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A nonsputum triage test to rule out tuberculosis (TB) disease is a WHO high-priority diagnostic, and a combinatory score based on a 3-gene host signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge assay (“Xpert MTB Host Response” or Xpert-MTB-HR-Prototype) of this 3-gene signature on biobanked blood samples from people living with HIV (PLHIV) against a comprehensive microbiological reference standard (CMRS) and against Xpert MTB/RIF on the first sputum sample alone., A nonsputum triage test to rule out tuberculosis (TB) disease is a WHO high-priority diagnostic, and a combinatory score based on a 3-gene host signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge assay (“Xpert MTB Host Response” or Xpert-MTB-HR-Prototype) of this 3-gene signature on biobanked blood samples from people living with HIV (PLHIV) against a comprehensive microbiological reference standard (CMRS) and against Xpert MTB/RIF on the first sputum sample alone. We depict results based on performance targets set by the WHO in comparison with a laboratory-based C-reactive protein (CRP) assay. Of 201 patients included, 67 were culture positive for Mycobacterium tuberculosis. The areas under the concentration-time curve (AUCs) for Xpert-MTB-HR-Prototype were 0.89 (confidence interval [CI], 0.83 to 0.94) against the CMRS and 0.94 (CI, 0.89 to 0.98) against Xpert MTB/RIF. Considering Xpert-MTB-HR-Prototype as a triage test (at the nearest upper value of sensitivity to 90%), specificities were 55.8% (CI, 47.2 to 64.1%) compared to the CMRS and 85.9% (CI, 79.3 to 90.7%) compared to Xpert MTB/RIF as confirmatory tests. Considering Xpert-MTB-HR-Prototype as a stand-alone diagnostic test, at a specificity near 95%, the test achieved a sensitivity of 65.7% (CI, 53.7 to 75.9%), while the CRP assay achieved a sensitivity of only 13.6% (CI, 7.3 to 23.4%). In this first accuracy study of a prototype blood-based host marker assay, we show the possible value of the assay for triage and diagnosis in PLHIV.

A non-sputum triage test to rule out TB disease is a WHO high-priority diagnostic and a combinatory score based on a 3-gene host-signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge-assay (“Xpert MTB Host Response”, or Xpert-MTB-HR-Prototype) of this 3-gene signature on bio-banked blood-samples from PLHIV against a comprehensive microbiological reference standard (CMRS) and against Xpert® MTB/RIF on first sputum alone. We depict results based on performance targets set by WHO in comparison with a laboratory-based CRP assay. Of 201 patients included, 67 were culture-positive for Mycobacterium tuberculosis. AUC for the Xpert-MTB-HR-Prototype was 0·89 (CI 0·83-0·94) against the CMRS and 0·94 (CI 0·89-0·98) against Xpert MTB/RIF. Considering Xpert-MTB-HR-Prototype as a triage test (at nearest upper value of sensitivity to 90%), specificity was 55·8% (CI 47·2-64·1) compared to the CMRS and 85·9% (CI 79·3-90·7) compared to Xpert MTB/RIF as confirmatory tests. Considering Xpert-MTB-HR-Prototype as a stand-alone diagnostic test, at a specificity near 95%, the test achieved a sensitivity of 65·7% (CI 53·7-75·9) while CRP achieved a sensitivity of only 13·6% (CI 7·3-23·4). In this first accuracy study of a prototype blood-based host-marker assay, we show the possible value of the assay for triage and diagnosis in PLHIV.One Sentence SummaryThis is an accuracy study of a blood-based host-marker assay demonstrating its potential in triage and diagnosis of active tuberculosis (TB) in people living with HIV (PLHIV).

