Predictive factors for identifying macrolide responder in treating chronic rhinosinusitis.

BACKGROUND: Low-dose macrolides (LDM) are anti-inflammatory agents with antineutrophilic activity, but patient selection for LDM therapy in treating chronic rhinosinusitis (CRS) is controversial. This study aimed to assess factors which predict LDM responders. METHODOLOGY: A prospective cohort study was performed. Patients with CRS received roxithromycin (150 mg) once daily for 12 weeks. Nasal secretions and serology were collected. Nine predictors for LDM response were assessed: nasal secretion IgE, nasal secretion IL-5, serum IgE, serum eosinophils, serum neutrophils, nasal polyps, asthma, allergy, and aspirin hypersensitivity, using receiver-operating curve analysis and multivariable logistic regression. Macrolide responders were those with sino-nasal outcome test-22 improvement, symptoms visual analogue scale decreased to ≤ ≤ ≤5, and no rescue medication. RESULTS: One hundred CRS patients (mean age 47.4 +- 14.1 years, 45% male) were enrolled. Univariable logistic regression showed local total IgE less than 5.21; and serum eosinophils less than 2.2% associated with macrolide response. Multivariate models showed local total IgE maintained an independent association with macrolide response, with an ability to discriminate between responders and non-responders of 63%. Serum total IgE, nasal secretion IL-5, serum neutrophil, nasal polyp, asthma, allergy, and aspirin hypersensitivity showed no association with LDM response. CONCLUSIONS: Low total IgE level in the nasal secretion but not in the serum, predict LDM response.

Performance characteristics and optimal cut-off value of triple adenylate nucleotides test versus adenosine triphosphate test as point-of-care testing for predicting inadequacy of duodenoscope reprocessing.

BACKGROUND: Adenosine triphosphate (ATP) test based on one nucleotide has been applied as point-of-care testing (POCT) for bacterial contamination in the medical and food industries. Hypothetically, testing three adenylate nucleotides (A3) may provide better detection of duodenoscope bacterial contamination than ATP test. AIM: To evaluate performance characteristics and optimal cut-off value of A3 and ATP tests in predicting bacterial contamination of duodenoscopes. METHODS: Four hundred duodenoscope samples obtained after 100 endoscopic retrograde cholangiopancreatography procedures were randomized into group A (A3 test) or B (ATP test). Samples were collected from the elevator at the four-step cleaning process of duodenoscope. We defined the new cut-off value of the test for reaching 100% negative predictive value (NPV) from our receiver operating characteristic (ROC). FINDINGS: Using the cultures from the four-step cleaning process as the reference, the areas under ROC (AUROC) were 0.83 and 0.84 for group A (N = 200) and group B (N = 200), respectively. Using the cultures from post-high-level disinfection (HLD) as the reference, the AUROC were 0.35 and 0.74 for group A (N = 50) and group B (N = 50), respectively. We investigated ATP as a POCT after HLD with a new cut-off value of 40 RLU. However, this threshold did not allow detection of low numbers of bacteria. CONCLUSION: A3 and ATP tests provide good performances in predicting bacterial contamination of duodenoscopes for the four-step cleaning process. The ATP <40 RLU is helpful as a POCT after HLD; however, the limitation of this cut-off value is its inability to detect low numbers of bacteria.

Chronic occupational exposure to lead leads to significant mucocutaneous changes in lead factory workers.

