Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

RATIONALE: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia and primary immunodeficiency disorders), but most cases remain idiopathic. OBJECTIVES: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. METHODS: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived, cells, cell cultures and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. MEASUREMENTS AND MAIN RESULTS: We identified bi-allelic pathogenic variants in WFDC2 in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and thus secretion of mature WFDC2. CONCLUSIONS: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

The role of SPAG1 in the assembly of axonemal dyneins in human airway epithelia.

Mutations in SPAG1, a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium, result in primary ciliary dyskinesia (PCD), a genetically heterogenous disorder characterized by chronic oto-sino-pulmonary disease, infertility and laterality defects. To further elucidate the role of SPAG1 in dynein assembly, we examined its expression, interactions and ciliary defects in control and PCD human airway epithelia. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains and canonical components of the R2TP complex. Protein levels of dynein heavy chains (DHCs) and interactions between DHCs and dynein intermediate chains (DICs) were reduced in SPAG1 mutants. We also identified a previously uncharacterized 60 kDa SPAG1 isoform, through examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants, that can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In summary, our data show that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding and/or binding of the DHCs to the DIC complex.

Expression of a Truncated Form of ODAD1 Associated with an Unusually Mild Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a rare lung disease caused by mutations that impair the function of motile cilia, resulting in chronic upper and lower respiratory disease, reduced fertility, and a high prevalence of situs abnormalities. The disease is genetically and phenotypically heterogeneous, with causative mutations in > 50 genes identified, and clinical phenotypes ranging from mild to severe. Absence of ODAD1 (CCDC114), a component of the outer dynein arm docking complex, results in a failure to assemble outer dynein arms (ODAs), mostly immotile cilia, and a typical PCD phenotype. We identified a female (now 34 years old) with an unusually mild clinical phenotype who has a homozygous non-canonical splice mutation (c.1502+5G>A) in ODAD1. To investigate the mechanism for the unusual phenotype, we performed molecular and functional studies of cultured nasal epithelial cells. We demonstrate that this splice mutation results in the expression of a truncated protein that is attached to the axoneme, indicating that the mutant protein retains partial function. This allows for the assembly of some ODAs and a significant level of ciliary activity that may result in the atypically mild clinical phenotype. The results also suggest that partial restoration of ciliary function by therapeutic agents could lead to significant improvement of disease symptoms.

Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study.

BACKGROUND: International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. METHODS: In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0-16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3-16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. FINDINGS: Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant; n=65) and childhood patients (≥4·3 years; MBSHH-Child; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR [n=65] and MBGrp4-HR [n=85]) and low-risk (MBGrp3-LR [n=50] and MBGrp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82-100]); standard risk (50 [23%] patients; 81% survival [70-94]); high-risk (82 [38%] patients; 42% survival [31-56]); and very high-risk (29 [13%] patients; 28% survival [14-56]). INTERPRETATION: The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. FUNDING: Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

Hepatitis B virus (HBV) infection, immunity and susceptibility among men who have sex with men (MSM), Los Angeles County, USA.

Men who have sex with men (MSM) bear a disproportionate burden of hepatitis B virus (HBV) infections. We used serologic data from the National HIV Behavioral Surveillance (NHBS) system to determine the prevalence and correlates of HBV infection, immunization, and susceptibility in a sample of Los Angeles County MSM. Approximately 19 % (95 % CI 15-24 %) had serologic evidence of current or past infection, while 35 % (95 % CI 30-40 %) were susceptible. Compared with the youngest age group, MSM ages 40-49 years had a lower prevalence of immunization (aPR 0.28, 95 % CI 0.17-0.45) and a higher prevalence of infection (aPR 8.53, 95 % CI 3.95-18.4) and susceptibility (aPR 2.02, 95 % CI 1.13-3.63). We also observed poor concordance between self-reported and serologic measures of vaccination. Our results indicate the possibility of missed opportunities to vaccinate MSM. Gaps in implementing existing vaccination strategies must be addressed to increase hepatitis B vaccination coverage for MSM, especially in older age groups.

A catalytic process enables efficient and programmable access to precisely altered indole alkaloid scaffolds.

