Effects of location, classroom orientation, and air change rate on potential aerosol exposure: an experimental and computational study.

This study examined the dispersion of potentially infectious aerosols in classrooms by means of both a CO2 tracer gas, and multizone contaminant transport modeling. A total of 20 tests were conducted in three different university classrooms at multiple air change rates (4.4-9.7/h), each with two different room orientations: one with the tracer gas released from six student desks toward the air return, and one with the same tracer gas released away from it. Resulting tracer concentrations were measured by 19 different monitors arrayed throughout the room. Steady-state, mean tracer gas concentrations were calculated in six instructor zones (A-F) around the periphery of the room, with the results normalized by the concentration at the return, which was assumed to be representative of the well-mixed volume of the room. Across all classrooms, zones farthest from the return (C, D) had the lowest mean normalized concentrations (0.75), while those closest to the return (A, F) had the highest (0.95). This effect was consistent across room orientations (release both toward and away from the return), and air change rates. In addition, all zones around the periphery of the room had a significantly lower concentration than those adjacent to the sources. Increasing the ventilation rate reduced tracer gas concentrations significantly. Similar trends were observed via a novel approach to CONTAM modeling of the same rooms. These results indicate that informed selection of teaching location within the classroom could reduce instructor exposure.

Attitudes towards HIV testing via home-sampling kits ordered online (RUClear pilots 2011-12).

BACKGROUND: The burden of disease relating to undiagnosed HIV infection is significant in the UK. BHIVA (British HIV Association) recommends population screening in high prevalence areas, expanding outside traditional antenatal/GUM settings. METHODS: RUClear 2011-12 piloted expanding HIV testing outside traditional settings using home-sampling kits (dry-blood-spot testing) ordered online. Greater Manchester residents (≥age 16) could request testing via an established, online chlamydia testing service (www.ruclear.co.uk). Participant attitudes towards this new service were assessed. Qualitative methods (thematic analysis) were used to analyse free-text data submitted by participants via hard copy questionnaires issued in all testing kits. RESULTS: 79.9% (2447/3062) participants completed questionnaires, of which 30.9% (756/2447) provided free-text data. Participants overwhelmingly supported the service, valuing particularly accessibility and convenience, allowing individuals to order tests any time of day and self-sample comfortably at home; avoiding the invasive nature of venipuncture and avoiding the need for face-to-face interaction with health services. The pilot was also clinically and cost-effective. CONCLUSION: Testing via home-sampling kits ordered online (dry-blood-spot testing) was felt to be an acceptable and convenient method for accessing a HIV test. Many individuals undertook HIV testing where they would otherwise not have been tested at all. Expansion of similar services may increase the uptake of HIV testing.

The messenger between worlds: the regulation of innate and adaptive type-2 immunity by innate lymphoid cells.

Although type-2 immune responses evolved primarily to defend against extracellular helminths, in part through the co-opting of tissue repair and remodeling mechanisms, they are often inappropriately directed towards relatively innocuous allergens resulting in conditions including asthma, allergic rhinitis, food allergy, and atopic dermatitis. The recent discovery of group 2 innate lymphoid cells (ILC2) has increased our understanding of the initiation of these responses and the roles played by CD4(+) T helper (Th) 2 cells in their modulation. This review focuses on the important messenger role of ILC2 in translating epithelial-derived alarmins into downstream adaptive type-2 responses via dendritic cells and T cells, with special emphasis on their roles in allergic disease.

Diarrhetic shellfish poisoning toxin esters in Danish blue mussels and surf clams.

Until recently, little focus was given to the presence of diarrhetic shellfish poisoning (DSP) toxin esters in seafood products. However, during the last few years, the occurrence of a high percentage of esters of the total amount of DSP toxins present in some seafood products has been observed. Samples of Danish surf clams (Spisola spp.) and blue mussels (Mytilus edulis) from 1999-2004 were analysed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the presence of DSP toxin esters. The samples contained only okadaic acid and esters of okadaic acid. The level of total okadaic acid equivalents ranged from 224 to 2516 microg kg-1 in surf clams. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 83 to 98%, mean 95%. The level of total okadaic acid equivalents ranged from 43 to 1631 microg kg-1 in blue mussels. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 21 to 86%, mean 59%. The probability of a high percentage of okadaic acid esters seems to increase with higher amounts of total okadaic acid equivalents in the bivalves. The large prevalence of DSP toxin esters are of particular importance because of the increased use of chemical methods instead of mouse bioassay for the detection of DSP toxicity.

Membrane cholesterol modulates serotonin transporter activity.