BACKGROUND: Chronic lead toxicity is a worldwide public health problem. Lead possesses deleterious effects on many organ systems. However, little is known regarding its clinical and biophysical effects on the skin. OBJECTIVE: To investigate mucocutaneous signs and biophysical property changes in skin after chronic lead toxicity. METHODS: One hundred and eighty-seven patients who were car battery workers participated in the study. Complete history and physical examination were performed. Blood was collected for laboratory analyses. Thorough skin examination by dermatologists was carried out in 134 subjects. Additionally, 96 patients with blood lead levels (BLL) >70 µg/dL were further evaluated for skin elasticity, sebum content, transepidermal water loss (TEWL), hydration, pH and pigmentation. An equal number of age-, sex- and skin-type-matched subjects were recruited as controls. RESULTS: The mean BLL of all subjects was 74.15 ± 11.58 µg/dL. The most frequently observed signs were gingival brown pigmentation in 112 (83.6%), gingivitis in 111 (82.8%) and lead line in 66 (49.3%) patients. The lead line was found in subjects with significantly higher BLLs (adjusted mean difference 6.45, 95% CI 2.30-10.60 µg/dL, P = 0.003) and in association with gingivitis (adjusted OR 7.32, 95% CI 2.08-25.74, P = 0.002). Mean BLL of the patients who underwent biophysical assessment was 82.77 ± 9.80 µg/dL. Patients exhibited a statistically significant lower skin hydration observed by corneometer as well as elasticity. The adjusted ORs of having dry skin and lower elasticity were 15.32 (95% CI 4.41-53.24), P < 0.001) and 1.96 (95% CI 1.06-3.60), P = 0.031), respectively. These differences were not significant for sebum content, TEWL, pH and pigmentation. CONCLUSION: Importantly, even in normal-appearing skin, level of hydration and elasticity decreased in lead-intoxicated patients. These results suggest that lead might possess harmful effects on the skin at measurable levels.

Cervical cytological abnormalities and HPV infection in perinatally HIV-infected adolescents.

BACKGROUND: Behaviourally HIV-infected adolescent females are at higher risk for abnormal cervical cytology and HPV infection compared to those who are uninfected, but data on perinatally HIV-infected adolescent females are lacking. METHODS: Cervical cytology, HPV infection and E6/E7 mRNA were assessed in sexually active 12-24-year-old adolescent females: perinatally HIV-infected (group 1, n = 40), behaviourally HIV-infected (group 2, n = 10), and HIV-uninfected (group 3, n = 10). RESULTS: Median age was lower in group 1 (18 years) than in groups 2 (24 years) and 3 (20.5 years) (P < 0.001), and median time since sexual debut was shorter: 2 vs 5 vs 4 years (P < 0.001). More trial participants in group 1 than group 2 were on antiretrovirals (90% vs 70%; P <0.001). Abnormal cervical cytology (atypical squamous cells of undetermined significance and higher) was observed in 30% (group 1), 40% (group 2) and 30% (group 3) (P = 0.92), whereas high-risk HPV infection was observed in 45%, 45% and 40%, respectively (P = 1.00). Positive E6/E7 mRNA was found in 28% of group 1, but not in other groups. High-risk HPV infection predicted abnormal cytology in all groups [OR 6.77, 95% confidence interval (CI) 1.99-23.0; P = 0.001). Additionally, plasma HIV RNA ≥50 copies/mL (OR 13.3, 95% CI 1.16-153.06; P = 0.04) predicted abnormal cytology in HIV-infected adolescent females. CONCLUSIONS: Despite the younger age and shorter time since sexual debut, cervical cytological abnormalities and HPV infection were as common in perinatally HIV-infected as in behaviourally infected and uninfected adolescents. HPV vaccination, pre-cancer screening and antiretroviral treatment in HIV-infected female adolescents should be implemented to minimise the risk of cervical cancer.

Concurrent quantification of quinolinic, picolinic, and nicotinic acids using electron-capture negative-ion gas chromatography-mass spectrometry.

Quinolinic, picolinic, and nicotinic acids and nicotinamide are end products of the kynurenine pathway from l-tryptophan and are intermediates in the biosynthesis of nicotinamide adenine dinucleotide. These compounds are involved in complex interrelationships with inflammatory and apoptotic responses associated with neuronal cell damage and death in the central nervous system. To facilitate the study of these compounds, we have utilized gas chromatography-mass spectrometry in electron capture negative ionization mode for their concurrent trace quantification in a single sample. Deuterium-labeled quinolinic, picolinic, and nicotinic acids were used as internal standards and the compounds were converted to their hexafluoroisopropyl esters prior to chromatography. Nicotinamide was readily quantified after conversion to nicotinic acid using gas-phase hydrolysis-a process which did not affect the deuterated internal standards. The on-column limit of quantification was less than 1 fmol for each of the analytes and calibration curves were linear. A packed column liner was used in the gas chromatograph inlet to effectively eliminate sample interference effects in the analysis of trace (femtomolar) levels of quinolinic acid. The method enables rapid and specific concurrent quantification of quinolinic, picolinic, and nicotinic acids in tissue extracts and physiological and culture media.

