for liver disease, on the PRS effect. We also evaluated the PRS with the two subcomponents of QTc, the QRS and JTc intervals. RESULTS: Out of 26 candidate SNPs, five risk SNPs were identified for QTc duration under the recessive model. For every unit increase in PRS, the QTc interval prolonged by 4.0 ms (95% CI: [2.0, 6.1]; p-value: <0.001) in the additive model and 9.4 ms in the recessive model (95% CI: [4.6, 14.1]; p-value: <0.001). Serum ALT showed a modification effect on PRS-QTc prolongation under the recessive model. In the normal ALT group, each PRS unit increased QTc interval by 11.7 ms (95% CI: [6.3, 17.1]; p-value: 2.60E-5), whereas this effect was not observed in the elevated ALT group (0.9 ms; 95% CI: [-7.0, 8.8]; p-value: 0.823). CONCLUSION: Several candidate genetic variants are associated with QTc interval prolongation in SCD patients, and serum ALT acts as a modifying factor. The association of a CPS1 gene variant in both QTc and JTc duration adds to NOS1AP as evidence of involvement of the urea cycle and nitric oxide metabolism in cardiac repolarization in SCD. Larger replication studies are needed to confirm these findings and elucidate the underlying mechanisms.

Mammalian MutS homologue 5 is required for chromosome pairing in meiosis.

MSH5 (MutS homologue 5) is a member of a family of proteins known to be involved in DNA mismatch repair. Germline mutations in MSH2, MLH1 and GTBP (also known as MSH6) cause hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome. Inactivation of Msh2, Mlh1, Gtmbp (also known as Msh6) or Pms2 in mice leads to hereditary predisposition to intestinal and other cancers. Early studies in yeast revealed a role for some of these proteins, including Msh5, in meiosis. Gene targeting studies in mice confirmed roles for Mlh1 and Pms2 in mammalian meiosis. To assess the role of Msh5 in mammals, we generated and characterized mice with a null mutation in Msh5. Msh5-/- mice are viable but sterile. Meiosis in these mice is affected due to the disruption of chromosome pairing in prophase I. We found that this meiotic failure leads to a diminution in testicular size and a complete loss of ovarian structures. Our results show that normal Msh5 function is essential for meiotic progression and, in females, gonadal maintenance.

A frame shift mutation in the PMP22 gene in hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) has been a associated with a deletion of 1.5 megabases of chromosome 17p. One of four biopsy proven HNPP families that we have studied did not possess this deletion. As the deleted DNA region includes the coding region for a peripheral myelin gene (PMP22), we used single strand conformation analysis to examine this gene for mutations in the non-deleted HNPP family. An abnormal fragment in exon 1 was identified, and sequencing revealed a two base pair deletion in all affected family members. The deletion results in a frame shift, providing strong evidence that this gene has an important role in the pathogenesis of the disease.

Clinical and Laboratory Correlates of QTc Duration in Adult and Pediatric Sickle Cell Disease.

Background: Sickle cell disease, a common genetic disorder in African Americans, manifests an increased risk of sudden death, the basis of which is incompletely understood. Prolongation of heart rate-corrected QT (QTc) interval on the electrocardiogram, a standard clinical measure of cardiac repolarization, may contribute to sudden death by predisposing to torsades de pointes ventricular tachycardia. Methods: We established a cohort study of 293 adult and 121 pediatric sickle cell disease patients drawn from the same geographic region as the Jackson Heart Study (JHS) cohort, in which significant correlates of QT duration have been characterized and quantitatively modeled. Herein, we establish clinical and laboratory correlates of QTc duration in our cohort using stepwise multivariate linear regression analysis. We then compared our adult sickle cell disease data to effect-size predictions from the published JHS statistical model of QT interval duration. Results: In adult sickle cell disease, gender, diuretic use, QRS duration, serum ALT levels, anion gap, and diastolic blood pressure show positive correlation; hemoglobin levels show inverse correlation; in pediatric sickle cell disease, age, hemoglobin levels, and serum bicarbonate and creatinine levels show inverse correlation. The mean QTc in our adult sickle cell disease cohort is 7.8 milliseconds longer than in the JHS cohort, even though the JHS statistical model predicts that the mean QTc in our cohort should be > 11 milliseconds shorter than in the much older JHS cohort, a differential of > 18 milliseconds. Conclusion: Sickle cell disease patients have substantial QTc prolongation relative to their age, driven by factors some overlapping, in adult and pediatric sickle cell disease, and distinct from those that have been defined in the general African American community.