A compound's overall contour impacts its ability to elicit biological response, rendering access to distinctly shaped molecules desirable. A natural product's framework can be modified, but only if it is abundant and contains suitably modifiable functional groups. Here we introduce a programmable strategy for concise synthesis of precisely altered scaffolds of scarce bridged polycyclic alkaloids. Central to our approach is a scalable catalytic multi-component process that delivers diastereo- and enantiomerically enriched tertiary homoallylic alcohols bearing differentiable alkenyl moieties. We used one product to launch progressively divergent syntheses of a naturally occurring alkaloid and its precisely expanded, contracted and/or distorted framework analogues (average number of steps/scaffold of seven). In vitro testing showed that a skeleton expanded by one methylene in two regions is cytotoxic against four types of cancer cell line. Mechanistic and computational studies offer an account for several unanticipated selectivity trends.

Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications.

Artificial lipid bilayers have revolutionized biochemical and biophysical research by providing a versatile interface to study aspects of cell membranes and membrane-bound processes in a controlled environment. Artificial bilayers also play a central role in numerous biosensing applications, form the foundational interface for liposomal drug delivery, and provide a vital structure for the development of synthetic cells. But unlike the envelope in many living cells, artificial bilayers can be mechanically fragile. Here, we develop prototype scaffolds for artificial bilayers made from multiple chemically linked tiers of actin filaments that can be bonded to lipid headgroups. We call the interlinked and layered assembly a multiple minimal actin cortex (multi-MAC). Construction of multi-MACs has the potential to significantly increase the bilayer's resistance to applied stress while retaining many desirable physical and chemical properties that are characteristic of lipid bilayers. Furthermore, the linking chemistry of multi-MACs is generalizable and can be applied almost anywhere lipid bilayers are important. This work describes a filament-by-filament approach to multi-MAC assembly that produces distinct 2D and 3D architectures. The nature of the structure depends on a combination of the underlying chemical conditions. Using fluorescence imaging techniques in model planar bilayers, we explore how multi-MACs vary with electrostatic charge, assembly time, ionic strength, and type of chemical linker. We also assess how the presence of a multi-MAC alters the underlying lateral diffusion of lipids and investigate the ability of multi-MACs to withstand exposure to shear stress.

NR0B2 re-educates myeloid immune cells to reduce regulatory T cell expansion and progression of breast and other solid tumors.

Although survival from breast cancer has dramatically increased, many will develop recurrent, metastatic disease. Unfortunately, survival for this stage of disease remains very low. Activating the immune system has incredible promise since it has the potential to be curative. However, immune checkpoint blockade (ICB) which works through T cells has been largely disappointing for metastatic breast cancer. One reason for this is a suppressive myeloid immune compartment that is unaffected by ICB. Cholesterol metabolism and proteins involved in cholesterol homeostasis play important regulatory roles in myeloid cells. Here, we demonstrate that NR0B2, a nuclear receptor involved in negative feedback of cholesterol metabolism, works in several myeloid cell types to impair subsequent expansion of regulatory T cells (Tregs); Tregs being a subset known to be highly immune suppressive and associated with poor therapeutic response. Within myeloid cells, NR0B2 serves to decrease many aspects of the inflammasome, ultimately resulting in decreased IL1ß; IL1ß driving Treg expansion. Importantly, mice lacking NR0B2 exhibit accelerated tumor growth. Thus, NR0B2 represents an important node in myeloid cells dictating ensuing Treg expansion and tumor growth, thereby representing a novel therapeutic target to re-educate these cells, having impact across different solid tumor types. Indeed, a paper co-published in this issue demonstrates the therapeutic utility of targeting NR0B2.

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB.

Disseminated cutaneous mast cell tumors with epitheliotropism and systemic mastocytosis in a domestic cat.

A 15-year-old female domestic, medium-haired cat presented to the referring veterinarian with a 2-month history of multiple, raised, disseminated, nodular skin lesions. A biopsy of 1 of the lesions was submitted to the Oklahoma Animal Disease Diagnostic Laboratory for evaluation. Histologically, there were multiple dermal nodules composed of sheets of neoplastic round cells. Multifocally, the neoplastic cells formed multiple small clusters of 3 to 5 cells within the epidermis. Distinct cytoplasmic granules were evident within the neoplastic cells with toluidine blue and Giemsa stains. The neoplastic cells were immunoreactive for c-KIT and lacked immunoreactivity for cluster of differentiation 3 with immunohistochemistry. Based on these findings, multiple epitheliotropic cutaneous mast cell tumors were diagnosed. The cat's health declined rapidly despite aggressive treatment, and the animal was humanely euthanatized. A complete necropsy revealed sheets of similar neoplastic mast cells within the spleen, liver, and individual cells scattered within the bone marrow. Exon 11 of the c-KIT messenger RNA from 1 of the cutaneous masses and the spleen was amplified with reverse transcription polymerase chain reaction, sequenced, and compared with the published c-KIT messenger RNA sequence from fetal cat tissues. The maximum identity was 100% for both tissue samples. To the authors' knowledge, the present report is the first to describe disseminated cutaneous mast cell tumors with epitheliotropism and systemic mastocytosis in a domestic cat.

