Subcutaneous lysophosphatidylcholine administration promotes a febrile and immune response in Holstein heifer calves.

Lysophosphatidylcholine (LPC) is immunomodulatory in nonruminants; however, the actions of LPC on immunity in cattle are undefined. Our objective was to study the effects of LPC administration on measures of immunity, liver health, and growth in calves. Healthy Holstein heifer calves (n = 46; age 7 ± 3 d) were randomly assigned to 1 of 4 treatments (n = 10 to 11 calves/treatment): a milk replacer diet unsupplemented with lecithin in the absence (CON) or presence of subcutaneously (s.c.) administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other) or pure LPC (pLPC; 99% LPC-18:0), or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c.-administered LPC (LYSO) for 5 wk. Calves received 5 s.c. injections of vehicle (10 mL of phosphate-buffered saline containing 20 mg of bovine serum albumin/mL; CON and LYSO) or vehicle containing mLPC or pLPC to provide 10 mg of total LPC per kilogram of BW per injection every 12 h during wk 2 of life. Calves were fed a milk replacer containing 27% crude protein and 24% fat at 1.75% of BW per day (dry matter basis) until wk 6 of life (start of weaning). Starter grain and water were provided ad libitum. Body measurements were recorded weekly, and clinical observations were recorded daily. Blood samples were collected weekly before morning feeding and at 0, 5, and 10 h, relative to the final s.c. injection of vehicle or LPC. Data were analyzed using a mixed model, with repeated measures including fixed effects of treatment, time, and their interaction. Dunnett's test was used to compare treatments to CON. Peak rectal temperatures were higher in mLPC or pLPC, relative to CON. Plasma LPC concentrations were greater in mLPC and LYSO calves 5 h and 10 h after the final injection, relative to CON. Calves receiving mLPC and pLPC also had higher circulating serum amyloid A concentrations, relative to CON. Calves receiving mLPC had greater serum aspartate aminotransferase, γ-glutamyltransferase, and glutamate dehydrogenase concentrations, relative to CON. Calves provided mLPC experienced lower average daily gain (ADG) after weaning, relative to CON. The LYSO treatment did not modify rectal temperatures, ADG, or measures of liver health, relative to CON. We conclude that LPC administered as s.c. injections induced an acute febrile response, modified measures of liver and immune function, and impaired growth in calves.

Anakinra for palmoplantar pustulosis: results from a randomized, double-blind, multicentre, two-staged, adaptive placebo-controlled trial (APRICOT).

BACKGROUND: Palmoplantar pustulosis (PPP) is a rare, debilitating, chronic inflammatory skin disease that affects the hands and feet. Clinical, immunological and genetic findings suggest a pathogenic role for interleukin (IL)-1. OBJECTIVES: To determine whether anakinra (an IL-1 receptor antagonist) delivers therapeutic benefit in PPP. METHODS: This was a randomized (1 : 1), double-blind, two-staged, adaptive, UK multicentre, placebo-controlled trial [ISCRTN13127147 (registered 1 August 2016); EudraCT number: 2015-003600-23 (registered 1 April 2016)]. Participants had a diagnosis of PPP (> 6 months) requiring systemic therapy. Treatment was 8 weeks of anakinra or placebo via daily, self-administered subcutaneous injections. Primary outcome was the Palmoplantar Pustulosis Psoriasis Area and Severity Index (PPPASI) at 8 weeks. RESULTS: A total of 374 patients were screened; 64 were enrolled (31 in the anakinra arm and 33 in the placebo arm) with a mean (SD) baseline PPPASI of 17·8 (10·5) and a PPP investigator's global assessment of severe (50%) or moderate (50%). The baseline adjusted mean difference in PPPASI favoured anakinra but did not demonstrate superiority in the intention-to-treat analysis [-1·65, 95% confidence interval (CI) -4·77 to 1·47; P = 0·30]. Similarly, secondary objective measures, including fresh pustule count (2·94, 95% CI -26·44 to 32·33; favouring anakinra), total pustule count (-30·08, 95% CI -83·20 to 23·05; favouring placebo) and patient-reported outcomes, did not show superiority of anakinra. When modelling the impact of adherence, the PPPASI complier average causal effect for an individual who received ≥ 90% of the total treatment (48% in the anakinra group) was -3·80 (95% CI -10·76 to 3·16; P = 0·285). No serious adverse events occurred. CONCLUSIONS: No evidence for the superiority of anakinra was found. IL-1 blockade is not a useful intervention for the treatment of PPP.

