High burden of viral respiratory co-infections in a cohort of children with suspected pulmonary tuberculosis.

BACKGROUND: The presentation of pulmonary tuberculosis (PTB) in young children is often clinically indistinguishable from other common respiratory illnesses, which are frequently infections of viral aetiology. As little is known about the role of viruses in children with PTB, we investigated the prevalence of respiratory viruses in children with suspected PTB at presentation and follow-up. METHODS: In an observational cohort study, children < 13 years were routinely investigated for suspected PTB in Cape Town, South Africa between December 2015 and September 2017 and followed up for 24 weeks. Nasopharyngeal aspirates (NPAs) were tested for respiratory viruses using multiplex PCR at enrolment, week 4 and 8. RESULTS: Seventy-three children were enrolled [median age 22.0 months; (interquartile range 10.0-48.0); 56.2% male and 17.8% HIV-infected. Anti-tuberculosis treatment was initiated in 54.8%; of these 50.0% had bacteriologically confirmed TB. At enrolment, ≥1 virus were detected in 95.9% (70/73) children; most commonly human rhinovirus (HRV) (74.0%). HRV was more frequently detected in TB cases (85%) compared to ill controls (60.6%) (p = 0.02). Multiple viruses were detected in 71.2% of all children; 80% of TB cases and 60.6% of ill controls (p = 0.07). At follow-up, ≥1 respiratory virus was detected in 92.2% (47/51) at week 4, and 94.2% (49/52) at week 8. CONCLUSIONS: We found a high prevalence of viral respiratory co-infections in children investigated for PTB, irrespective of final PTB diagnosis, which remained high during follow up. Future work should include investigating the whole respiratory ecosystem in combination with pathogen- specific immune responses.

A Sendai virus-derived RNA agonist of RIG-I as a virus vaccine adjuvant.

The innate immune system is responsible for recognizing invading pathogens and initiating a protective response. In particular, the retinoic acid-inducible gene 1 protein (RIG-I) participates in the recognition of single- and double-stranded RNA viruses. RIG-I activation leads to the production of an appropriate cytokine and chemokine cocktail that stimulates an antiviral state and drives the adaptive immune system toward an efficient and specific response against the ongoing infection. One of the best-characterized natural RIG-I agonists is the defective interfering (DI) RNA produced by Sendai virus strain Cantell. This 546-nucleotide RNA is a well-known activator of the innate immune system and an extremely potent inducer of type I interferon. We designed an in vitro-transcribed RNA that retains the type I interferon stimulatory properties, and the RIG-I affinity of the Sendai virus produced DI RNA both in vitro and in vivo. This in vitro-synthesized RNA is capable of enhancing the production of anti-influenza virus hemagglutinin (HA)-specific IgG after intramuscular or intranasal coadministration with inactivated H1N1 2009 pandemic vaccine. Furthermore, our adjuvant is equally effective at increasing the efficiency of an influenza A/Puerto Rico/8/34 virus inactivated vaccine as a poly(I·C)- or a squalene-based adjuvant. Our in vitro-transcribed DI RNA represents an excellent tool for the study of RIG-I agonists as vaccine adjuvants and a starting point in the development of such a vaccine.

Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design.

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD(+) interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N(6)-(1-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (6e), N(6)-(substituted-naphthalenemethyl)-2'-deoxy-2'-(substituted-benzamido)adenosine analogues were investigated. N(6)-(1-Naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (6m), N(6)-[1-(3-hydroxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (7m), N(6)-[1-(3-methoxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (9m), N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (11e), and N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC(50)'s of these compounds were in the range 2-12 microM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N(6)-(substituted)-2'-deoxy-2'-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N(6) and C2' substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5'-amido substituents and found that 2',5'-dideoxy-2'-(3,5-dimethoxybenzamido)-5'-(diphenylacetamido)adenosine (49) displays an IC(50) of 60-100 microM against the three parasite GAPDH's.

S-alk(en)yl-L-cysteine sulfoxides, alliinase and aroma in Leucocoryne.

