A Transcriptomic Meta-Analysis Shows Lipid Metabolism Dysregulation as an Early Pathological Mechanism in the Spinal Cord of SOD1 Mice.

Amyotrophic lateral sclerosis (ALS) is a multifactorial and complex fatal degenerative disorder. A number of pathological mechanisms that lead to motor neuron death have been identified, although there are many unknowns in the disease aetiology of ALS. Alterations in lipid metabolism are well documented in the progression of ALS, both at the systemic level and in the spinal cord of mouse models and ALS patients. The origin of these lipid alterations remains unclear. This study aims to identify early lipid metabolic pathways altered before systemic metabolic symptoms in the spinal cord of mouse models of ALS. To do this, we performed a transcriptomic analysis of the spinal cord of SOD1G93A mice at an early disease stage, followed by a robust transcriptomic meta-analysis using publicly available RNA-seq data from the spinal cord of SOD1 mice at early and late symptomatic disease stages. The meta-analyses identified few lipid metabolic pathways dysregulated early that were exacerbated at symptomatic stages; mainly cholesterol biosynthesis, ceramide catabolism, and eicosanoid synthesis pathways. We present an insight into the pathological mechanisms in ALS, confirming that lipid metabolic alterations are transcriptionally dysregulated and are central to ALS aetiology, opening new options for the treatment of these devastating conditions.

A genetic modifier suggests that endurance exercise exacerbates Huntington's disease.

Polyglutamine expansions in the huntingtin gene cause Huntington's disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed 'draggen' mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration.

A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways.

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.

Genetic Analysis Reveals a Hierarchy of Interactions between Polycystin-Encoding Genes and Genes Controlling Cilia Function during Left-Right Determination.

During mammalian development, left-right (L-R) asymmetry is established by a cilia-driven leftward fluid flow within a midline embryonic cavity called the node. This 'nodal flow' is detected by peripherally-located crown cells that each assemble a primary cilium which contain the putative Ca2+ channel PKD2. The interaction of flow and crown cell cilia promotes left side-specific expression of Nodal in the lateral plate mesoderm (LPM). Whilst the PKD2-interacting protein PKD1L1 has also been implicated in L-R patterning, the underlying mechanism by which flow is detected and the genetic relationship between Polycystin function and asymmetric gene expression remains unknown. Here, we characterize a Pkd1l1 mutant line in which Nodal is activated bilaterally, suggesting that PKD1L1 is not required for LPM Nodal pathway activation per se, but rather to restrict Nodal to the left side downstream of nodal flow. Epistasis analysis shows that Pkd1l1 acts as an upstream genetic repressor of Pkd2. This study therefore provides a genetic pathway for the early stages of L-R determination. Moreover, using a system in which cultured cells are supplied artificial flow, we demonstrate that PKD1L1 is sufficient to mediate a Ca2+ signaling response after flow stimulation. Finally, we show that an extracellular PKD domain within PKD1L1 is crucial for PKD1L1 function; as such, destabilizing the domain causes L-R defects in the mouse. Our demonstration that PKD1L1 protein can mediate a response to flow coheres with a mechanosensation model of flow sensation in which the force of fluid flow drives asymmetric gene expression in the embryo.

Symptomatic secondary syphilis: empirical antimicrobial treatment or await microbiology?

Men who have sex with men (MSM) with symptomatic secondary syphilis present with characteristic symptoms of rash, fever and lymphadenopathy; due to delays in microbiology results and some patients failing to return for treatment, empirical treatment is sometimes offered. We reviewed all patients presenting with secondary syphilis diagnosed on the basis of clinical symptoms and signs and reviewed treatment. Of the 36 patients, 16 MSM (44%; 95% confidence interval (CI) 27.8-60.2%) were treated empirically, whereas treatment was delayed for 20 (56%; 95% CI 39.8-72.2%) waiting for microbiology results. Treatment delays were longer for HIV-negative than HIV-positive MSM (10 vs 5 days respectively; P = 0.01); the cumulative treatment delay was 143 days. Concerns over antimicrobial resistance and overuse of antimicrobials should drive the use of point-of-care testing for syphlis.

Chronic Kidney Disease Risk in African and Caribbean Populations With HIV.

We conducted an observational cohort study of end-stage kidney disease (ESKD) in >7000 African and Caribbean people with HIV in the UK. Using Poisson regression and East Africans as the reference group, the adjusted incidence rate ratio (95% confidence interval) of ESKD was 3.14 (1.26-7.84) in Southern Africans, 6.35 (2.53-15.96) in West Africans, and 5.26 (1.91-14.43) in Caribbeans. Higher CD4 cell count and suppressed HIV replication were associated with reduced risk of ESKD. The risk of ESKD varied among HIV-positive people of African heritage, with the highest rates observed in those of West African descent.

