An alternative downstream translation start site in the non-TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages.

Toll-like receptor (TLR) signaling relies on Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor proteins that recruit downstream signaling molecules to generate tailored immune responses. In addition, the palmitoylated transmembrane adaptor protein family member Scimp acts as a non-TIR-containing adaptor protein in macrophages, scaffolding the Src family kinase Lyn to enable TLR phosphorylation and proinflammatory signaling responses. Here we report the existence of a smaller, naturally occurring translational variant of Scimp (Scimp TV1), which is generated through leaky scanning and translation at a downstream methionine. Scimp TV1 also scaffolds Lyn, but in contrast to full-length Scimp, it is basally rather than lipopolysaccharide (LPS)-inducibly phosphorylated. Macrophages from mice that selectively express Scimp TV1, but not full-length Scimp, have impaired sustained LPS-inducible cytokine responses. Furthermore, in granulocyte macrophage colony-stimulating factor-derived myeloid cells that express high levels of Scimp, selective overexpression of Scimp TV1 enhances CpG DNA-inducible cytokine production. Unlike full-length Scimp that localizes to the cell surface and filopodia, Scimp TV1 accumulates in intracellular compartments, particularly the Golgi. Moreover, this variant of Scimp is not inducibly phosphorylated in response to CpG DNA, suggesting that it may act via an indirect mechanism to enhance TLR9 responses. Our findings thus reveal the use of alternative translation start sites as a previously unrecognized mechanism for diversifying TLR responses in the innate immune system.

High sensitivity and specificity of a 5-analyte protein and microRNA biosignature for identification of active tuberculosis.

OBJECTIVES: Non-sputum-based tests to accurately identify active tuberculosis (TB) disease and monitor response to therapy are urgently needed. This study examined the biomarker capacity of a panel of plasma proteins alone, and in conjunction with a previously identified miRNA signature, to identify active TB disease. METHODS: The expression of nine proteins (IP-10, MCP-1, sTNFR1, RANTES, VEGF, IL-6, IL-10, TNF and Eotaxin) was measured in the plasma of 100 control subjects and 100 TB patients, at diagnosis (treatment naïve) and over the course of treatment (1-, 2- and 6-month intervals). The diagnostic performance of the nine proteins alone, and with the miRNA, was assessed. RESULTS: Six proteins were significantly up-regulated in the plasma of TB patients at diagnosis compared to controls. Receiver operator characteristic curve analysis demonstrated that IP-10 with an AUC = 0.874, sensitivity of 75% and specificity of 87% was the best single biomarker candidate to distinguish TB patients from controls. IP-10 and IL-6 levels fell significantly within one month of commencing treatment and may have potential as indicators of a positive response to therapy. The combined protein and miRNA panel gave an AUC of 1.00. A smaller panel of only five analytes (IP-10, miR-29a, miR-146a, miR-99b and miR-221) showed an AUC = 0.995, sensitivity of 96% and specificity of 97%. CONCLUSIONS: A novel combination of miRNA and proteins significantly improves the sensitivity and specificity as a biosignature over single biomarker candidates and may be useful for the development of a non-sputum test to aid the diagnosis of active TB disease.

Frontline Science: LPS-inducible SLC30A1 drives human macrophage-mediated zinc toxicity against intracellular Escherichia coli.