Levels of S-alk(en)yl-L-cysteine sulfoxides, alliinase and enzymatically generated pyruvic acid were determined in the bulb, leaf and scape of five species and a natural hybrid of Leucocoryne (Liliaceae), a genus of ornamental geophytes indigenous to Chile. (+)-S-Methyl-L-cysteine sulfoxide (MCSO) was present in all plant parts of all species at levels between 0.09 and 1.41 mg g(-1) fr. wt. Trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide (PRENCSO) was present in plant parts of three species only (L. angustipetala, L. oadorata and L. purpurea) at levels between 0.12 and 1.82 mg g(-1) fr. wt. No other S-alk(en)yl-L-cysteine sulfoxides were detected. Alliinase (EC 4.4.1.4) was detected in the leaf, bulb and scape of L. angustipetala and L. purpurea, only in the leaves of L. coquimbensis and L. purpurea x L. coquimbensis, and only in the bulb of L. odorata. Enzymatically generated pyruvic acid was detected in all plant parts of all species at levels between trace amounts and 5.33 micromol g(-1) fr. wt. As PRENCSO is produced only in Leucocoryne species exhibiting a strong and unpleasant onion-like aroma, it is probable that the enzymatic degradation of PRENCSO is the main cause of that aroma. Consequently, Leucocoryne cultivars should be selected in species and hybrids that lack the ability to synthesise PRENCSO.

Optimal allocation of cognitive resources to spatial locations.

From experiments showing that subjects differentially attend to parts of the visual field, psychologists have inferred a limitation on human visual information processing capacity. The model presented describes an optimal way to allocate a limited quantity of "cognitive resources," "attention" or "mental effort". For this model, the sense of optimality is maximizing the probability of finding a target. In an experiment to test the model, subjects searched for a single target letter in an otherwise blank field. Two probability distributions were used to select a target location for a trial, but only one distribution was used for a session. For both distributions, the fit of the model was quite good. It is suggested that the model provides a promising way to test the assumption limited visual processing capacity.

Hematologic characteristics of avian malaria cases in African black-footed penguins (Spheniscus demersus) during the first outdoor exposure season.

Twenty-nine juvenile, captive-reared African black-footed penguins (Spheniscus demersus) were hematologically monitored every 2 wk over the period of 24 wk during their first outdoor exposure. Blood samples taken from the penguins were screened for 12 blood evaluation parameters. Parasitemic penguins were medically treated. Eighteen birds (62.1%) experienced naturally acquired malaria and 11 birds (37.9%) remained nonparasitemic. A total of 32 avian malaria episodes were noted; 25 (78.1%) were identified as Plasmodium elongatum, 5 (15.6%) as Plasmodium relictum, and 2 (6.3%) as Plasmodium spp. One P. elongatum (3.4%) and 3 P. relictum (10.3%) infections were fatal. All deaths occurred during the first episode of parasitemia. Gross lesions of the birds that died included hepatomegaly and splenomegaly. Interstitial pneumonia with schizonts was observed on histological examinations. The range, mean, and SD of 12 hematological parameters were determined for nonparasitemic and parasitemic penguins. Differences between these groups in total white blood cell (WBC) counts and relative lymphocytosis (LYMPHS) were not significant. The combined classes of total WBC counts (> 20.0 x 10(3)/microliters) and LYMPHS (> 60.0%) are not indicative of avian malaria infection in African penguins. No correlations were found between changes in the values of blood parameters with season or age of penguins. Treatment of parasitemic birds significantly reduced expected mortality from 50.0% to 13.8%.

An ELISA for detecting anti-Plasmodium spp. antibodies in African black-footed penguins (Spheniscus demersus).

An enzyme-linked immunosorbent assay (ELISA) utilizing 3 Plasmodium falciparum antigens, R32tet32, P.F.R27, and crude red blood cell extract, was developed for the detection of circulating anti-Plasmodium relictum or anti-Plasmodium elongatum antibodies in sera from naturally infected adult African black-footed penguins (Spheniscus demersus) at The Baltimore Zoo, Maryland. A concentration of 2.0 micrograms/ml of each antigen was optimal in terms of specificity, sensitivity, and test speed. It was possible to detect anti-Plasmodium spp. antibodies at a dilution of 10(-4.11). Low absorbance values (less than 0.050) of nonspecific background were observed. The binding efficacy of anti-penguin IgG coupled to alkaline phosphatase to antibodies in the penguin sera was significantly higher than the binding efficacy of anti-chicken IgG. All penguins, bled in the winter time, in controlled mosquito-free conditions had anti-Plasmodium spp. antibodies reactive with P. falciparum antigens. The penguins showed age-dependent variation in antibody levels. There was a decrease in antibody titration units that was significantly correlated with the number of outdoor exposure years experienced by the birds, despite the season-comparable epizootiologic conditions in their summer open-air habitat. We concluded that the decrease of anti-malarial antibodies could be explained by an antibody-mediated equilibrium of immunity in naturally immunized birds harboring endothelial-stage parasites. The ELISA described is sensitive, and it requires a minimal amount of equipment to collect the blood samples. The assay can be used for detecting and monitoring levels of anti-Plasmodium spp. antibodies in selected groups of penguins.