ObjectivesTo elucidate the views of intermittent catheter (IC) users regarding urinary tract infection (UTI) symptom presentation, terminology for describing signs and symptoms, the cause of UTI and management strategies.DesignQualitative study with semi-structured interviews. The transcribed text was analysed thematically.Setting12 general practitioner (GP) surgeries in Hampshire and Dorset, UK.ParticipantsA convenience sample of 30 IC users, aged over 18, using IC for at least 3 months who had at least one self-reported UTI since starting IC.ResultsParticipants reported a variety of signs and symptoms, such as urine cloudiness and smell, as indicators of UTI. The terms used often differed from those in the modified National Institute on Disability and Rehabilitation Research (NIDRR) symptom set. IC users had difficulty distinguishing possible UTI symptoms from those of their comorbidities. They expressed uncertainty about the cause of UTI, often attributing it to poor hygiene and lifestyle behaviours. Whereas some viewed UTI as an expected consequence of IC use that could be self-managed, others felt more concerned and were more reliant on their GP for support. A range of management strategies was described, including drinking more fluids, increased attention to personal hygiene and self-medicating with antibiotics.ConclusionsThere is uncertainty among IC users about UTI signs and symptoms and when to seek help. Individual accounts of UTI fitted generally within the modified NIDRR descriptors but adopted less technical and more ‘lay’ language. IC users’ descriptions of UTI signs and symptoms can lack precision, owing partly to the presence of underlying health conditions. This, together with differing levels of concern about the need to seek help and self-medication with antibiotics, presents challenges for the GP. This study provides the basis for developing a self-help tool which may aid identification of UTI and enhance communication with healthcare professionals.

Studies have shown that people who require single room isolation while in hospital often feel lonely, sad, worried, bored and in need of information. A literature review identified only 16 papers reporting on the child's experience of isolation. Findings indicate that children feel lonely, are scared of the personal protective equipment and feel bored. Patients' parents feel guilty, worried and under increased pressure to visit their children. It is also suggested that isolation may affect child development.

Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 106 International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.

ObjectivesTo explore the views of intermittent catheter (IC) users regarding the advantages and disadvantages of single-use or reuse of catheters.DesignQualitative study with semi-structured interviews. The interviews were recorded, transcribed and analysed thematically.SettingParticipant’s own homes in Hampshire and Dorset, UK.ParticipantsA convenience sample of 39 IC users, aged 23–86 years, using IC for at least 3 months.ResultsThe analysis revealed four main themes: concerns regarding risk of urinary tract infection (UTI); cleaning, preparation and storage; social responsibility; practicalities and location. The main concern was safety, with the fear that reuse could increase risk of UTI compared with single-use sterile catheters. If shown to be safe then around half of participants thought they might consider reusing catheters. The practicalities of cleaning methods (extra products, time and storage) were considered potentially burdensome for reuse; but for single-use, ease of use and instant usability were advantages. Always having a catheter without fear of ‘running out’ was considered an advantage of reuse. Some participants were concerned about environmental impact (waste) and cost of single-use catheters. The potential for reuse was usually dependent on location. The analysis showed that often the disadvantages of single-use could be off-set by the advantages of reuse and vice versa, for example, the need to take many single-use catheters on holiday could be addressed by reuse, while the burden of cleaning would be obviated by single-use.ConclusionsIf shown to be safe with a practical cleaning method, some participants would find reuse an acceptable option, alongside their current single-use method. The choice to use a mixture of single-use and reuse of catheters for different activities (at home, work or holiday) could optimise the perceived advantages and disadvantages of both. The safety and acceptability of such an approach would require testing in a clinical trial.

The striatum is known to possess high levels of D1-like and D2-like receptors (D1Rs and D2Rs, respectively). We have previously shown that selective inhibition of D1Rs increases the dopaminergic metabolic response and proposed that this effect is associated with the concomitant activation of postsynaptic D2Rs by endogenous dopamine (DA). Here, we examined whether activation of D2Rs modulates the metabolism and synthesis of DA in the striatum. We used male Wistar rats to evaluate the effects of the systemic administration of a D2R agonist (bromocriptine), a D1R antagonist (SCH-23390), and the co-administration of these compounds with pargyline on the inhibition of monoamine oxidase. DA and l-3,4-dihidroxyphenylacetic acid (DOPAC) levels and 3,4-dihydroxy-l-phenylalanine (l-DOPA) content were measured using high performance liquid chromatography. The systemic administration of SCH-23390 alone, at 0.25, 0.5, 1 or 2 mg/kg, significantly (P

