High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

Greatly reduced risk of EBV reactivation in rituximab-experienced recipients of alemtuzumab-conditioned allogeneic HSCT.

EBV-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed the incidence and risk factors for EBV reactivation in 186 adult patients undergoing consecutive allo-HSCT with alemtuzumab T-cell depletion at a single centre. The cumulative incidence of EBV reactivation was 48% (confidence interval (CI) 41-55%) by 1 year, with an incidence of high-level EBV reactivation of 18% (CI 13-24%); 8 patients were concurrently diagnosed with PTLD. Amongst patients with high-level reactivation 31/38 (82%) developed this within only 2 weeks of first EBV qPCR positivity. In univariate analysis age⩾50 years was associated with significantly increased risk of EBV reactivation (hazard ratio (HR) 1.54, CI 1.02-2.31; P=0.039). Furthermore, a diagnosis of non-Hodgkin lymphoma (NHL) was associated with greatly reduced risk of reactivation (HR 0.10, CI 0.03-0.33; P=0.0001) and this was confirmed in multivariate testing. Importantly, rituximab therapy within 6 months prior to allo-HSCT was also highly predictive for lack of EBV reactivation (HR 0.18, CI 0.07-0.48; P=0.001) although confounding with NHL was apparent. Our data emphasise the risk of PTLD associated with alemtuzumab. Furthermore, we report the clinically important observation that rituximab, administered in the peri-transplant period, may provide effective prophylaxis for PTLD.

COPD-related morbidity and mortality after smoking cessation: status of the evidence.

The evidence base for the benefit of quitting smoking as regards morbidity and mortality outcomes in patients with moderate-to-severe chronic obstructive pulmonary disease (COPD) is limited. The present article is a review of the existing literature. A systematic literature search in medical databases was performed until March 2006, and subsequently until September 1, 2007. The outcomes examined were COPD-related morbidity and mortality (including all-cause mortality) in COPD patients in connection with smoking cessation. A total of 21 and 27 published articles on morbidity and mortality, respectively, were identified and reviewed. For both outcomes, only a few of the studies included patients with severe COPD. Most of the studies reported a beneficial effect of smoking cessation compared with continued smoking, whereas a few found no improvement. Methodological problems, including small study sizes, poor data quality, possibility of reverse causality and incomplete ascertainment of cause of death, limit interpretation of some of the studies. The evidence as a whole supports the conclusion that, even in severe chronic obstructive pulmonary disease, smoking cessation slows the accelerated rate of lung function decline and improves survival compared with continued smoking.

Keratoconus: an analysis of corneal asymmetry.

BACKGROUND: Keratoconus, a non-inflammatory corneal ectasia, is reported to have bilateral involvement in over 90% of patients. The purpose of this study was to quantify the extent of asymmetry of disease at presentation to a regional corneal clinic. METHODS: Eighty three patients diagnosed at presentation, using a combination of videokeratography, slit lamp examination, and refractive findings were retrospectively selected. On this basis, 73 patients were designated as having evidence of keratoconus in both eyes. In order to quantify the degree of asymmetry between fellow eyes in these bilateral patients, intraclass correlation was calculated for best spectacle corrected visual acuity (BSCVA) and for 13 different topographical indices generated using videokeratography. In order to examine the link between each index and visual function, the intrapatient differences in each index were compared to the intrapatient differences in BSCVA using Pearson's correlation. RESULTS: BSCVA showed a high degree of asymmetry between fellow eyes with a correlation coefficient of r = 0.006. With the exception of area analysed, all of the topographical indices also showed disparity between paired eyes (r = 0.01 to r = 0.25). Pearson's analysis found that the intrapatient differences in the standard deviation of the power (SDP), average corneal power (ACP), central corneal power (K), as well as the composite keratoconus prediction index (KPI) inversely correlated with the intrapatient differences in best spectacle corrected acuity (r = -0.76,-0.75,-0.69, and -0.73 respectively). CONCLUSIONS: This study demonstrates, quantitatively, the asymmetry of disease found in patients at the point of initial diagnosis of keratoconus. It also suggests that increases in indices which reflect various aspects of corneal power as well as the composite index KPI correlate with a decrease in BSCVA.

Health impact of "reduced yield" cigarettes: a critical assessment of the epidemiological evidence.