Maternal antibodies against Plasmodium spp. in African black-footed penguin (Spheniscus demersus) chicks.

Anti-Plasmodium spp. antibody titers of mating pairs of adult, captive-reared, African black-footed penguins (Spheniscus demersus) and their chicks were determined using the enzyme-linked-immunosorbent assay (ELISA). Two Plasmodium falciparum antigens were used for the ELISA: R32tet32 (sporozoite antigen), and crude red blood cell extract (CRBCE). Eighteen chicks were bled weekly for ten weeks starting with their day of hatching. The yolk sacs of two penguin eggs were biopsed for ELISA-detectable maternal antibodies (MAB). None of the 28 adult penguins were parasitemic by Giemsa-stained thin blood smear; however, all had anti-Plasmodium spp. immunoglobulins reacting with P. falciparum antigens. All 18 newly hatched chicks had anti-Plasmodium spp. MAB while housed in a mosquito-free environment. The level of MAB in the newly hatched chicks was correlated significantly (P < 0.001) with antibody level detected in their female parents (R32tet32: r = 0.87, CRBCE: r = 0.89). No correlation was found between antibody titers of the newly hatched chicks and their male parents. The level of maternal-fetal antibodies was regressed significantly (P < 0.001) over the 10-week period. Penguin chicks over 10 weeks of age had no anti-Plasmodium spp. MAB. Egg-yolk samples had significantly (P < 0.03) higher MAB titers than female parents that laid these eggs.

Subclinical avian malaria infections in African black-footed penguins (Spheniscus demersus) and induction of parasite recrudescence.

The subclinical and clinical Plasmodium elongatum and Plasmodium relictum infections of captive-reared African black-footed penguins (Spheniscus demersus) were evaluated in nine adult and 29 juvenile penguins in the Baltimore Zoo (Maryland, USA) during summer 1988 and winter 1989. Two diagnostic methods were used: Giemsa-stained thin blood films, and subinoculation of penguin blood into 1-day-old Peking ducklings. Chloroquine and primaquine treatment was applied to all parasitemic juvenile penguins. Twenty-nine parasite-free, juvenile penguins were monitored for parasitemia by Giemsa-stained thin blood films every two weeks for 26 weeks of their first outdoor exposure. Eighteen of 29 penguins experienced naturally acquired malaria; 14 were infected with P. elongatum, three with P. relictum, and one bird had a mixed P. relictum and P. elongatum infection. Eleven of 18 juveniles became parasitemic again after chloroquine and primaquine treatments. Based on Giemsa-stained thin blood smears and subinoculation of penguin blood into 1-day-old ducklings, performed in a mosquito-free environment in winter, nine adult penguins had no evidence of Plasmodium spp. infection. After dexmethasone-induced immunosuppression, four of six of these nonparasitemic adult penguins were found to be infected with P. relictum by the blood inoculation method.

Complications with the use of carfentanil citrate and xylazine hydrochloride to immobilize domestic horses.

Carfentanil citrate, the only opioid approved in the United States for immobilizing large exotic animals, increasingly has been used to chemically restrain exotic horses, such as Prezwalski's horses (Equus przewalskii) and wild horses (E caballus). Because carfentanil's duration of action is long and renarcotization may develop 2 to 24 hours after administration of antagonists, a study was designed to compare the physiologic effects of opioid antagonists, using domestic horses chemically restrained with xylazine hydrochloride and carfentanil. The study was terminated after the initial 3 horses developed severe tachycardia and hypertension, which resulted in the death of 1 horse from pulmonary edema. Although it was possible that the clinical findings in these horses may have resulted from use of an inadequate dosage of carfentanil or xylazine, or both, analysis of the results more likely indicated that domestic and exotic horses may respond differently to carfentanil, and domestic horses may not be a good model for use in studies of carfentanil.

The analysis of a repertory grid.