TLR-inducible zinc toxicity is an antimicrobial mechanism utilized by macrophages, however knowledge of molecular mechanisms mediating this response is limited. Here, we show that E. coli exposed to zinc stress within primary human macrophages reside in membrane-bound vesicular compartments. Since SLC30A zinc exporters can deliver zinc into the lumen of vesicles, we examined LPS-regulated mRNA expression of Slc30a/SLC30A family members in primary mouse and human macrophages. A number of these transporters were dynamically regulated in both cell populations. In human monocyte-derived macrophages, LPS strongly up-regulated SLC30A1 mRNA and protein expression. In contrast, SLC30A1 was not LPS-inducible in macrophage-like PMA-differentiated THP-1 cells. We therefore ectopically expressed SLC30A1 in these cells, finding that this was sufficient to promote zinc-containing vesicle formation. The response was similar to that observed following LPS stimulation. Ectopically expressed SLC30A1 localized to both the plasma membrane and intracellular zinc-containing vesicles within LPS-stimulated THP-1 cells. Inducible overexpression of SLC30A1 in THP-1 cells infected with the Escherichia coli K-12 strain MG1655 augmented the zinc stress response of intracellular bacteria and promoted clearance. Furthermore, in THP-1 cells infected with an MG1655 zinc stress reporter strain, all bacteria contained within SLC30A1-positive compartments were subjected to zinc stress. Thus, SLC30A1 marks zinc-containing compartments associated with TLR-inducible zinc toxicity in human macrophages, and its ectopic over-expression is sufficient to initiate this antimicrobial pathway in these cells. Finally, SLC30A1 silencing did not compromise E. coli clearance by primary human macrophages, suggesting that other zinc exporters may also contribute to the zinc toxicity response.

A transcriptional blood signature distinguishes early tuberculosis disease from latent tuberculosis infection and uninfected individuals in a Vietnamese cohort.

OBJECTIVES: Global tuberculosis (TB) control is restricted by the failure to detect an estimated 3.3 million TB cases annually. In the majority of TB endemic settings, sputum smear microscopy is used to diagnose TB, but this test is insensitive for TB in its early stages. The objective of this study is to establish a concise gene signature that discriminates between individuals with early TB disease, latent TB infection (LTBI) and those without infection. METHODS: This is a case control study nested within a cluster-randomised trial of population screening for active TB using Xpert MTB/RIF. Whole blood samples from 303 participants with active TB (97), LTBI (92) and uninfected individuals (114) were subject to transcriptomic analysis of selected target genes based on a systematic review of previous studies. RESULTS: Analysis of 82 genes identified a pattern of differentially expressed genes in TB disease. A seven gene signature was identified that distinguished between TB disease and no TB disease with an AUC of 0.86 (95% CI: 0.80-0.91), and between TB disease from LTBI with an AUC of 0.88 (95% CI: 0.82-0.93). CONCLUSION: This gene signature accurately distinguishes early TB disease from those without TB disease or infection, in the context of community-wide TB screening. It could be used as a non-sputum based screening tool or triage test to detect prevalent cases of TB in the community.

High-resolution longitudinal N- and O-glycoprofiling of human monocyte-to-macrophage transition.

Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon-liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0-1, 22.1-30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8-35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6-39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.

Developing new TB biomarkers, are miRNA the answer?

Efforts to reduce the global TB burden are hindered by the lack of simple, reliable non-sputum based diagnostics. To date studies investigating the biomarker potential of circulating host proteins and mRNA have not shown sufficient diagnostic utility. Recently, there has been increasing interest in circulating miRNA as a biomarker of TB disease. This review examined all published miRNA-TB biomarker studies to determine if a reproducible miRNA signature of TB disease could be elucidated. From 15 miRNA profiling studies, 894 miRNA differentially expressed between TB patients and healthy controls were identified in at least one study. Of these, 143 miRNA were validated by qPCR with 53 differentially expressed between TB patients and controls. Interestingly, only 8 of these miRNA were identified in 2 or more studies, and no consensus on a reproducible miRNA signature for identification of TB disease could be identified. TB disease is clearly associated with a wide breadth of differentially expressed miRNA. This review highlights our recent progress and the multiple factors, including environment, source of tissue, ethnicity and extent of TB disease that may influence miRNA expression. Coordinated efforts are required to validate identified targets in multiple populations to progress miRNA biomarker development.

SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages.