Cigarette Smoke Exposure Induces Retrograde Trafficking of CFTR to the Endoplasmic Reticulum.

Chronic obstructive pulmonary disease (COPD), which is most commonly caused by cigarette smoke (CS) exposure, is the third leading cause of death worldwide. The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane anion channel that is widely expressed in epithelia throughout the body. In the airways, CFTR plays an important role in fluid homeostasis and helps flush mucus and inhaled pathogens/toxicants out of the lung. Inhibition of CFTR leads to mucus stasis and severe airway disease. CS exposure also inhibits CFTR, leading to the decreased anion secretion/hydration seen in COPD patients. However, the underlying mechanism is poorly understood. Here, we report that CS causes CFTR to be internalized in a clathrin/dynamin-dependent fashion. This internalization is followed by retrograde trafficking of CFTR to the endoplasmic reticulum. Although this internalization pathway has been described for bacterial toxins and cargo machinery, it has never been reported for mammalian ion channels. Furthermore, the rapid internalization of CFTR is dependent on CFTR dephosphorylation by calcineurin, a protein phosphatase that is upregulated by CS. These results provide new insights into the mechanism of CFTR internalization, and may help in the development of new therapies for CFTR correction and lung rehydration in patients with debilitating airway diseases such as COPD.

The proline repeat domain of p53 binds directly to the transcriptional coactivator p300 and allosterically controls DNA-dependent acetylation of p53.

The transcription coactivator p300 cannot acetylate native p53 tetramers, thus revealing intrinsic conformational constraints on p300-catalyzed acetylation. Consensus site DNA is an allosteric effector that promotes acetylation of p53, suggesting that p300 has an undefined conformationally flexible interface within the p53 tetramer. To identify such conformationally responsive p300-binding sites, p300 was subjected to peptide selection from a phage-peptide display library, a technique that can define novel protein-protein interfaces. The enriched p300-binding peptides contained a proline repeat (PXXP/PXPXP) motif, and five proline repeat motifs actually reside within the p53 transactivation domain, suggesting that this region of p53 may harbor the second p300 contact site. p300 binds in vitro to PXXP-containing peptides derived from the proline repeat domain, and PXXP-containing peptides inhibit sequence-specific DNA-dependent acetylation of p53, indicating that p300 docking to both the LXXLL and contiguous PXXP motif in p53 is required for p53 acetylation. Deletion of the proline repeat motif of p53 prevents DNA-dependent acetylation of p53 by occluding p300 from the p53-DNA complex. Sequence-specific DNA places an absolute requirement for the proline repeat domain to drive p53 acetylation in vivo. Chromatin immunoprecipitation was used to show that the proline repeat deletion mutant p53 is bound to the p21 promoter in vivo, but it is not acetylated, indicating that proline-directed acetylation of p53 is a post-DNA binding event. The PXXP repeat expands the basic interface of a p300-targeted transactivation domain, and proline-directed acetylation of p53 at promoters indicates that p300-mediated acetylation can be highly constrained by substrate conformation in vivo.

Incident Infection and Resistance Mutation Analysis of Dried Blood Spots Collected in a Field Study of HIV Risk Groups, 2007-2010.