ATMIN is a transcriptional regulator of both lung morphogenesis and ciliogenesis.

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmin(gpg6/gpg6), Atmin(H210Q/H210Q) and Dynll1(GT/GT), revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1(GT/GT) embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.

Clinical characteristics and outcomes of HIV-associated immune complex kidney disease.

BACKGROUND: The pathogenesis and natural history of HIV-associated immune complex kidney disease (HIVICK) is not well understood. Key questions remain unanswered, including the role of HIV infection and replication in disease development and the efficacy of antiretroviral therapy (ART) in the prevention and treatment of disease. METHODS: In this multicentre study, we describe the renal pathology of HIVICK and compare the clinical characteristics of patients with HIVICK with those with IgA nephropathy and HIV-associated nephropathy (HIVAN). Poisson regression models were used to identify risk factors for each of these pathologies. RESULTS: Between 1998 and 2012, 65 patients were diagnosed with HIVICK, 27 with IgA nephropathy and 70 with HIVAN. Black ethnicity and HIV RNA were associated with HIVICK, receipt of ART with IgA nephropathy and black ethnicity and CD4 cell count with HIVAN. HIVICK was associated with lower rates of progression to end-stage kidney disease compared with HIVAN and IgA nephropathy (P < 0.0001). Patients with HIVICK who initiated ART and achieved suppression of HIV RNA experienced improvements in estimated glomerular filtration rate and proteinuria. CONCLUSIONS: These findings suggest a pathogenic role for HIV replication in the development of HIVICK and that ART may improve kidney function in patients who have detectable HIV RNA at the time of HIVICK diagnosis. Our data also suggest that IgA nephropathy should be viewed as a separate entity and not included in the HIVICK spectrum.

Probing Cancer Cell Metabolism Using NMR Spectroscopy.

Altered cellular metabolism is now accepted to be at the core of many diseases including cancer. Over the past 20 years, NMR has become a core technology to study these metabolic perturbations in detail. This chapter reviews current NMR-based methods for steady-state metabolism and, in particular, the use of non-radioactive stable isotope-enriched tracers. Opportunities and challenges for each method, such as 1D (1)H NMR spectroscopy and (13)C carbon-based NMR spectroscopic methods, are discussed. Ultimately, the combination of NMR and mass spectra as orthogonal technologies are required to compensate for the drawbacks of each technique when used singly are discussed.

Myofibrillar myopathy caused by a mutation in the motor domain of mouse MyHC IIb.

Ariel is a mouse mutant that suffers from skeletal muscle myofibrillar degeneration due to the rapid accumulation of large intracellular protein aggregates. This fulminant disease is caused by an ENU-induced recessive mutation resulting in an L342Q change within the motor domain of the skeletal muscle myosin protein MYH4 (MyHC IIb). Although normal at birth, homozygous mice develop hindlimb paralysis from Day 13, consistent with the timing of the switch from developmental to adult myosin isoforms in mice. The mutated myosin (MYH4(L342Q)) is an aggregate-prone protein. Notwithstanding the speed of the process, biochemical analysis of purified aggregates showed the presence of proteins typically found in human myofibrillar myopathies, suggesting that the genesis of ariel aggregates follows a pathogenic pathway shared with other conformational protein diseases of skeletal muscle. In contrast, heterozygous mice are overtly and histologically indistinguishable from control mice. MYH4(L342Q) is present in muscles from heterozygous mice at only 7% of the levels of the wild-type protein, resulting in a small but significant increase in force production in isolated single fibres and indicating that elimination of the mutant protein in heterozygotes prevents the pathological changes observed in homozygotes. Recapitulation of the L342Q change in the functional equivalent of mouse MYH4 in human muscles, MYH1, results in a more aggregate-prone protein.

MicroRNAs 296 and 298 are imprinted and part of the GNAS/Gnas cluster and miR-296 targets IKBKE and Tmed9.