IFN-beta1b induces kynurenine pathway metabolism in human macrophages: potential implications for multiple sclerosis treatment.

Interferon-beta(1b) (IFN-beta(1b)) has limited efficacy in the treatment of relapsing-remitting multiple sclerosis (RRMS). The kynurenine pathway (KP) is chiefly activated by IFN-gamma and IFN-alpha, leading to the production of a variety of neurotoxins. We sought to determine whether IFN-beta(1b) induces the KP in human monocyte-derived macrophages, as one explanation for its limited efficacy. Serial dilutions of IFN-beta(1b) (at concentrations comparable to those found in the sera of IFN-beta(1b)-treated patients) were added to human macrophage cultures. Supernatants were collected at various time points and assayed for the KP end product, quinolinic acid (QUIN). The effect of IFN-beta(1b) on the KP enzymes indoleamine 2,3-dioxygenase (IDO), 3-hydroxyanthranilate dioxygenase (3HAO), and quinolinate phosphoribosyltransferase (QPRTase) mRNA expression was assessed by semiquantitative RT-PCR. IFN-beta(1b) (> or =10 IU/ml) led to increased mRNA expression of both IDO and QUIN production (7901 +/- 715 nM) after 72 h at 50 IU/ml IFN-beta(1b) (p < 0.0001). This study demonstrates that IFN-beta(1b), in pharmacologically relevant concentrations, induces KP metabolism in human macrophages and may be a limiting factor in its efficacy in the treatment of MS. Inhibitors of the KP may be able to augment the efficacy of IFN-beta in MS.

Characterisation of kynurenine pathway metabolism in human astrocytes and implications in neuropathogenesis.

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.

Kynurenine pathway inhibition reduces neurotoxicity of HIV-1-infected macrophages.

The AIDS dementia complex (ADC) is a consequence of excessive immune activation driven at least in part by systemic HIV infection and probably brain infection. Quinolinic acid (QUIN) is a neurotoxic tryptophan metabolite produced by macrophages in response to stimulation with cytokines or infection with HIV-1. Consequently it has been implicated in ADC pathogenesis. However, macrophages infected with HIV-1 synthesize numerous neurotoxic substances. Therefore we conducted experiments using human fetal brain tissue to determine the relative importance of QUIN as a neurotoxin in ADC. Human macrophages were infected with HIV-1 in vitro using a viral isolate from a demented patient. 6-Chloro-D-tryptophan, an inhibitor of QUIN biosynthesis, was added to half the macrophage cultures to block formation of QUIN. Supernatants containing QUIN (SQpos) or in which QUIN biosynthesis had been inhibited (SQneg) were then added to human fetal brain aggregate cultures. Toxicity was evaluated using lactate dehydrogenase efflux, trypan blue exclusion, immunohistochemistry, image analysis, and electron microscopy. Each technique showed a reduction of toxicity in SQneg-treated cultures. These studies confirm the significance of QUIN as a neurotoxin in ADC and suggest that neuroprotective strategies may have a place in the treatment of this disease.

Quinolinic acid production by macrophages stimulated with IFN-gamma, TNF-alpha, and IFN-alpha.