OBJECTIVE: To assess the utility of cost-effective dried blood spot (DBS) field sampling for incidence and drug resistance surveillance of persons at high risk for HIV infection. METHODS: We evaluated DBS collected in 2007-2010 in non-clinical settings by finger-stick from HIV-positive heterosexuals at increased risk of HIV infection (n = 124), men who have sex with men (MSM, n = 110), and persons who inject drugs (PWID, n = 58). Relative proportions of recent-infection findings among risk groups were assessed at avidity index (AI) cutoffs of ≤25%, ≤30%, and ≤35%, corresponding to an infection mean duration of recency (MDR) of 220.6, 250.4, and 278.3 days, respectively. Drug resistance mutation prevalence was compared among the risk groups and avidity indices. RESULTS: HIV antibody avidity testing of all self-reported ARV-naïve persons (n = 186) resulted in 9.7%, 11.3% and 14.0% with findings within the 221, 250, and 278-day MDRs, respectively. The proportion of ARV-naïve MSM, heterosexuals, and PWID reporting only one risk category who had findings below the suggested 30% AI was 23.1%, 6.9% and 3.6% (p<0.001), respectively. MSM had the highest prevalence of drug resistance and the only cases of transmitted multi-class resistance. Among the ARV-experienced, MSM had disproportionately more recent-infection results than did heterosexuals and PWID. CONCLUSIONS: The disproportionately higher recent-infection findings for MSM as compared to PWID and heterosexuals increased as the MDR window increased. Unreported ARV use might explain greater recent-infection findings and drug resistance in this MSM population. DBS demonstrated utility in expanded HIV testing; however, optimal field handling is key to accurate recent-infection estimates.

In vitro activities of Daptomycin, Linezolid, and Quinupristin-Dalfopristin against a challenge panel of Staphylococci and Enterococci, including vancomycin-intermediate staphylococcus aureus and vancomycin-resistant Enterococcus faecium.

We assessed the in vitro activities of daptomycin, linezolid, and quinupristin-dalfopristin (QD) against a contemporary challenge panel of 88 staphylococcal and 90 enterococcal isolates. The staphylococci selected included vancomycin-intermediate Staphylococcus aureus (VISA), methicillin-resistant S. aureus, and coagulasenegative staphylococci. Enterococcal isolates included vancomycin-resistant Enterococcus faecium (VREF) containing either vanA, vanB1, or vanD. The MICs of daptomycin, linezolid, and QD were determined using commercial broth microdilution panels. All three VISA isolates were susceptible to daptomycin, linezolid, and QD. QD was the most active agent against staphylococcal isolates (MIC50 < or = 0.5 microg/ml and MIC90 = 1 microg/ml), including those with decreased susceptibility to vancomycin. QD was also the most active agent against VREF (MIC90 < or = 0.5 microg/ml). No differences were seen for susceptibility of vanA, vanB1, and vanD VREF strains for daptomycin, linezolid, or QD. Daptomycin was the most effective against E. faecalis. On the basis of manufacturer-suggested interpretive criteria, 92% of isolates were susceptible (MIC90 = 4 microg/ml). All isolates tested were susceptible to at least one antimicrobial agent for which interpretive criteria have been defined. Population analysis of three S. aureus isolates for which the daptomycin MICs were 8 microg/ml showed a pattern of homogeneous resistance.

Multidisciplinary care of skin problems in stoma patients.

Skin integrity is essential for the normal usage of a stoma appliance. There is little published on the prevalence, prevention or management of stoma skin problems. Allergic contact dermatitis is often cited as the cause, usually without evidence from formal investigations. The authors approached, by postal questionnaire, 525 patients who had had a stoma formation in the last 10 years. A total of 325 responded. All those who described a skin problem were invited to attend a multidisciplinary clinic for further investigations and appropriate treatment of their peristomal skin. This may be severe and debilitating as well as socially restricting. However, with a multidisciplinary approach a number of conditions can be recognized and easily treated, thus improving the quality of life for stoma patients.

HIV infection and awareness among men who have sex with men-20 cities, United States, 2008 and 2011.

Over half of HIV infections in the United States occur among men who have sex with men (MSM). Awareness of infection is a necessary precursor to antiretroviral treatment and risk reduction among HIV-infected persons. We report data on prevalence and awareness of HIV infection among MSM in 2008 and 2011, using data from 20 cities participating in the 2008 and 2011 National HIV Behavioral Surveillance System (NHBS) among MSM. Venue-based, time-space sampling was used to recruit men for interview and HIV testing. We analyzed data for men who reported ≥ 1 male sex partner in the past 12 months. Participants who tested positive were considered to be aware of their infection if they reported a prior positive HIV test. We used multivariable analysis to examine differences between results from 2011 vs. 2008. HIV prevalence was 19% in 2008 and 18% in 2011 (p = 0.14). In both years, HIV prevalence was highest among older age groups, blacks, and men with lower education and income. In multivariable analysis, HIV prevalence did not change significantly from 2008 to 2011 overall (p = 0.51) or in any age or racial/ethnic category (p>0.15 in each category). Among those testing positive, a greater proportion was aware of their infection in 2011 (66%) than in 2008 (56%) (p<0.001). In both years, HIV awareness was higher for older age groups, whites, and men with higher education and income. In multivariable analysis, HIV awareness increased from 2008 to 2011 overall (p<0.001) and for all age and racial/ethnic categories (p<0.01 in each category). In both years, black MSM had the highest HIV prevalence and the lowest awareness among racial/ethnic groups. These findings suggest that HIV-positive MSM are increasingly aware of their infections.