Danger signals activate Toll-like receptors (TLRs), thereby initiating inflammatory responses. Canonical TLR signalling, via Toll/Interleukin-1 receptor domain (TIR)-containing adaptors and proinflammatory transcription factors such as NF-κB, occurs in many cell types; however, additional mechanisms are required for specificity of inflammatory responses in innate immune cells. Here we show that SCIMP, an immune-restricted, transmembrane adaptor protein (TRAP), promotes selective proinflammatory cytokine responses by direct modulation of TLR4. SCIMP is a non-TIR-containing adaptor, binding directly to the TLR4-TIR domain in response to lipopolysaccharide. In macrophages, SCIMP is constitutively associated with the Lyn tyrosine kinase, is required for tyrosine phosphorylation of TLR4, and facilitates TLR-inducible production of the proinflammatory cytokines IL-6 and IL-12p40. Point mutations in SCIMP abrogating TLR4 binding also prevent SCIMP-mediated cytokine production. SCIMP is, therefore, an immune-specific TLR adaptor that shapes host defence and inflammation.

Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.

We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled carefully controlled characterization of this novel innate immune antimicrobial pathway. In summary, our study provides new insights at the cellular level into the well-documented effects of zinc in promoting host defense against infectious disease, as well as the complex host subversion strategies employed by S Typhimurium to combat this pathway.-Kapetanovic, R., Bokil, N. J., Achard, M. E. S., Ong, C.-L. Y., Peters, K. M., Stocks, C. J., Phan, M.-D., Monteleone, M., Schroder, K., Irvine, K. M., Saunders, B. M., Walker, M. J., Stacey, K. J., McEwan, A. G., Schembri, M. A., Sweet, M. J. Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.

Co-transcriptomic Analysis by RNA Sequencing to Simultaneously Measure Regulated Gene Expression in Host and Bacterial Pathogen.

Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. Here we describe the application of RNA sequencing using samples obtained from in vitro infection assays to simultaneously quantify both host and bacterial pathogen gene expression changes, as well as general approaches that can be undertaken to interpret the RNA sequencing data that is generated. These methods can be used to provide insights into host TLR-regulated transcriptional responses to microbial challenge, as well as pathogen subversion mechanisms against such responses.

Using the MCoTI-II Cyclotide Scaffold To Design a Stable Cyclic Peptide Antagonist of SET, a Protein Overexpressed in Human Cancer.

The SET protein is a promising drug target in cancer therapy, because of its ability to inhibit the function of the tumor suppressor gene protein phosphatase 2A (PP2A). COG peptides, derived from apolipoprotein E (apoE), are potent antagonists of SET; they induce cytotoxicity in cancer cells upon binding to intracellular SET and modulate the nuclear factor kappa B (NF-κB) signaling pathway. However, the therapeutic potential of COG peptides is limited, because of their poor proteolytic stability and low bioavailability. In this study, the COG peptide, COG1410, was stabilized by grafting it onto the ultrastable cyclic peptide scaffold, Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The grafted MCoTI-II peptides were cytotoxic to a cancer cell line and showed high stability in human serum. The most potent grafted MCoTI-II peptide inhibited lipopolysaccharide (LPS)-mediated activation of NF-κB in murine macrophages. Overall, this study demonstrates the application of the MCoTI-II scaffold for the development of stable peptide drugs for cancer therapy.

Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death.

The pseudokinase mixed lineage kinase domain-like (MLKL) is an essential effector of necroptotic cell death. Two distinct human MLKL isoforms have previously been reported, but their capacities to trigger cell death have not been compared directly. Herein, we examine these two MLKL isoforms, and further probe the features of the human MLKL N-terminal domain that are required for cell death. Expression in HEK293T cells of the N-terminal 201 amino acids (aa) of human MLKL is sufficient to cause cell death, whereas expression of the first 154 aa is not. Given that aa 1-125 are able to initiate necroptosis, our findings indicate that the helix that follows this region restrains necroptotic activity, which is again restored in longer constructs. Furthermore, MLKL isoform 2 (MLKL2), which lacks much of the regulatory pseudokinase domain, is a much more potent inducer of cell death than MLKL isoform 1 (MLKL1) in ectopic expression studies in HEK293T cells. Modelling predicts that a C-terminal helix constrains the activity of MLKL1, but not MLKL2. Although both isoforms are expressed by human monocyte-derived macrophages at the mRNA level, MLKL2 is expressed at much lower levels. We propose that it may have a regulatory role in controlling macrophage survival, either in the steady state or in response to specific stimuli.