Quinolinic acid (QUIN) has been associated with several inflammatory neurologic disorders, including AIDS dementia complex (ADC). Recent studies suggest that activation of macrophages with either HIV-1 or interferon-gamma (IFN-gamma) can lead to QUIN production. However, the importance of other cytokines, especially those related to the macrophage and that are especially important in ADC pathogenesis, remains unclear. We, therefore, sought to determine the role of tumor necrosis factor-alpha (TNF-alpha) and IFN-alpha in the production of QUIN. Primary human macrophages were stimulated with two different concentrations of these cytokines alone, in combination with each other, and with IFN-gamma. QUIN concentrations in the supernatants were then measured by mass spectrometry at 24, 48, and 72 hs. Results at 72 h showed significant increases in QUIN production in the cells stimulated with IFN-gamma (10297 +/- 170 nmol/L) and also in those stimulated with IFN-alpha (3600 +/- 113 nmol/L), whereas TNF-alpha-stimulated macrophages produced low levels of QUIN (1108 +/- 23 nmol/L). Macrophages stimulated with the cytokine combinations TNF-alpha and IFN-gamma, IFN-alpha, and IFN-gamma, and TNF-alpha and IFN-alpha also resulted in increases in QUIN production (11471 +/- 77.6 nmol/L, 16656 +/- 184 nmol/L, and 3369 +/- 120.5 nmol/L, respectively). The increases in QUIN production in all of the cytokine treatments approached or exceeded in vivo concentrations of QUIN that have been shown to be neurotoxic. These data further support a role for QUIN in cytokine-mediated neuronal death in inflammatory disorders of the brain, especially ADC.

Role of ECMO in the treatment of respiratory syncytial virus bronchiolitis: a collaborative report.

AIM: To report the collaborative experience of extracorporeal membrane oxygenation (ECMO) in the treatment of respiratory syncytial virus (RSV) bronchiolitis between April 1989 and January 1995. METHODS: The medical records of patients with confirmed RSV bronchiolitis referred to three centres (Leicester, Glasgow, and Great Ormond Street) were reviewed. RESULTS: Twenty four infants were identified. Seventeen had been born prematurely (gestational range 23-40 weeks, median 30 weeks). Thirteen infants had been mechanically ventilated after birth and seven of these had evidence of bronchopulmonary dysplasia (BPD). The age of onset of RSV infection varied from three to 64 weeks (mean 17.4 weeks, median 12 weeks). Ventilation before ECMO ranged from one to 16 days and oxygenation indices at the time of referral ranged from 21-73 (mean 39). Ribavirin was used in eight of the 24 patients. Sixteen patients received venoarterial and eight veno-venous ECMO. ECMO hours ranged from 32-647 (median 196 hours). One infant died (survival rate 96%). Cranial ultrasound abnormalities were detected in three patients. However, at follow up only one of the 23 survivors had evidence of developmental delay. CONCLUSION: A group of paediatric patients in whom ECMO can be of benefit has been identified. The use of ECMO should be considered when other means of support prove unsuccessful.

Elevated levels of eukaryotic translation initiation factor eIF-4E, mRNA in a broad spectrum of transformed cell lines.

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Over-expression of eIF-4E in fibroblasts results in their malignant transformation. However, no information on eIF-4E expression in established transformed cell lines has been available. We report here that a variety of tumor cell lines, chemically, virally and oncogenically transformed, exhibit elevated levels of eIF-4E mRNA expression as compared to their normal counterparts. Overexpression of eIF-4E, which is normally rate-limiting in protein synthesis, may stimulate the translation of regulatory and oncogenic proteins involved in transformation.

Methylated oxypurines and induction of differentiation of murine erythroleukemia cells.

Murine erythroleukemia cell lines derived from Friend virus infected mice can be induced to differentiate in vitro by numerous agents. Among these compounds are certain naturally occurring purines such as hypoxanthine or 1-methylhypoxanthine. We have extended these studies to other modified oxypurines and have identified some areas of cell regulation with which they may be interacting. Monomethylated derivatives of guanine, hypoxanthine or xanthine are active as inducers of differentiation. Excluding hypoxanthine, the parent oxypurines guanine and xanthine are ineffective in inducing differentiation. The dimethyl- and trimethylxanthine derivatives are also inactive as inducers. The methylated oxypurines are not metabolized to nucleotides by the cell and, therefore, probably do not interact with nucleic acid synthesis directly. We have investigated one cellular process of possible regulatory significance with which they do interact. ADP-ribosylation has been implicated in control of gene expression and differentiation. The methylated oxypurines inhibit this reaction, as measured in permeabilized cells, in the same concentration range at which they are effective as inducers of differentiation. Additionally, 1-methylguanine and 7-methylguanine decrease incorporation of mannose and glucosamine into glycoprotein and into dolichol-oligosaccharide precursors. These effects may be related to cell surface alterations observed during differentiation.