Genomic imprinting is the phenomenon whereby a subset of genes is differentially expressed according to parental origin. Imprinted genes tend to occur in clusters, and microRNAs are associated with the majority of well-defined clusters of imprinted genes. We show here that two microRNAs, miR-296 and miR-298, are part of the imprinted Gnas/GNAS clusters in both mice and humans. Both microRNAs show imprinted expression and are expressed from the paternally derived allele, but not the maternal allele. They arise from a long, noncoding antisense transcript, Nespas, with a promoter more than 27 kb away. Nespas had been shown previously to act in cis to regulate imprinted gene expression within the Gnas cluster. Using microarrays and luciferase assays, IKBKE, involved in many signaling pathways, and Tmed9, a protein transporter, were verified as new targets of miR-296. Thus, Nespas has two clear functions: as a cis-acting regulator within an imprinted gene cluster and as a precursor of microRNAs that modulate gene expression in trans. Furthermore, imprinted microRNAs, including miR-296 and miR-298, impose a parental specific modulation of gene expression of their target genes.

HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants.

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.

Pleiotropic brain function of whirlin identified by a novel mutation.

Despite some evidence indicating diverse roles of whirlin in neurons, the functional corollary of whirlin gene function and behavior has not been investigated or broadly characterized. A single nucleotide variant was identified from our recessive ENU-mutagenesis screen at a donor-splice site in whirlin, a protein critical for proper sensorineural hearing function. The mutation (head-bob, hb) led to partial intron-retention causing a frameshift and introducing a premature termination codon. Mutant mice had a head-bobbing phenotype and significant hyperactivity across several phenotyping tests. Lack of complementation of head-bob with whirler mutant mice confirmed the head-bob mutation as functionally distinct with compound mutants having a mild-moderate hearing defect. Utilizing transgenics, we demonstrate rescue of the hyperactive phenotype and combined with the expression profiling data conclude whirlin plays an essential role in activity-related behaviors. These results highlight a pleiotropic role of whirlin within the brain and implicate alternative, central mediated pathways in its function.

A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy.

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.

Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) reveals a requirement for MAPK signalling in mouse sex determination.

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans.

Mouse hitchhiker mutants have spina bifida, dorso-ventral patterning defects and polydactyly: identification of Tulp3 as a novel negative regulator of the Sonic hedgehog pathway.

The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data.

Upregulation of PKD1L2 provokes a complex neuromuscular disease in the mouse.

Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

Zfhx3-mediated genetic ablation of the SCN abolishes light entrainable circadian activity while sparing food anticipatory activity.

Circadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor Zfhx3 in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were ablated in vivo although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still anticipate food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity although activity levels in mutants were altered. This work highlights a critical role for Zfhx3 in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioral responses to environmental signals.

Ciliary exclusion of Polycystin-2 promotes kidney cystogenesis in an autosomal dominant polycystic kidney disease model.

The human PKD2 locus encodes Polycystin-2 (PC2), a TRPP channel that localises to several distinct cellular compartments, including the cilium. PKD2 mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD) and affect many cellular pathways. Data underlining the importance of ciliary PC2 localisation in preventing PKD are limited because PC2 function is ablated throughout the cell in existing model systems. Here, we dissect the ciliary role of PC2 by analysing mice carrying a non-ciliary localising, yet channel-functional, PC2 mutation. Mutants develop embryonic renal cysts that appear indistinguishable from mice completely lacking PC2. Despite not entering the cilium in mutant cells, mutant PC2 accumulates at the ciliary base, forming a ring pattern consistent with distal appendage localisation. This suggests a two-step model of ciliary entry; PC2 first traffics to the cilium base before TOP domain dependent entry. Our results suggest that PC2 localisation to the cilium is necessary to prevent PKD.

Transcript Analysis Reveals a Hypoxic Inflammatory Environment in Human Chronic Otitis Media With Effusion.

Chronic otitis media with effusion (COME) is the most common cause of childhood hearing loss in the developed world. Underlying pathophysiology is not well understood, and in particular the factors that lead to the transition from acute to chronic inflammation. Here we present the first genome-wide transcript analysis of white blood cells in the effusion of children with COME. Analysis of microarray data for enriched pathways reveals upregulation of hypoxia pathways, which is confirmed using real-time PCR and determining VEGF protein titres. Other pathways upregulated in both mucoid and serous effusions include Toll-like receptor signaling, complement, and RANK-RANKL. Cytology reveals neutrophils and macrophages predominated in both serous and mucoid effusions, however, serous samples had higher lymphocyte and eosinophil differential counts, while mucoid samples had higher neutrophil differential counts. Transcript analysis indicates serous fluids have CD4+ and CD8+ T-lymphocyte, and NK cell signatures. Overall, our findings suggest that inflammation and hypoxia pathways are important in the pathology of COME, and targets for potential therapeutic intervention, and that mucoid and serous COME may represent different immunological responses.