Lymphocytes adhere to cells or extracellular matrices to perform functions relating to cytotoxicity, extravasation and tissue localization, as well as modulation of lymphocyte growth and maturation. This adherence is mainly mediated by 3 families of cell-surface adhesion molecules: integrins, immunoglobulin-related molecules and selectins. Since variations in the degree of adherence may affect the pathophysiology of lymphoproliferative disorders, the expression of a large number of adhesion molecules was analysed on Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), and on EBV-positive or EBV-negative Burkitt's lymphoma (BL) lines, by immunofluorescence flow cytometry and immunoprecipitation with monoclonal antibodies. With regard to the beta 1, beta 2 and beta 3 integrin subfamilies, LCLs strongly expressed CD49d/CD29 (VLA-4), CD11a/CD18 (Leu-CAMa, LFA-1) and CD51/CD61 (vitronectin receptor). These cells also abundantly expressed CD54 (ICAM-1) and CD58 (LFA-3) as well as the "homing receptors" L-selectin (LECAM-1) and CD44. BL lines had considerably lower amounts of VLA-4 than LCLs, and ICAM-1 was expressed only by some of the tumor lines. All other adhesion molecules were absent or minimally expressed in the BL cells.

In humans the glycoprotein complexes CD11 CD18 mediate leukocyte adhesion to cells. Mouse monoclonal antibodies (mAb) 60.3, 7E4, and IB4 to human CD18, found to cross-react with rabbit white blood cells, were used to identify the antigen in rabbit cells and to study adherence of rabbit leukocytes in vitro and in vivo . These antibodies labeled almost all unfractionated rabbit blood leukocytes and immunoprecipitated surface glycopolypeptides with apparent molecular weights of 85,000 and 150,000 from these cells. Adhesion of purified rabbit polymorphonuclear cells (PMNs) to cultured vascular endothelial cells in the presence of phorbol ester was blocked by the antibodies in a dose-dependent manner. The acute inflammatory response characterized by local accumulation of PMNs and concomitant plasma extravasation following intradermal injections of zymosan-activated serum (ZAS) in rabbits was inhibited in animals pretreated intravenously with anti-CD18 mAb. Intravital microscopy of the rabbit tenuissimus muscle demonstrated that anti-CD18 treatment specifically blocked the adhesion of activated leukocytes to the venular endothelium and thereby the subsequent diapedesis of these cells into the extravascular space. The lymphocyte-dependent tissue swelling resulting from a delayed-type hypersensitivity reaction in the rabbit ear was partially inhibited by anti-CD18 mAb. Systemic anti-CD18 treatment induced a pronounced increase in the number of circulating mononuclear and polymorphonuclear cells with a maximum at 24 hr after injection of the antibody. It is concluded that GP150 GP85 is the rabbit homologue of human CD11 CD18 , and that leukocyte-cell adhesion mediated by these glycoprotein complexes participates in acute and delayed inflammatory responses and leukocyte distribution in vivo .

Hematogenous spread of tumor cells and metastasis formation in secondary organs are insidious aspects of cancer. In the present intravital microscopic study in the rabbit mesentery, we examined the in vivo flow behavior of six human tumor cell lines of different histological origin. The tumor cells and human neutrophils were injected locally into a side branch of the superior mesenteric artery upstream of the observed microvascular area in the mesentery. None of the tumor cells behaved similar to the leukocytes of which a substantial fraction rolled along the endothelium of small venules. Thus, the tumor cells passed the same venular segments without interacting with the endothelial lining. Yet, three of the tumor cell lines (HT-29, DLD-1, and HCT-8) were strongly positive for the oligosaccharides Lewis x , sialyl-Lewis x , and sialyl-Lewis a which are recognized by the endothelial selectins that mediate leukocyte rolling. On the other hand, some tumor cells were trapped in the smallest vessels and remained so throughout the experimental period, apparently due to a discrepancy in size between tumor cells and microvessel lumen. Taken together, our in vivo findings suggest that initial microvascular arrest of metastasizing tumor cells is dependent primarily on mechanical factors rather than on receptor-mediated leukocyte-like adhesive interactions.