The problems of repertory grid analysis fall into two main groups: (i) general scaling problems; (ii) specific methods of the simplification of the grid data. Some problems of psychological scaling are considered including 'ranking' versus 'rating', equal interval assumption, and comparison of constructs. The grid analysis methods of principal component analysis, multidimensional scaling and cluster analysis are described briefly, whilst a hand-sorting cluster-type method is demonstrated. Methods for processing pairs and groups of grids are also considered.

Molecular distribution within collagen gel columns.

Purified collagen columns with collagen concentrations of 5 and 10% were constructed by modification of the procedure for the 1% gel as described by Shaw and Schy. The resulting columns were calibrated by elution of various tracers, and the observations compared with those of the 1% collagen gel column. The data were fitted to the Ogston gel model as formulated by Laurent and Killander, but the model was found to be not fully applicable. The thermodynamic treatment of Hjertén was applied to the data with more satisfactory agreement.

The pathology of the appendix in ulcerative colitis.

Sixty-five patients who had the documented histologic diagnosis of chronic ulcerative colitis were selected in order to study appendiceal involvement in this disease. There were 28 males and 37 female patients with an average age of 30 years. The incidence of appendiceal involvement involving the right colon was found to be 47 per cent. In all of these cases the appendiceal involvement occurred in association with the disease process in the cecum or the right colon. In seven patients a classic picture of ulcerative appendicitis in active phase was found. In 78 per cent of patients a similarity between the disease stages in the colon and in the appendix was observed.

Measurement of beryllium in lung tissue of a chronic beryllium disease case and cases with sarcoidosis.

BACKGROUND: The clinical features of chronic beryllium disease (CBD) are similar to many other chronic lung diseases. In particular, it may be difficult to distinguish it from pulmonary sarcoidosis since the two conditions may be very alike in clinical, pathological and radiological features. Aim To determine if the amount of beryllium found in the lungs could be used to differentiate CBD from sarcoidosis and controls. METHODS: Analyses for beryllium in the autopsied lung tissues of 29 cases and controls were carried out. The cases included one CBD, three confirmed sarcoidosis and 25 controls. Blocks of formalin-fixed tissues were analysed by an atomic absorption spectrophotometer equipped with a graphite furnace. A method for analysis of beryllium in air was modified to permit tissue analysis. RESULTS: The CBD case had a much higher average beryllium level, but some individual results were similar to controls and patients with sarcoidosis. CONCLUSION: The CBD case had beryllium levels within the range of values reported in the literature. The differentiation between CBD and sarcoidosis could not be made with reasonable assurance based only on the analytic result. Occupational history is very important in making a diagnosis of CBD, along with the analysis of tissues. Tissue analysis helped confirm the diagnosis of compensatable CBD in this particular case.

Effects of motor activity on children's intentional and incidental memory for spatial locations.

Kindergartners (means = 57) and third graders (means = 8-7) encountered a large model town in 1 of 3 conditions of motor involvement with the environment: standing, riding, or walking. Half the children in each motor condition were instructed to remember the location of the buildings (international memory), while the remaining children were not given specific memory instructions (incidental memory). Only the kindergartners' accuracy increased as a function of the amount of motor activity. There was no difference between intentional and incidental memory conditions. It was concluded that: (1) Kindergartners depend on motor activity more than third graders to learn about the location of objects in an unfamiliar environment; and (2) the complexity of the spatial task was primarily responsible for equivalent performance in the intentional and incidental memory conditions.

The amino acid sequence of the PKR-eIF2alpha phosphorylation homology domain of hepatitis C virus envelope 2 protein and response to interferon-alpha.

A region of the hepatitis C virus (HCV) envelope 2 protein, the protein kinase, PKR and early initiation factor 2alpha phosphorylation homology domain (PePHD), may be important in interferon (IFN)-alpha resistance. The PePHD was amplified by polymerase chain reaction and sequenced, and the amino acid sequence derived from pretreatment serum of 14 genotype 3-infected patients with a range of responses to IFN-alpha therapy. Only 1 patient had a PePHD variant. IFN-resistant PePHD variants present at low titers in pretreatment serum should be selected by therapy; therefore, the PePHD amino acid sequence was also obtained from serum collected during or after treatment in 5 patients with breakthrough or relapse of HCV RNA positivity. No difference was found between the pre- and posttreatment PePHD sequences. Thus, it appears that pretreatment sequencing of the PePHD would not enable clinicians to predict the treatment response. There was no evidence that IFN therapy exerts selection pressure in this region.