The synaptic actions of the neurotransmitter serotonin are terminated by a selective high-affinity reuptake mediated by the serotonin transporter (SERT). To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we stably expressed the rat SERT in human embryonic kidney 293 cells and, upon altering the cholesterol content of these cells by different means, analyzed SERT activity. Depletion of the level of membrane cholesterol by treatment with either the cholesterol chelating agent methyl-beta-cyclodextrin (MbetaCD), cholesterol oxidase, or the cholesterol-binding fluorochrome filipin resulted in a decrease in SERT activity due to both a loss of affinity of substrate and ligand binding and a concomitant reduction of the maximal transport rate. In cholesterol-depleted membranes, cholesterol levels could be restored to those found in untreated membranes by incubation of the membranes with an MbetaCD-cholesterol complex, which correlated with a reversal of the cholesterol depletion-mediated decrease in the level of high-affinity binding. This was not the case when other steroids, such as ergosterol, 5-cholestene, or pregnenolone, were substituted into cholesterol-depleted membranes. These results suggest that membrane cholesterol modulates the functional properties of the SERT by specific molecular interactions which are needed to stabilize the transporter in its optimally active form.

Aspergillus fumigatus-induced allergic airway inflammation alters surfactant homeostasis and lung function in BALB/c mice.

The differential regulation of pulmonary surfactant proteins (SPs) is demonstrated in a murine model of Aspergillus fumigatus (Af )-induced allergic airway inflammation and hyperresponsiveness. BALB/c mice were sensitized intraperitoneally and challenged intranasally with Af extract. Enzyme-linked immunosorbent assay analysis of serum immunoglobulin (Ig) levels in these mice showed markedly increased total IgE and Af-specific IgE and IgG1. This was associated with peribronchial/perivascular tissue inflammation, airway eosinophilia, and secretion of interleukin (IL)-4 and IL-5 into the bronchoalveolar lavage fluid (BALF). Functional analysis revealed that in comparison with nonsensitized mice, allergic sensitization and challenge resulted in significant increases in acetylcholine responsiveness. To analyze levels of SPs, the cell-free supernate of the BALF was further fractionated by high-speed (20,000 x g) centrifugation. After sensitization and challenges, the pellet (large-aggregate fraction) showed a selective downregulation of hydrophobic SPs SP-B and SP-C by 50%. This reduction was reflected by commensurate decreases in SP-B and SP-C messenger RNA (mRNA) expression of the lung tissue of these animals. In contrast, there was a 9-fold increase in SP-D protein levels in the 20,000 x g supernate without changes in SP-D mRNA. The increased levels of SP-D showed a significant positive correlation with serum IgE (r = 0.85, P < 0.001). Tissue mRNA and protein levels of SP-A in either the large- or the small-aggregate fractions were unaffected. Our data indicate that allergic airway inflammation induces selective inhibition of hydrophobic SP synthesis accompanied by marked increases in the lung collectin SP-D protein content of BALF. These changes may contribute significantly to the pathophysiology of Af-induced allergic airway hyperresponsiveness.

Pneumocystis carinii pneumonia alters expression and distribution of lung collectins SP-A and SP-D.

Surfactant proteins SP-A and SP-D, members of the collectin family, have been shown to play a significant role in lung host defense. Both proteins selectively bind Pneumocystis carinii (PC) organisms and modulate the interaction of this pathogen with alveolar macrophages. We hypothesized that the expression and distribution of lung collectins SP-A and SP-D is altered by PC lung infection. PC organisms (2 x 10(5)) were inoculated intratracheally into C.B-17 scid/scid mice that do not require steroids for immunosuppression. Four weeks after inoculation, bronchoalveolar lavage (BAL) fluid was fractionated into three fractions-cell pellet, large aggregate (LA), and small aggregate (SA) surfactant-and each fraction was analyzed for the expression of surfactant components. In uninfected mice, the majority of SP-A (62% +/- 10%) was found in association with lipids in the LA fraction, while 55% +/- 14% of SP-D was distributed in the SA fraction. In contrast, both hydrophobic proteins SP-B and SP-C were associated exclusively with LA. PC infection resulted in major changes in the expression of all surfactant components. Total protein content of LA was unchanged by PC infection (115% +/- 18% of control), whereas SA protein content markedly increased (240% +/- 18% of control level, P <.001). In contrast, the phospholipid content of LA was significantly decreased (53% +/- 5% of control level, P <.001), whereas the SA phospholipid content of infected mice was increased (172% +/- 16% of control level, P <.001). By Western blotting, PC pneumonia (PCP) induced a 3-fold increase in the total alveolar SP-D protein that was reflected mainly in increases in SA SP-D (454% +/- 135% of control, P <.05). The total alveolar SP-A protein content was also increased in PCP because of a large increase in SP-A in SA (720% +/- 115% of control, P <.05); SP-A levels in LA were unchanged. The increases in lung collectin expression were selective, because PCP resulted in the down-regulation of both SP-B and SP-C in LA (5% +/- 2% and 13% +/- 2% of control, respectively, P <.001). We conclude that PCP induces marked elevations in alveolar collectin levels because of increased expression and accumulation of SP-A and SP-D protein in SA surfactant.