Monocyte/macrophages adhere to cells (lymphocytes, vascular endothelial and other cell types) and to extracellular matrix components (fibronectin and laminin) by using specific cell surface adhesive structures. In the present study we have analyzed expression of integrins, immunoglobulin (Ig)-related, selectins, and other adhesion molecules on blood monocytes, in vitro differentiated macrophages (ivMs), and alveolar macrophages (AMs), obtained from healthy nonsmokers by bronchoalveolar lavage (BAL). We have also investigated expression of adhesion molecules on myelomonocytic cell lines HL-60, THP-1, KG-1, and U937 before and after tetradecanoyl phorbol acetate (TPA)-induced differentiation. With regard to the integrin family, monocytes expressed beta 1 (CD29), alpha 4, alpha 5, alpha 6, beta 2 (CD18), CD11a, CD11b, and CD11c subunits, but not alpha V (CD51). Some reactivity with mAbs against the platelet antigens CD41b (IIb) and CD61 (beta 3) was detected. The Ig-related molecules CD54 (ICAM-1), ICAM-2, and CD58 (LFA-3) were expressed, as well as L-selectin and the carbohydrate ligands Le(x) (CD15) and sialyl Le(x). Immunolabeling for the structurally unrelated molecules CD44 and CD36 was strongly positive. In comparison to monocytes, AMs showed much lower expression of alpha 4, alpha 6, beta 2, CD11a, CD11b, L-selectin, Le(x), and sialyl Le(x). Moreover, ICAM-2 and CD36 were practically absent whereas expression of alpha 3, but not of CD11c, was higher. Similar results were obtained with ivMs. All four myelomonocytic cell lines showed down-regulation of alpha 4 and up-regulation of CD11c after TPA treatment. These findings indicate that maturation of monocytes into macrophages is accompanied by characteristic changes in adhesion molecule expression. The particular array of adhesion molecules on monocytes and macrophages may account for differences in the functional properties of these cells.

Insulin-dependent diabetes mellitus (IDDM) results from a T cell-dependent autoimmune destruction of insulin-producing pancreatic beta cells. In the present study, expression of adhesion molecule ICAM-1 (CD54) on pancreatic beta cells was studied in normal, obese hyperglycemic (ob/ob), and nonobese diabetic (NOD) mice. Freshly isolated pancreatic beta cells from ob/ob mice did not express ICAM-1, but treatment of the cells with IL-1-beta, TNF-alpha, or INF-gamma strongly induced its expression as measured by immunofluorescence flow cytometry. The cytokines acted in a dose- and time-dependent manner. Maximal induction by either cytokine occurred at 24 hr and thereafter expression decreased, except for INF-gamma. Immunoprecipitation from IL-1-beta-treated beta cells demonstrated a cell-surface glycoprotein with an apparent molecular weight of 95 kDa. ICAM-1 expression was undetectable on pancreatic beta cells of normal and ob/ob mice as measured by immunohistochemistry. In NOD mice at different ages (1 to 6 months) ICAM-1 was also undetectable on beta cells, in contrast to the strong expression on infiltrating mononuclear cells. The present study indicates that mouse pancreatic beta cells, under certain conditions, can express ICAM-1.

The beta 2-integrin CD11a/CD18 binds to the intercellular adhesion molecules (ICAM)-1 (CD54) and ICAM-2. ICAM-1 has a wide distribution, and its expression is up-regulated by various cytokines. In contrast, ICAM-2 has a more restricted distribution, and is mainly expressed on endothelial cells. In the present study we show that it is not induced by inflammatory cytokines or other treatments on any of several cells studied. Moreover, antibodies to the intercellular adhesion ligands were not able to block all CD11a/CD18-dependent adhesion, indicating the presence of additional CD11a/CD18 ligands.