Cigarettes with lower machine measured "tar" and nicotine yields have been marketed as "safer" than high tar products over the last four decades, but there is conflicting evidence about the impact of these products on the disease burden caused by smoking. This paper critically examines the epidemiological evidence relevant to the health consequences of "reduced yield" cigarettes. Some epidemiological studies have found attenuated risk of lung cancer but not other diseases, among people who smoke "reduced yield" cigarettes compared to smokers of unfiltered, high yield products. These studies probably overestimate the magnitude of any association with lung cancer by over adjusting for the number of cigarettes smoked per day (one aspect of compensatory smoking), and by not fully considering other differences between smokers of "high yield" and "low yield" cigarettes. Selected cohort studies in the USA and UK show that lung cancer risk continued to increase among older smokers from the 1950s to the 1980s, despite the widespread adoption of lower yield cigarettes. The change to filter tip products did not prevent a progressive increase in lung cancer risk among male smokers who began smoking during and after the second world war compared to the first world war era smokers. National trends in vital statistics data show declining lung cancer death rates in young adults, especially males, in many countries, but the extent to which this is attributable to "reduced yield" cigarettes remains unclear. No studies have adequately assessed whether health claims used to market "reduced yield" cigarettes delay cessation among smokers who might otherwise quit, or increase initiation among non-smokers. There is no convincing evidence that past changes in cigarette design have resulted in an important health benefit to either smokers or the whole population. Tobacco control policies should not allow changes in cigarette design to subvert or distract from interventions proven to reduce the prevalence, intensity, and duration of smoking.

The Escherichia coli orthologue of the Salmonella ushB gene (ushB(c)) produces neither UDP-sugar hydrolase activity nor detectable protein, but has an identical sequence to that of Escherichia coli cdh.

Salmonella ushB, which encodes a membrane-bound UDP-sugar hydrolase, has an Escherichia coli orthologue (ushB(c)) which does not detectably produce this activity. In this report, we show that ushB(c) does not produce any detectable protein either, despite being transcribed normally. Remarkably, ushB(c) is shown to have 100% sequence identity with E. coli cdh, previously characterised as encoding an active CDP-diglyceride hydrolase, an apparent contradiction with implications regarding enzyme evolution. We suggest that a useful gene designation is cdh (ushB(c)) rather than either ushB(c) or cdh, alone.

Utility of measurements of oxygen cost of breathing in predicting success or failure in trials of reduced mechanical ventilatory support.

OBJECTIVE: Test whether a change in oxygen consumption produced by a reduction in level of mechanical ventilatory support predicts failure to tolerate the reduction in level of support. DESIGN: Prospective study of the sensitivity and specificity of increased oxygen cost of breathing as a predictor of failure to tolerate a reduction in ventilatory support in patients undergoing weaning, using a protocol that incrementally reduces the level of mechanical ventilatory support. SETTING: University medical center. METHODS: We studied 228 trials in 30 patients who had required mechanical ventilatory support for at least 72 hours and who were being weaned using a standardized protocol that provided for three 30-minute trials of reduced mechanical ventilatory support per day, followed by ventilatory muscle rest. Using a metabolic monitor, we monitored oxygen consumption (V(O(2))) prior to and during 228 incremental reductions in level of mechanical ventilatory support conducted as part of a standardized weaning protocol. Oxygen cost of breathing was defined as the difference in V(O(2)) (Delta V(O(2))) during the trial of reduced mechanical ventilatory support, compared to a 30-minute resting period immediately before the trial. A successful trial was defined as one that could be continued for 30 minutes without development of clinical signs of ventilatory failure. Changes in V(O(2)) and the ratio of respiratory frequency to tidal volume (f/V(T)) during a weaning trial were evaluated as predictors for failure of a 30-minute trial of reduced ventilatory support. RESULTS: A 15% increase in oxygen cost of breathing predicted failure in the trial, with a sensitivity of 96.6%, specificity of 85.7%, positive predictive value of 98.5%, and negative predictive value of 72.0%. Neither change in V(O(2)) measured early in the trial nor f/V(T) proved to be as successful in predicting failure to tolerate an incremental reduction in ventilatory support. CONCLUSION: Change in V(O(2)) following an incremental reduction in level of mechanical ventilatory support may be a useful predictor for determining which patients will rapidly fail to tolerate that level of reduction.

State-specific trends in smoke-free workplace policy coverage: the current population survey tobacco use supplement, 1993 to 1999.

We examined trends in smoke-free workplace policies among all indoor workers in the United States using the National Cancer Institute's Tobacco Use Supplement to the Census Bureau's Current Population Survey (total n = 270,063). Smoke-free was defined as smoking not permitted in public or common areas or in work areas of a worksite. Nationally, we found that nearly 70% of the US workforce worked under a smoke-free policy in 1999. At the state level, a greater than 30-percentage-point differential existed in the proportion of workers with such policies. Although significant progress has been made to reduce worker exposure to environmental tobacco smoke on the job, we predict further progress may be difficult unless comprehensive regulations to protect all workers are implemented at the national, state, or local level.