P. carinii induces selective alterations in component expression and biophysical activity of lung surfactant.

Studies of Pneumocystis carinii pneumonia (PCP) suggest an important role for the surfactant system in the pathogenesis of the hypoxemic respiratory insufficiency associated with this infection. We hypothesized that PCP induces selective alterations in alveolar surfactant component expression and resultant biophysical properties. PCP was induced by intratracheal inoculation of 2 x 10(5) P. carinii organisms into C.B-17 scid/scid mice. Six weeks after inoculation, large (LA)- and small (SA)-aggregate surfactant fractions were prepared from bronchoalveolar lavage fluids and analyzed for expression of surfactant components and for biophysical activity. Total phospholipid content was significantly reduced in LA surfactant fractions from mice infected with PCP (53 +/- 15% of uninfected mice; P < 0.05). Quantitation of hydrophobic surfactant protein (SP) content demonstrated significant reductions of alveolar SP-B and SP-C protein levels in mice with PCP compared with those in uninfected mice (46 +/- 7 and 19 +/- 6%, respectively; P < 0.05 for both). The reductions in phospholipid, SP-B, and SP-C in LA fractions measured during PCP were associated with an increase in the minimum surface tension of LAs as measured by pulsating bubble surfactometer (13.1 +/- 1.1 vs. 5.4 +/- 1.8 mN/m; P < 0.05). In contrast to decreases in the hydrophobic SPs, SP-D content in the SA fraction was markedly increased (343 +/- 30% of control value; P < 0. 05) and SP-A levels in LA surfactant were maintained (93 +/- 26% of control value) during P. carinii infection. In all cases, the changes in SP content were reflected by commensurate changes in the levels of mRNA. We conclude that PCP induces selective alterations in surfactant component expression, including profound decreases in hydrophobic protein contents and resultant increases in surface tension. These changes, demonstrated in an immunologically relevant animal model, suggest that alterations in surfactant could contribute to the hypoxemic respiratory insufficiency observed in PCP.

The PerFect lipid optimizer kit for maximizing lipid-mediated transfection of eukaryotic cells.

The transfection efficiencies of a panel of eight uniquely different lipid reagents has been evaluated with two other commercially available lipids for use in transfecting a diversity of eukaryotic cell lines. The PerFect lipids are available individually or together in an optimization panel format that can be tested in any given cell line, enabling one to evaluate the optimal lipid for transfecting each individual cell line. Our results demonstrate that no single lipid is optimal for plasmid transfection over a broad range of cell types, thus emphasizing the need for multiple unique lipid reagents and a simple format for testing their transfection efficiency on a given cell type.

Fish hypersensitivity. I. In vitro and oral challenge results in fish-allergic patients.

The purpose of this study was to determine whether patients allergic to one fish species can safely eat other fish species. Eleven atopic, food-allergic children and young adults with histories consistent with IgE-mediated fish hypersensitivity were skin prick tested to 10 fish species. Skin prick tests (SPTs) were positive to all 10 fish in eight of the 11 patients, and the remaining three patients had at least two positive fish SPTs. Positive oral challenges occurred to only one fish in seven of the patients, to two fish species in one patient, and to three fish species in two patients. One patient did not react to any of the fish tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses were performed on raw and cooked protein extracts from nine of the 10 fish species used in SPTs. Several protein bands in the raw-fish extracts appeared to denature with cooking and form high molecular weight conglomerates. Immunoblot analyses with sera from documented fish-allergic patients demonstrated specific IgE binding to protein bands from fish to which patients were not clinically allergic, as determined by oral challenge. In ELISA-inhibition assays, the concentration of fish antigen required to achieve 50% inhibition was similar for fish to which the patients were clinically allergic as compared to fish to which they were clinically tolerant. SPT and in vitro evidence of IgE-specific cross-reactivity does not necessarily correlate with symptomatic fish allergy. In addition, these fish-hypersensitive patients were able to consume one or more other fish species without adverse allergic reactions.

Nuclease Bal-31 mapping of proteins bound to a tRNA(tyr) gene in SV40 minichromosomes.

We have analyzed proteins bound to active and to inactive tRNA(tyr) genes imbedded in the late coding region of SV40 minichromosomal DNA. Bal-31 nuclease resection from the 5' and 3' sides of the active tRNA(tyr) gene reveals proteins bound to the 5' flank, to the promoter 'A' block, to an intragenic sequence, to the promoter 'B' block and to a 3' downstream terminator/pause sequence. The proteins bound near the promoter 'B' block and the downstream terminator/pause sequence are reduced or eliminated by an inactivating deletion in the tRNA(tyr) 'B block'. That proteins are detected in the 5' flank and over the promoter 'A block' of the inactive gene contrasts with current notions regarding the requirement for a functional 'B' block for binding of transcription factors.