Innate immune perturbations, accumulating DAMPs and inflammasome dysregulation: A ticking time bomb in ageing.

Ageing has pronounced effects on the immune system, including on innate immune cells. Whilst most studies suggest that total numbers of different innate immune cell populations do not change dramatically during ageing, many of their functions such as phagocytosis, antigen presentation and inflammatory molecule secretion decline. In contrast, many endogenous damage-associated molecular patterns (DAMPs) accumulate during ageing. These include reactive oxygen species (ROS) released from damaged mitochondria, extracellular nucleotides like ATP, high mobility group box (HMGB) 1 protein, oxidized low density lipoprotein, amyloid-beta (Aß), islet amyloid polypeptide and particulates like monosodium urate (MSU) crystals and cholesterol crystals. Some of these DAMPs trigger the activation of inflammasomes, cytosolic danger sensing signalling platforms that drive both the maturation of specific pro-inflammatory mediators such as IL-1ß, as well as the initiation of pro-inflammatory pyroptotic cell death. Herein, we review the evidence that dysregulated inflammasome activation, via altered innate immune cell functions and elevated levels of DAMPs, contributes to the establishment of chronic, low-grade inflammation (characterized by elevated levels of IL-6 and C-reactive protein) and the development of age-related pathological processes.

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions.

Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host-pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow-derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up-regulated at 24 h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration.

AIM: To develop a model of stress-induced senescence to study the hepatocyte senescence associated secretory phenotype (SASP). METHODS: Hydrogen peroxide treatment was used to induce senescence in the human HepG2 hepatocyte cell line. Senescence was confirmed by cytochemical staining for a panel of markers including Ki67, p21, heterochromatin protein 1ß, and senescence-associated-ß-galactosidase activity. Senescent hepatocytes were characterised by gene expression arrays and quantitative polymerase chain reaction (qPCR), and conditioned media was used in proteomic analyses, a human chemokine protein array, and cell migration assays to characterise the composition and function of the hepatocyte SASP. RESULTS: Senescent hepatocytes induced classical markers of senescence (p21, heterochromatin protein 1ß, and senescence-associated-ß-galactosidase activity); and downregulated the proliferation marker, Ki67. Hepatocyte senescence induced a 4.6-fold increase in total secreted protein (P = 0.06) without major alterations in the protein profile. Senescence-induced genes were identified by microarray (Benjamini Hochberg-corrected P < 0.05); and, consistent with the increase in secreted protein, gene ontology analysis revealed a significant enrichment of secreted proteins among inducible genes. The hepatocyte SASP included characteristic factors such as interleukin (IL)-8 and IL-6, as well as novel components such as SAA4, IL-32 and Fibrinogen, which were validated by qPCR and/or chemokine protein array. Senescent hepatocyte-conditioned medium elicited migration of inflammatory (granulocyte-macrophage colony stimulating factor, GM-CSF-derived), but not non-inflammatory (CSF-1-derived) human macrophages (P = 0.022), which could contribute to a pro-inflammatory microenvironment in vivo, or facilitate the clearance of senescent cells. CONCLUSION: Our novel model of hepatocyte senescence provides insights into mechanisms by which senescent hepatocytes may promote chronic liver disease pathogenesis.

Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper.

The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases in vivo, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells.

Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages.

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.

Copper redistribution in murine macrophages in response to Salmonella infection.