Targeting radiation-resistant hypoxic tumour cells through ATR inhibition.

BACKGROUND: Most solid tumours contain regions of sub-optimal oxygen concentration (hypoxia). Hypoxic cancer cells are more resistant to radiotherapy and represent the most aggressive fraction of a tumour. It is therefore essential that strategies continue to be developed to target hypoxic cancer cells. Inhibition of the DNA damage response (DDR) might be an effective way of sensitising hypoxic tumour cells to radiotherapy. METHODS: Here, we describe the cellular effects of pharmacological inhibition of the apical DDR kinase ATR (Ataxia Telangiectasia and Rad 3 related) with a highly selective inhibitor, VE-821, in hypoxic conditions and its potential as a radiosensitiser. RESULTS: VE-821 was shown to inhibit ATR-mediated signalling in response to replication arrest induced by severe hypoxia. In these same conditions, VE-821 induced DNA damage and consequently increased Ataxia Telangiectasia Mutated-mediated phosphorylation of H2AX and KAP1. Consistently, ATR inhibition sensitised tumour cell lines to a range of oxygen tensions. Most importantly, VE-821 increased radiation-induced loss of viability in hypoxic conditions. Using this inhibitor we have also demonstrated for the first time a link between ATR and the key regulator of the hypoxic response, HIF-1. HIF-1 stabilisation and transcriptional activity were both decreased in response to ATR inhibition. CONCLUSION: These findings suggest that ATR inhibition represents a novel strategy to target tumour cells in conditions relevant to pathophysiology and enhance the efficacy of radiotherapy.

Massive astrocyte destruction in neuromyelitis optica despite natalizumab therapy.

Auto-antibody mediated astrocyte injury is implicated as a primary event in neuromyelitis optica (NMO) by biomarker, post-mortem and experimental studies that differentiate the condition from multiple sclerosis. We describe the clinical, radiological and neuropathological features of a severe cerebral attack in a natalizumab-treated patient with relapsing myelitis and serum aquaporin-4 antibodies. Our findings support autopsy evidence that abrupt astrocyte destruction precedes demyelination in NMO, and emphasize the importance of serological testing in patients with limited disease. Adherence to current NMO diagnostic criteria may delay treatment, or lead to inappropriate therapy with beta-interferon or natalizumab.

Hypoxia-driven metabolic reprogramming of adipocytes fuels cancer cell proliferation.

Objective: Obesity increases the risk of certain cancers, especially tumours that reside close to adipose tissue (breast and ovarian metastasis in the omentum). The obesogenic and tumour micro-environment share a common pathogenic feature, oxygen deprivation (hypoxia). Here we test how hypoxia changes the metabolome of adipocytes to assist cancer cell growth. Methods: Human and mouse breast and ovarian cancer cell lines were co-cultured with human and mouse adipocytes respectively under normoxia or hypoxia. Proliferation and lipid uptake in cancer cells were measured by commercial assays. Metabolite changes under normoxia or hypoxia were measured in the media of human adipocytes by targeted LC/MS. Results: Hypoxic cancer-conditioned media increased lipolysis in both human and mouse adipocytes. This led to increased transfer of lipids to cancer cells and consequent increased proliferation under hypoxia. These effects were dependent on HIF1α expression in adipocytes, as mouse adipocytes lacking HIF1α showed blunted responses under hypoxic conditions. Targeted metabolomics of the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes media revealed that culture with hypoxic-conditioned media from non-malignant mammary epithelial cells (MCF10A) can alter the adipocyte metabolome and drive proliferation of the non-malignant cells. Conclusion: Here, we show that hypoxia in the adipose-tumour microenvironment is the driving force of the lipid uptake in both mammary and ovarian cancer cells. Hypoxia can modify the adipocyte metabolome towards accelerated lipolysis, glucose deprivation and reduced ketosis. These metabolic shifts in adipocytes could assist both mammary epithelial and cancer cells to bypass the inhibitory effects of hypoxia on proliferation and thrive.