The role of the intracellular inhibitor of periplasmic UDP-sugar hydrolase (5'-nucleotidase) in Escherichia coli: cytoplasmic localisation of 5'-nucleotidase is conditionally lethal.

E. coli UshA, a bifunctional enzyme with UDP-sugar hydrolase and 5'-nucleotidase activities, is secreted to the periplasm but has a specific protein inhibitor located in the cytoplasm. It has been previously suggested that some 5'-nucleotidase, or a folded domain of this enzyme, may be active in the cytoplasm prior to export. If true, the intracellular inhibitor may have a role in protecting the cell from the likely deleterious effects of any intracellular UshA activity. Using deletion mutagenesis to remove the UshA signal peptide, we have shown that the resulting UshA derivative is an active cytoplasmic 5'-nucleotidase, and causes conditional lethality. Our results support the hypothesis that the physiological role of the UshA inhibitor is to protect the intracellular nucleotide pool from any cytoplasmic 5'-nucleotidase activity.

Enamel matrix derivative (EMDOGAIN) rapidly stimulates phosphorylation of the MAP kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells.

In our previous study, we demonstrated that porcine enamel matrix derivative (EMD) induces p21WAF1/cip1 within 8 hours and subsequently arrests the cell cycle of human oral epithelial cells in G1 phase. In contrast, EMD markedly stimulates the proliferation of gingival fibroblasts without inducing p21WAF1/cip1. To investigate the mechanism of how EMD produces these differential effects, we have focused on the initial response of these two cell types to EMD. In epithelial cell cultures, EMD stimulated cytoskeletal actin polymerization within 30 min and promoted cell adhesion in our experimental system. EMD failed to stimulate either intracellular Ca2+ mobilization or cAMP production in either cell type. In both epithelial and fibroblastic cells, EMD (25-100 microgram/ml) rapidly produced dose-dependent phosphorylation of the mitogen-activated protein kinase (MAPK) family: extracellular signal response kinase (ERK), p38-MAPK (p38-K), and c-Jun-terminal kinase/stress-activated protein kinase (JNK). However, neither inhibitors of MEK (ERK kinase) nor p38-K could block EMD's anti-proliferative action on epithelial cells. On the other hand, EMD rapidly stimulated translocation of smad2 into the nucleus in both cell types. Spurred by this finding, we assayed for TGF-beta1, a ligand for one receptor associated with smad2 activation, and detected significant levels in EMD preparations. The sum of these pharmacological findings indicates that EMD contains at least one bioactive factor, which is most probably TGF-beta1 (or TGF-beta-like substances). In conjunction with the similarities in the differential growth-modulating actions between EMD and what is known for TGF-beta, we suggest that TGF-beta might act as the principal growth regulating agent of oral fibroblastic and epithelial cell types in EMD despite being present in only low levels.

Sulfinimine-mediated asymmetric synthesis of 1,3-disubstituted tetrahydroisoquinolines: a stereoselective synthesis of cis- and trans-6,8-dimethoxy-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline.

[reaction: see text] The highly diastereoselective addition of lateral lithiated o-tolunitriles to sulfinimines followed by treatment of the resulting sulfinamide with MeLi, hydrolysis, and reduction represents a concise new methodology for the asymmetric synthesis of 1,3-disubstituted tetrahydroisoquinolines.

Sex differences in workplace smoking policies: results from the current population survey.

OBJECTIVE: To determine the prevalence of smoking policies in indoor work environments in the United States, with a special focus on sex differences in the provision of these policies. METHOD: Information on the prevalence and restrictiveness of workplace smoking policies was obtained from 86,490 currently employed indoor workers (50,865 women and 35,625 men) 15 years of age and older who responded to the National Cancer Institute's Tobacco Use Supplement to the Current Population Survey, a cross-sectional survey of households in all 50 states and the District of Columbia conducted between 1995 and 1996. RESULTS: Eighty-six percent of respondents reported that their workplaces had official smoking policies, and 63% reported that their workplaces were smoke free. Women reported significantly higher rates of both official smoking policies and smoke-free workplaces than men, regardless of racial/ethnic or age group. CONCLUSION: The overall rates of worksite smoking restrictions, including the establishment of smoke-free workplaces, were higher than those reported in earlier surveys. Disparities in coverage will need to be reduced if all workers, regardless of sex, race, age, or industry of employment, are to be protected from the demonstrated hazards of environmental tobacco smoke.