Safety of casein hydrolysate formula in children with cow milk allergy.

The purpose of this study was to determine whether a new casein hydrolysate infant formula, Alimentum, could be administered safely to children with cow milk hypersensitivity. The formula was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and with a sensitive enzyme-linked inhibition immunoassay, and it was judged to be safe for clinical trials in children allergic to cow milk. Twenty-five such children underwent double-blind placebo-controlled oral food challenges with 10 gm of powdered cow milk and casein hydrolysate formula. All children were highly atopic and had positive skin prick reactions to cow milk. No patient reacted to placebo during a double-blind, placebo-controlled food challenge. Two patients lost their allergy to cow milk and did not react during the challenge; the remaining patients reacted with a variety of cutaneous, respiratory, and gastrointestinal symptoms within 15 to 90 minutes of challenge. All children tolerated the blinded challenge to the casein hydrolysate and were fed the hydrolysate openly without difficulty. We conclude that this casein hydrolysate is generally safe to feed to children with immediate hypersensitivity to cow milk. We recommend that all infant formulas promoted as "hypoallergenic" be tested in milk-allergic patients to assess their allergenic potential, in addition to standard nutritional evaluation and animal testing for antigenicity.

Natural history of food hypersensitivity in children with atopic dermatitis.

Patients with atopic dermatitis and food hypersensitivity who were adhering to an elimination diet underwent repeat double-blind, placebo-controlled oral food challenges annually for follow-up of their food allergy. After 1 year, 19 of 75 patients lost all signs of clinical food hypersensitivity (15 of 45 patients allergic to one food, and 4 of 21 allergic to two foods). Of the individual foods, 38 of 121 no longer elicited symptomatic responses. After 2 years, patients underwent a second rechallenge; 4 of 44 patients tested lost their clinical food hypersensitivity. In 20 patients undergoing a third rechallenge, no food hypersensitivity was lost. Loss rate of food hypersensitivity varied among foods; after 1 year, there was a 26% loss of symptomatic food allergy to five major allergens (egg, milk, soy, wheat, and peanut) compared with a 66% loss rate to other food allergens. Loss of symptomatic allergy was not affected by the patient's age at diagnosis, except with milk allergy, for which older patients were more likely to lose clinical food hypersensitivity (p less than 0.05). Total serum IgE and prick skin tests were not useful for predicting loss of symptomatic food hypersensitivity. There was no significant decrease in skin test wheal size corresponding to loss of clinical food hypersensitivity. Patients developing only skin symptoms during the initial challenge were most likely to lose symptomatic food hypersensitivity.

Hepatitis B in a residential population with a mental handicap.

Hepatitis serology was used to assess the nature and extent of hepatitis B virus (HBV) infection in the population of a residential centre for people with a mental handicap comprising 56 with Down's Syndrome (DS) and 193 with other mental handicaps (OMH). Markers of HBV were present in 170 cases (68%), of which 25 (10%) were HBsAg positive and potential carriers and 145 IgG anti-HBc positive (58%) indicating exposure to but not necessarily immunity from the virus. Of 25 who were HBsAg positive five were HBeAg positive, 17 were anti-HBe positive, two were HBsAg positive only and one was anti-HBe plus IgM anti-HBc positive. Age and length of residence were significantly associated with HBsAg, IgG anti-HBc and total markers of infection but the degree of mental handicap was not and the DS/OMH ratio only with HBsAg. The results indicated widespread HBV infection, no acute disease cases, relatively few infectious cases and a pre-disposition of DS residents to retain HBsAg in their sera. Vaccination of staff and at risk residents is recommended.

Identification and characterization of the adenosine 3',5'-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum.

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3',5'-[32P]monophosphate (8-N3-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5'-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.

Probabilistic behavior of DNA segregation in Escherichia coli.

The pattern of segregation of DNA in Escherichia coli B/rK was analyzed by using the Methocel technique for forming chains of cells and the membrane binding elution method. Strain B/rK was shown to have a relatively high degree of nonrandom segregation and was used in a critical experiment to test the proposal that only one DNA strand acts nonrandomly during segregation. Thymidine-labeled cells were bound to a nitrocellulose membrane, and newly dividing cells were eluted from the membrane for six generations. The segregation of DNA in the eluted cells as well as in the cells bound to the membrane was examined by the Methocel technique. No difference in segregation was found between the two populations of cells, a result which indicates that the two strands are equivalent in segregation and that the pattern of segregation is not the result of a permanent binding of any strand to a pole of a cell.