Benzene and total hydrocarbons exposures in the downstream petroleum industries.

A review of studies, including both articles published in peer-reviewed journals and reports that were not peer reviewed, regarding occupational exposure to benzene and total hydrocarbons in the downstream petroleum industry operations was performed. The objective was to provide a broad estimate of exposures by compiling exposure data according to the following categories: refinery, pipeline, marine, rail, bulk terminals and trucks, service stations, underground storage tanks, tank cleaning, and site remediations. The data in each category was divided into personal occupational long-term and short-term samples. The summarized data offers valuable assistance to hygienists by providing them with an estimate and range of exposures. The traditional 8-hour time-weighted average (TWA) exposure and the 40-hour workweek do not generally coincide with exposure periods applicable to workers in marine, pipeline, railcar, and trucking operations. They are more comparable with short-term exposure or task-based exposure assessments. The marine sector has a large number of high exposures. Although relatively few workers are exposed, their exposures to benzene and total hydrocarbons are sometimes an order of magnitude higher than the respective exposure limits. It is recommended that in the future, it would be preferable to do more task-based exposure assessments and fewer traditional TWA long-term exposure assessments within the various sectors of the downstream petroleum industry.

A novel alliinase from onion roots. Biochemical characterization and cDNA cloning.

We have purified a novel alliinase (EC 4.4.1.4) from roots of onion (Allium cepa L.). Two isoforms with alliinase activity (I and II) were separated by concanavalin A-Sepharose and had molecular masses of 52.7 (I) and 50.5 (II) kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 51 (I) and 57.5 (II) kD by gel filtration fast-protein liquid chromatography. Isoform I had an isoelectric point of 9.3, while isoform II had isoelectric points of 7.6, 7.9, 8.1, and 8.3. The isoforms differed in their glycosylation. Both contained xylose/fucose containing complex-type N-linked glycans, and isoform II also contained terminal mannose structures. Both isoforms had activity with S-alk(en)yl-L-cysteine sulfoxides. Unlike other allium alliinases, A. cepa root isoforms had cystine lyase activity. We cloned a gene from A. cepa root cDNA and show that it codes for A. cepa root alliinase protein. Homology to other reported allium alliinase genes is 50%. The gene coded for a protein of mass 51.2 kD, with two regions of deduced amino acid sequence identical to a 25- and a 40-amino acid region, as determined experimentally. The A. cepa root alliinase cDNA was expressed mainly in A. cepa roots. The structure and function of the alliinase gene family is discussed.

Preclinical safety testing of DISC-hGMCSF to support phase I clinical trials in cancer patients.

BACKGROUND: DISC-hGMCSF is a gH-deleted HSV-2 based vector expressing human GM-CSF that is being developed for cancer immunotherapy. To support first clinical use, a range of preclinical safety studies were performed using DISC-hGMCSF in addition to DISC-murine-GMCSF and the backbone vector, TA-HSV. METHODS: The toxicity of the DISC vectors was assessed by repeated dose, neurovirulence and neuroinvasiveness studies in mice, and by safety studies in rabbits, guinea pigs and athymic nude mice. Studies were also conducted to determine whether the vector could establish latency in local ganglia in mice following intradermal injection, and whether it could reactivate from the latent state. The vector biodistribution following intravenous administration was also investigated in mice, using PCR to detect vector DNA. RESULTS: The DISC vectors were essentially non-toxic in all the systems studied. No adverse reactions were seen in mice receiving four intravenous doses of DISC-mGMCSF and the results from studies of neurovirulence, neuroinvasiveness, local tolerance in rabbit, general safety in mice and guinea pigs and safety in athymic nude mice were consistent with DISC being unable to replicate and cause disease. The vector could establish latency in local ganglia in mice, but at low efficiency, and could not reactivate infectious virions. Following intravenous administration, vector DNA was widely distributed up to Day 28, but by Day 56 had disappeared from gonads and brain and was only found in blood and liver. CONCLUSION: The panel of safety studies provided evidence that DISC-hGMCSF will be unable to replicate and cause disease, and has low toxicity in man. These data were presented to the Medicines Control Agency and the Gene Therapy Advisory Committee as part of the regulatory submissions for a clinical trial in melanoma patients. These submissions have been approved, and DISC-hGMCSF has now entered a phase I clinical trial in the UK by direct intratumoural injection.