Cigarette smoking among the elderly: disease consequences and the benefits of cessation.

The disease consequence of smoking occurs disproportionately among the elderly because of the long duration of cumulative injury or change that underlies the bulk of tobacco-caused disease. Older smokers are less likely than younger smokers to attempt quitting, but they are more likely to be successful in the attempts that they do make to quit. Excess absolute rates of disease incidence and mortality due to smoking increase steadily with increasing age and duration of smoking, and there is little evidence to suggest that the disease consequences of smoking diminish among the elderly. Although cardiovascular disease is the most common cause of excess mortality among younger smokers, lung cancer is the largest cause of excess smoking-related mortality over the age of 60 years; and at older ages the excess death rate from chronic obstructive lung disease equals that for cardiovascular disease. Because of the dramatic increases in smoking-related excess mortality with advancing age, approximately 70% of the 400,000 or more deaths occur among those over age 60 years. The benefits of cessation are proportionately somewhat less among the elderly and may manifest more slowly than among younger smokers, but cessation remains the most effective way of altering smoking-induced disease risks at all ages, including those over the age of 60 years.

Cytostatic action of enamel matrix derivative (EMDOGAIN) on human oral squamous cell carcinoma-derived SCC25 epithelial cells.

During surgical treatment of periodontal disease, enamel matrix derivative (EMD) is topically applied as a substitute for extracellular matrix in order to facilitate regeneration of damaged periodontal tissue. However, the mechanism for EMD action is poorly understood. We have now examined the effects of EMD on the proliferation of oral epithelial (SCC25) cells in vitro. After 3 days of treatments, EMD (25 100 microg/ml) dose-dependently inhibited cell division and concomitantly arrested cell cycle at the G1 phase. Prior to this inhibition, EMD significantly up-regulated p21WAF1/cip1, a cyclin-dependent kinase inhibitor, induced G1-arrest, and inhibited DNA synthesis. In addition, EMD down-regulated expression of cytokeratin-18 (CK18) protein, which was most due to decreased production, but less to increased degradation. However, EMD did not discernibly increase the number of apoptotic cells over 8 days of treatment. These findings indicate (1) that EMD acts as a cytostatic agent, rather than a cytotoxic agent, on epithelial cells, and (2) that this anti-proliferative action is probably due to p21WAF1/cip1-mediated G1-arrest. Furthermore, our in vitro cellular data clearly verify and provide an explanation for the clinical observation that EMD application suppresses the down-growth of junctional epithelium onto dental root surfaces, a process that frequently interferes with the formation of new connective tissue attachments.

The effect of nicotinamide on spontaneous and induced activity in smooth and skeletal muscle.

BACKGROUND AND PURPOSE: Nicotinamide (NA) is currently undergoing clinical trials as a tumour radiosensitizer. The dose that can be administered is currently 80 mg/kg per day, but this may be restricted to 60 mg/kg per day by the high incidence of nausea and vomiting. To investigate some of NA's underlying mechanisms of action, we have used an ex vivo system to study the direct effect of this drug, over a wide range of concentrations, on isolated spontaneously active rat ileum. Effects on the gut were compared with the action of NA on skeletal and vascular smooth muscle. MATERIALS AND METHODS: Isolated rat ileum rings were perfused with oxygenated Krebs' solution in an organ bath. NA (1 microM to 10 mM) was introduced to the perfusate and the change in amplitude of spontaneous peristaltic activity recorded. Dissected frog sartorius muscle was bathed in modified oxygenated Ringer's solution in an organ bath. The muscle was electrically stimulated to generate isometric contractions. Tension was then measured before and after the addition of a range of NA concentrations (8.2-24.6 mM) to the organ bath. RESULTS: NA inhibited peristalsis in the ileum in a dose-dependent manner. At a drug concentration of 1 mM the amplitude of contractions was reduced to <50% of the initial control value. NA had no effect on the electrically induced contractions in the isolated frog sartorius muscle. CONCLUSIONS: Gut smooth muscle is highly sensitive to the relaxant effect of NA producing 50% relaxation at a concentration approximately 10 fold lower than that required in rat arterial smooth muscle, while having no effect on non-mammalian skeletal smooth muscle. This may provide explanations for the occurrence of emesis in patients undergoing combined nicotinamide therapies and highlight possible alternatives available to counter this unwanted side-effect.

Health risks associated with cigar smoking.