The movement of key transition metal ions is recognized to be of critical importance in the interaction between macrophages and intracellular pathogens. The present study investigated the role of copper in mouse macrophage responses to Salmonella enterica sv. Typhimurium. The copper chelator BCS (bathocuproinedisulfonic acid, disodium salt) increased intracellular survival of S. Typhimurium within primary mouse BMM (bone-marrow-derived macrophages) at 24 h post-infection, implying that copper contributed to effective host defence against this pathogen. Infection of BMM with S. Typhimurium or treatment with the TLR (Toll-like receptor) 4 ligand LPS (lipopolysaccharide) induced the expression of several genes encoding proteins involved in copper transport [Ctr (copper transporter) 1, Ctr2 and Atp7a (copper-transporting ATPase 1)], as well as the multi-copper oxidase Cp (caeruloplasmin). Both LPS and infection with S. Typhimurium triggered copper accumulation within punctate intracellular vesicles (copper 'hot spots') in BMM as indicated by the fluorescent reporter CS1 (copper sensor 1). These copper hot spots peaked in their accumulation at approximately 18 h post-stimulation and were dependent on copper uptake into cells. Localization studies indicated that the copper hot spots were in discrete vesicles distinct from Salmonella containing vacuoles and lysosomes. We propose that copper hot spot formation contributes to antimicrobial responses against professional intracellular bacterial pathogens.

Intramacrophage survival of uropathogenic Escherichia coli: differences between diverse clinical isolates and between mouse and human macrophages.

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1(+) vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.

Molecular genetics of long QT syndrome.

Long QT syndrome (LQTS) is a cardiac disorder associated with sudden death especially in young, seemingly healthy individuals. It is characterised by abnormalities of the heart beat detected as lengthening of the QT interval during cardiac repolarisation. The incidence of LQTS is given as 1 in 2000 but this may be an underestimation as many cases go undiagnosed, due to the rarity of the condition and the wide spectrum of symptoms. Presently 12 genes associated with LQTS have been identified with differing signs and symptoms, depending on the locus involved. The majority of cases have mutations in the KCNQ1 (LQT1), KCNH2 (LQT2) and SCN5A (LQT3) genes. Genetic testing is increasingly used when a clearly affected proband has been identified, to determine the nature of the mutation in that family. Unfortunately tests on probands may be uninformative, especially if the defect does not lie in the set of genes which are routinely tested. Novel mutations in these known LQTS genes and additional candidate genes are still being discovered. The functional implications of these novel mutations need to be assessed before they can be accepted as being responsible for LQTS. Known epigenetic modification affecting KCNQ1 gene expression may also be involved in phenotypic variability of LQTS. Genetic diagnosis of LQTS is thus challenging. However, where a disease associated mutation is identified, molecular diagnosis can be important in guiding therapy, in family testing and in determining the cause of sudden cardiac death. New developments in technology and understanding offer increasing hope to families with this condition.

Mutations at KCNQ1 and an unknown locus cause long QT syndrome in a large Australian family: implications for genetic testing.

A large Australian family affected with long QT syndrome (LQTS) was studied. The medical characteristics of the 16 clinically affected members were consistent with LQT1. A previously identified mutation in KCNQ1 was found in 12 affected individuals and 1 unaffected infant but absent in 4 affected family members. A haplotype consisting of specific alleles for microsatellites flanking in KCNQ1 was associated with the mutation. This was absent from the four affected individuals without the mutation, who had three different haplotypes in this region, indicating that LQTS is unlikely to be segregating with KCNQ1 in these anomalous family members. A genome scan revealed 12 regions where all four of these individuals shared alleles. One region on chromosome 21 contained the KCNE1, KCNE2, KCNJ6, and KCNJ15 genes. A common variant of KCNE1 was segregating in the family but did not explain the anomalous cases. A candidate region on chromosome 7 contained the AKAP9 and KCND2 genes. A previously reported mutation in the N-terminal Yotiao region of AKAP9 was absent from the family. No evidence was found implicating any other known or suspected LQTS gene. This family shows that there remain unidentified genetic causes of LQTS which are clinically significant and highlights the difficulties associated with genetic testing in LQTS, since we cannot rule out risk in individuals who are negative for the known mutation in KCNQ1 without knowing the second disease locus.