Ribavirin induced differentiation of murine erythroleukemia cells.

The synthetic nucleoside, ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a broad spectrum antiviral agent currently being tested in clinical studies with AIDS patients; and mycophenolic acid, a non-nucleoside inhibitor of inosinate (IMP) dehydrogenase, are effective inducers of terminal differentiation of Friend virus transformed murine erythroleukemia cells. The inhibition of cell division and the induced maturation produced by these agents appears to be a consequence of inhibition of IMP dehydrogenase, since growth inhibition is reversed and differentiation is prevented by the simultaneous exposure of cells treated with the agents to exogenous guanine or guanosine, which circumvents the effects of blockage of IMP dehydrogenase. However, while the effects mycophenolic acid, a pure IMP dehydrogenase inhibitor with no other biochemical effects, were completely reversed by guanine salvage supplies, cells exposed to ribavirin responded in a different manner. At levels of guanine salvage supplies below 50 microM, growth inhibition and cell differentiation were partially reversed. At salvage supply concentrations greater than 50 microM, while differentiation was completely blocked, the toxicity of ribavirin was increased and cell division was greatly diminished. These results indicate additional biochemical effects for ribavirin unrelated to the inhibition of IMP dehydrogenase, which may be related to its antiviral properties.

Alterations in cell surface properties induced by modified purines.

Primary Chinese hamster embryo cultures exposed chronically to 1-methylguanine or 7-methylguanine, modified purines derived from nucleic acid turnover, exhibit a number of properties characteristic of transformed cell lines. One of the earliest effects observed following exposure of cells to the methylated purines is an alteration in cell surface properties as measured by the interaction of the cells with the lectin concanavalin A. Within sixteen hours following inclusion of the compounds in the culture medium, the cells exhibit an increase in concanavalin A mediated hemadsorption. The increase in hemadsorption is accompanied by an alteration in distribution of receptors within the cell population as measured by flow microfluorometry using fluorescin conjugated concanavalin A, and by a decrease in the total number of receptors as measured by binding of radiolabelled concanavalin A. Possible mechanisms for these alterations and their significance for growth control are discussed.

Induction of adipocyte formation in 10T1/2 cells by 1-methylguanine and 7-methylguanine.

1-Methylguanine and 7-methylguanine are naturally occurring modified purines derived from tRNA, found in elevated levels in the serum and urine of cancer patients. When C3H/10T1/2 clone-8 mouse cells are exposed to low levels of the methylated purines, they are induced to differentiate into adipocytes. Differentiation is induced in a dose-dependent manner and is similar in extent to that achieved by other inducing agents, such as 5-azacytidine. The methylated purines are not mutagenic, nor are they incorporated into DNA. They may exert their effect by modifying cellular regulatory processes, such as methylation of DNA. High levels of circulating methylated purines in cancer patients may play a role in tumor-host interactions.

An improved rapid assay for S-adenosyl-L-homocysteine hydrolase.

A coupled enzyme system was devised to assay S-adenosyl-L-homocysteine hydrolase activity spectrophotometrically and to stain the hydrolase selectively in polyacrylamide gels. The assay procedure monitors the formation of uric acid derived from the catabolism of the adenosine moiety of S-adenosylhomocysteine. The staining procedure allows the determination of the molecular weight of the hydrolase when extracts are electrophoresed on polyacrylamide gradient gels and compared to standard of known molecular weight. The specificity of the enzyme for the homocysteine moiety was also investigated by testing modified compounds as substrates. All the analogs tested were inactive as substrates for hydrolysis, indicating a strict specificity.