This article summarizes principal findings from a conference convened by the American Cancer Society in June 1998 to examine the health risks of cigar smoking. State-of-the-science reports were presented and 120 attendees (representing government and private agencies, academia, health educators, and tobacco control experts) participated in panels and summary development discussions. The following conclusions were reached by consensus: (1) rates of cigar smoking are rising among both adults and adolescents; (2) smoking cigars instead of cigarettes does not reduce the risk of nicotine addiction; (3) as the number of cigars smoked and the amount of smoke inhaled increases, the risk of death related to cigar smoking approaches that of cigarette smoking; (4) cigar smoke contains higher concentrations of toxic and carcinogenic compounds than cigarettes and is a major source of fine-particle and carbon monoxide indoor air pollution; and (5) cigar smoking is known to cause cancers of the lung and upper aerodigestive tract. JAMA. 2000;284:735-740

Patterns of adolescent smoking initiation rates by ethnicity and sex.

OBJECTIVE: To define US national sex specific rates of smoking initiation among Hispanic, non-Hispanic white, and African American adolescents aged 12-17 years for each calendar year from 1940 through 1992. METHODS: Adult survey data from the tobacco use supplement of the Current Population Survey in 1992-93 and 1995-96 were used to reconstruct the age at which individuals began to smoke and the calendar year in which they were that age. From these data, the number of individuals who began a calendar year as never smokers and who were aged 12-17 years during that year could be estimated and formed the denominator of the initiation rate. The number of these individuals who reported taking up smoking during that year formed the numerator of the initiation rate. RESULTS: Initiation rates among male adolescents in each of the three racial/ethnic groups have declined since 1945. However, since 1983, initiation rates among male adolescents overall have increased. Non-Hispanic white male adolescents generally initiated cigarette smoking at higher rates than Hispanic or African American male adolescents. Initiation rates among Hispanic male adolescents have not been statistically different from initiation rates among African American male adolescents. From 1978 to 1982, initiation rates among Hispanic and African American male adolescents experienced a sharp decline, and the rate of decline was steeper than that experienced by non-Hispanic white male adolescents. Initiation rates among female adolescents have increased since 1940, catching up to male adolescent initiation rates by the mid 1970s. Initiation rates among female adolescents appeared to level off or increased slightly again from the mid 1980s to 1990. Non-Hispanic white female adolescents generally initiate cigarette smoking at higher rates than Hispanic or African American female adolescents. Initiation rates among non-Hispanic white and African American female adolescents equalled the initiation rates of their male counterparts by the mid 1970s, but initiation rates among Hispanic female adolescents did not overlap with initiation rates of Hispanic male adolescents until 1990. From 1975 to 1980, initiation rates among African American female adolescents decreased sharply, but, unlike initiation rates among the two other ethnic groups, rates continued to decline from 1984 to 1990. CONCLUSIONS: Different patterns of increasing and decreasing smoking initiation among sex and ethnic adolescent groups suggest the effect of varying social and cultural influences. These findings support the importance of including ethnic factors in studies of smoking behaviour.

Contractile properties of human renal cell carcinoma recruited arteries and their response to nicotinamide.

BACKGROUND AND PURPOSE: The manipulation of tumour blood supply and thus oxygenation is a potentially important strategy for improving the treatment of solid tumours by radiation. Increased knowledge about the characteristics that distinguish the tumour vasculature from its normal counterparts may enable tumour blood flow to be more selectively modified. Nicotinamide (NA) causes relaxation of preconstricted normal and tumour-supply arteries in rats. It has also been shown to affect microregional blood flow in human tumours. Direct effects of NA on human tumour supply arteries have not previously been reported. This paper describes our evaluation of the effects of NA on two parameters: 'spontaneous', oscillatory contractile activity and agonist (phenylephrine)-induced constriction in the arteries supplying human renal cell carcinomas. MATERIALS AND METHODS: Isolated renal cell carcinoma feeder vessels were perfused in an organ bath with the alpha(1)-adrenoceptor agonist phenylephrine (PE). When the arteries had reached a plateau of constriction, nicotinamide (8.2 mM) was added to the perfusate and changes in perfusion pressure were measured. RESULTS: PE (10 microM) induced a sustained constriction in the majority of the renal cell carcinoma feeder vessels examined, demonstrating that they retain contractile characteristics, at least in response to this alpha(1)-adrenoceptor agonist. In combination with NA (8.2 mM) the constriction was significantly attenuated in half of the preparations. In addition, seven arteries exhibited spontaneous contractile activity which was significantly attenuated by NA in six of them. CONCLUSIONS: NA can significantly attenuate both 'spontaneous' and agonist-induced constrictions in tumour-recruited human arteries, though not all arteries are sensitive.