A pro forma and review process for the assessment of standards of care in stillbirths.

Stillbirth rates do not address deficiencies in care. We collected information on stillbirths from 2004 to 2009 using a standardised pro forma. A local panel used the pro forma to grade the level of care received by the Confidential Enquiry into Stillbirths and Deaths in Infancy (CESDI) categorisation. Comparison using kappa scores showed agreement between local and external multidisciplinary panels of similar referral patterns (n = 47, κ = 0.7), and that the categorisation was unaffected by the individual who fills out the pro forma (n = 17, κ = 0.5). There was less agreement between the local panel and adverse event review (n = 100, κ = 0.45). This report represents a validation of the pro forma and the review process for standard use in all units.

The Early External Cephalic Version (ECV) 2 Trial: an international multicentre randomised controlled trial of timing of ECV for breech pregnancies.

OBJECTIVE: To investigate whether initiating external cephalic version (ECV) earlier in pregnancy might increase the rate of successful ECV procedures, and be more effective in decreasing the rate of non-cephalic presentation at birth and of caesarean section. DESIGN: An unblinded multicentred randomised controlled trial. SETTING: A total of 1543 women were randomised from 68 centres in 21 countries. POPULATION: Women with a singleton breech fetus at a gestational age of 33(0/7) weeks (231 days) to 35(6/7) weeks (251 days) of gestation were included. METHODS: Participants were randomly assigned to having a first ECV procedure between the gestational ages of 34(0/7) (238 days) and 35(6/7) weeks of gestation (early ECV group) or at or after 37(0/7) (259 days) weeks of gestation (delayed ECV group). MAIN OUTCOME MEASURES: The primary outcome was the rate of caesarean section; the secondary outcome was the rate of preterm birth. RESULTS: Fewer fetuses were in a non-cephalic presentation at birth in the early ECV group (314/765 [41.1%] versus 377/768 [49.1%] in the delayed ECV group; relative risk [RR] 0.84, 95% CI 0.75, 0.94, P=0.002). There were no differences in rates of caesarean section (398/765 [52.0%] versus 430/768 [56.0%]; RR 0.93, 95% CI 0.85, 1.02, P=0.12) or in risk of preterm birth (50/765 [6.5%] versus 34/768 [4.4%]; RR 1.48, 95% CI 0.97, 2.26, P=0.07) between groups. CONCLUSION: External cephalic version at 34-35 weeks versus 37 or more weeks of gestation increases the likelihood of cephalic presentation at birth but does not reduce the rate of caesarean section and may increase the rate of preterm birth.

The trophoblast is a component of the innate immune system during pregnancy.