Leukocyte adhesion to cells is mediated by the cell-surface glycoprotein complex CDIIa-c/CD18 and, in some cases, the glycoprotein gp84. The process is associated with leukocyte activation and modulates lymphocyte proliferation and maturation. Epstein-Barr virus (EBV)-transformed normal B lymphocytes give rise to lymphoblastoid cell fines (LCLs) which grow as large clusters mediated by adhesion molecules. In contrast, newly explanted EBV-infected Burkitt lymphoma (BL) cells usually grow as single cells or as loose clusters. We now report that EBV positive- and EBV-negative BL lines lack the adhesive protein complex, or have only low levels of it, whereas LCLs, representing various stages of B-lymphocyte development, contain considerably higher amounts, as measured by immunofluorescence flow cytometry and immuno precipitation. The level of gp84 expression is of the same magnitude in both types of cells.

Monoclonal antibody LB-2 to a surface antigen on human B cells, lymphoblast, monocytes and vascular endothelial cells largely inhibited adhesion among Epstein Barr virus-immortalized normal B cells (EBV-B) and concanavalin A-stimulated blood mononuclear cells (Con A-BMC) before and after phorbol ester treatment. The antibody inhibited to a lesser extent phorbol ester-induced aggregation of monocytes, U937 cells and fresh BMC and had virtually no inhibitory effect on the adhesion among enriched T cells and granulocytes. A surface glycoprotein band of 84 kDa was obtained from EBV-B cells by immunoprecipitation and gel electrophoresis. Immunological and biochemical studies clearly distinguished this molecule from gp90 and associated glycoproteins which also mediate leukocyte adhesion.

In humans, lymphocyte adhesion to cells is mediated by the protein heterodimer CD11a/CD18 (Leu-CAMa, LFA-1) and its ligand CD54 (ICAM-1). Although the murine CD11a/CD18 is well characterized, the mouse homologue of human ICAM-1 has not been identified. In the present study a rat monoclonal antibody to the murine lymphocyte activation antigen MALA-2 was found to inhibit in a dose-dependent manner the phorbol ester-enhanced aggregation of mouse lymphoblasts, an adhesion-specific assay, and hence to define an adhesion molecule. By immunofluorescence flow cytometry the antigen expression was low on spleen cells but it largely increased after stimulation with mitogens. The antigen was expressed by some, but not all, lymphoid cell lines, and myelomonocytic and mastocytoma cells were also positive. In frozen tissue sections MALA-2 was mainly detected on germinal center B cells, dendritic cells, macrophages and vascular endothelium, including high endothelial venules. Cell surface labeling followed by immunoprecipitation and gel electrophoresis indicated that the antigen is a sialoglycoprotein which has a relative molecular mass of 95 kDa and displays a faster electrophoretic mobility under non-reducing conditions. The function, cellular distribution and molecular properties of MALA-2 are indistinguishable from those of human ICAM-1.

Adhesion of monocytes to each other and to T cells and substrates is increased by phorbol esters. In the presence of these compounds monocyte aggregation was almost completely inhibited (greater than 90%) by monoclonal antibody 60.3. This antibody recognizes GP90 (CD18), a leukocyte surface glycoprotein which is separately and noncovalently associated to either GP160 (CD11a), GP155 (CD11b), or GP130 (CD11c). Anti-LFA-1 antibody (CD11a) was only partially inhibitory (35%) while antibodies 60.1 (CD11b) and anti-Leu-M5 (CD11c) had a minimal inhibitory effect (10%). Antibody LB-2 recognizing a single glycoprotein distinct from the GP90-GP160 complex and expressed on activated B and T cells, monocytes, and vascular endothelial cells was partially inhibitory (22%). Monoclonal antibodies anti-C3bR (CD35), T29/33 (CD45, leukocyte common antigen 200). TA-1 (CD11a), OKM1 (CD11b), F10-44-2 (brain-leukocyte antigen), OKM5 (monocyte-endothelial cell antigen) and to class I or class II molecules exerted no inhibition on the monocyte aggregation. Fab fragments of antibody 60.3 efficiently inhibited not only monocyte aggregation in the absence or presence of phorbol esters but also adhesion of these cells to autologous or allogeneic T lymphocytes and, to a lesser extent, to plastic surfaces. It is thus concluded that GP90, either alone or associated to the larger glycoproteins, and LB-2 antigen mediate monocyte adhesion.