Evaluation of the CDC proposed laboratory HIV testing algorithm among men who have sex with men (MSM) from five US metropolitan statistical areas using specimens collected in 2011.

BACKGROUND: Until recently most testing algorithms in the United States (US) utilized Western blot (WB) as the supplemental test. CDC has proposed an algorithm for HIV diagnosis which includes an initial screen with a Combo Antigen/Antibody 4th generation-immunoassay (IA), followed by an HIV-1/2 discriminatory IA of initially reactive-IA specimens. Discordant results in the proposed algorithm are resolved by nucleic acid-amplification testing (NAAT). OBJECTIVES: Evaluate the results obtained with the CDC proposed laboratory-based algorithm using specimens from men who have sex with men (MSM) obtained in five metropolitan statistical areas (MSAs). STUDY DESIGN: Specimens from 992 MSM from five MSAs participating in the CDC's National HIV Behavioral Surveillance System in 2011 were tested at local facilities and CDC. The five MSAs utilized algorithms of various screening assays and specimen types, and WB as the supplemental test. At the CDC, serum/plasma specimens were screened with 4th generation-IA and the Multispot HIV-1/HIV-2 discriminatory assay was used as the supplemental test. NAAT was used to resolve discordant results and to further identify acute HIV infections from all screened-non-reactive missed by the proposed algorithm. Performance of the proposed algorithm was compared to site-specific WB-based algorithms. RESULTS: The proposed algorithm detected 254 infections. The WB-based algorithms detected 19 fewer infections; 4 by oral fluid (OF) rapid testing and 15 by WB supplemental testing (12 OF and 3 blood). One acute infection was identified by NAAT from all screened-non-reactive specimens. CONCLUSIONS: The proposed algorithm identified more infections than the WB-based algorithms in a high-risk MSM population. OF testing was associated with most of the discordant results between algorithms. HIV testing with the proposed algorithm can increase diagnosis of infected individuals, including early infections.

Identification of Escherichia coli O157 : H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis.

Enterohaemorrhagic Escherichia coli (EHEC) cause acute gastroenteritis in humans that may be complicated by life-threatening systemic sequelae. The predominant EHEC serotype affecting humans in the UK and North America is O157 : H7 and infections are frequently associated with contact with ruminant faeces. Strategies to reduce the carriage of EHEC in ruminants are expected to lower the incidence of human EHEC infections; however, the molecular mechanisms underlying persistence of EHEC in ruminants are poorly understood. This paper reports the first comprehensive survey for EHEC factors mediating colonization of the bovine intestines by using signature-tagged transposon mutagenesis. Seventy-nine E. coli O157 : H7 mutants impaired in their ability to colonize calves were isolated and 59 different genes required for intestinal colonization were identified by cloning and sequencing of the transposon insertion sites. Thirteen transposon insertions were clustered in the locus of enterocyte effacement (LEE), which encodes a type III protein secretion system required for the formation of attaching and effacing lesions on intestinal epithelia. A putative structural component of the apparatus (EscN) is essential for intestinal colonization; however, the type III secreted effector protein Map plays only a minor role. Other Type III secretion-associated genes were implicated in colonization of calves by E. coli O157 : H7, including z0990 (ecs0850), which encodes the non-LEE-encoded type III secreted effector NleD and the closely related z3023 (ecs2672) and z3026 (ecs2674) genes which encode homologues of Shigella IpaH proteins. We also identified a novel fimbrial locus required for intestinal colonization in calves by E. coli O157 : H7 (z2199-z2206; ecs2114-ecs2107/locus 8) and demonstrated that a mutant harbouring a deletion of the putative major fimbrial subunit gene is rapidly out-competed by the parent strain in co-infection studies. Our data provide valuable new information for the development of intervention strategies.

MtuA, a lipoprotein receptor antigen from Streptococcus uberis, is responsible for acquisition of manganese during growth in milk and is essential for infection of the lactating bovine mammary gland.

A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO(4) but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10(5) CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn(2+) during growth in bovine milk and infection of the lactating bovine mammary gland.