Systemic infection with Listeria monocytogenes, a Gram-positive intracellular bacterium, has been used extensively to analyze the innate immune response. Macrophages are central to this response, acting as both the host for and principal defense against this bacterium. During pregnancy L. monocytogenes has a predilection for replication at the maternal-placental interface and consequently is an important cause of fetal morbidity and mortality. However, macrophages are mostly excluded from the murine placenta with neutrophils acting as the main immune effector cell against this bacterium. Colony stimulating factor (CSF)-1, a macrophage growth factor, is synthesized in high concentrations by the uterine epithelium during pregnancy, where it is targeted to trophoblast bearing CSF-1-receptors. To define the involvement of CSF-1 in placental immunity, we infected pregnant mice either homozygous or heterozygous for an inactivating recessive mutation in the gene for CSF-1 (osteopetrotic; Csfmop) with L. monocytogenes. CSF-1 was required to recruit neutrophils to the site of listerial infection in the decidua basalis, and infection by Listeria remained unrestrained in its absence. CSF-1 acted by inducing the trophoblast to synthesize the neutrophil chemoattractants (KC) and macrophage inflammatory protein (MIP)-2. Thus, during pregnancy, trophoblast responsive to CSF-1 acts to organize the maternal immune response to bacterial infection at the utero-placental interface. This previously unknown function indicates that the trophoblast acts as a pregnancy-specific component of the innate immune system.

T-cell antigen receptor transmembrane peptides modulate T-cell function and T cell-mediated disease.

This study describes a novel method of inhibiting T-cell function by the use of peptides rationally designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence involved with TCR receptor assembly. The most effective peptide (core peptide, CP) modulating in vitro and in vivo T-cell function contained nine amino acids two of which, lysine and arginine, were hydrophilic and separated by four hydrophobic amino acids. CP without chemical modification or conjugation was able to enter non-T and T cells. Conjugation of CP at the carboxyl terminus with palmitic acid resulted in a greater inhibition of T-cell interleukin-2 (IL-2) production in vitro than peptide alone. When examined for effects in vivo, CP reduced clinical signs of inflammation in three T cell-mediated disease models including adjuvant-induced arthritis, experimental allergic neuritis, and cyclophosphamide-induced diabetes in NOD/Lt(F) mice. This peptide or its analogues has potential as a therapeutic agent in human inflammatory and autoimmune disorders.

Regulation of colony-stimulating factor 1 during pregnancy.

Pregnancy results in an elevation in serum and tissue concentrations of the mononuclear phagocytic growth factor, CSF-1 (colony-stimulating factor 1). These increases are associated with an increase in the number of monocytes in the circulation, and with increases in the number of splenic macrophage precursors. In contrast to the approximately 2-fold elevation of the CSF-1 concentrations in serum and most tissues, pregnancy results in a 1,000-fold increase in the concentration of uterine CSF-1. The roughly fivefold elevation in uterine CSF-1 concentration observed at day 5 of pregnancy could be mimicked by administration of chorionic gonadotrophin in intact but not ovariectomized mice. These dramatic changes in uterine CSF-1 concentrations may indicate a role for CSF-1 in the regulation of nonmononuclear phagocytic cell types.

Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.

In human breast carcinomas, overexpression of the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R) correlates with poor prognosis. To establish if there is a causal relationship between CSF-1 and breast cancer progression, we crossed a transgenic mouse susceptible to mammary cancer with mice containing a recessive null mutation in the CSF-1 gene (Csf1(op)) and followed tumor progression in wild-type and null mutant mice. The absence of CSF-1 affects neither the incidence nor the growth of the primary tumors but delayed their development to invasive, metastatic carcinomas. Transgenic expression of CSF-1 in the mammary epithelium of both Csf1(op)/Csf1(op) and wild-type tumor-prone mice led to an acceleration to the late stages of carcinoma and to a significant increase in pulmonary metastasis. This was associated with an enhanced infiltration of macrophages into the primary tumor. These studies demonstrate that the growth of mammary tumors and the development to malignancy are separate processes and that CSF-1 selectively promotes the latter process. CSF-1 may promote metastatic potential by regulating the infiltration and function of tumor-associated macrophages as, at the tumor site, CSF-1R expression was restricted to macrophages. Our data suggest that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects.

Delayed hematopoietic development in osteopetrotic (op/op) mice.

Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.

Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice.

Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTalpha-/- mice, TNF-/- mice, and a new LTalpha-/- line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTalpha gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTalpha with soluble TNF receptor in unmanipulated wild-type or TNF-/- mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.