Review of the 92nd Annual Meeting of the British Association of Dermatologists, 3-5 July 2012, Birmingham, U.K.

This is a synopsis of the main research and clinical findings presented at the 92nd Annual Meeting of the British Association of Dermatologists, held on 3-5 July 2012 in Birmingham, U.K. The conference highlighted the biological, epidemiological and therapeutic advances that have been made recently in the field of dermatology. This synopsis is a selection of the major findings from the meeting; it is not intended to be a substitute for reading the conference proceedings and related references quoted in this article.

The impact of dietary Black Soldier Fly larvae oil and meal on laying hen performance and egg quality.

Recently, the US FDA and Association of American Feed Control Officials approved Black Soldier Fly larvae (BSFL) as a feed ingredient for poultry. The objectives of this work were 1) to evaluate the nutritional profile of BSFL oil and meal in laying hens, and 2) measure the impact of the BSFL treatments on hen performance and egg quality. In 2 experiments, BSFL oil and meal were fed to replicate hens from 43 to 47 wk and from 51 to 55 wk of age. The hens were fed isocaloric, isonitrogenous diets with 3 treatment levels of BSFL oil (1.5, 3, and 4.5%, Exp. 1) or BSFL meal (8, 16 and 24%, Exp. 2). Data were analyzed by one-factor ANOVA for the main effect of diet and Tukey's multiple comparison for mean separation when significant. Exp. 1 results suggest BSFL oil could readily substituted for soybean oil with commercial hens at inclusion levels up to 4.5%. ADFI, BW, egg production, FCR, and egg weight were not impacted by the oil treatments (P > 0.05). Yolk color among hens fed the BSFL oil was greater averaging 7.88 compared to 7.37 from Control hen eggs (P = 0.0001). Exp. 2 diet formulation replaced soybean oil and meal with BSFL meal, and some additional corn was used in the higher BSFL diets. Diet amino acid balance at the highest level of inclusion (24% BSFL meal) indicates arginine and tryptophan are limiting and ADFI, BW and egg production were reduced (P < 0.05). Egg production averaged 85.14% for the Control, 8 and 16% BSFL meal hens and was significantly greater than hens fed 24% meal at 77.01%. However, 8 and 16% BSFL meal levels had no negative impact on performance and were not significantly different than the Controls. Yolk color was again higher among the meal treatments compared to the control (P = 0.0351). These experiments indicate that BSFL oil and meal can be used as dietary energy, protein and amino acids for hen maintenance, egg production and yolk coloration, although there may be upper limits of dietary inclusion.

Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide.

FhuA, the receptor for ferrichrome-iron in Escherichia coli, is a member of a family of integral outer membrane proteins, which, together with the energy-transducing protein TonB, mediate the active transport of ferric siderophores across the outer membrane of Gram-negative bacteria. The three-dimensional structure of FhuA is presented here in two conformations: with and without ferrichrome-iron at resolutions of 2.7 and 2.5 angstroms, respectively. FhuA is a beta barrel composed of 22 antiparallel beta strands. In contrast to the typical trimeric arrangement found in porins, FhuA is monomeric. Located within the beta barrel is a structurally distinct domain, the "cork," which mainly consists of a four-stranded beta sheet and four short alpha helices. A single lipopolysaccharide molecule is noncovalently associated with the membrane-embedded region of the protein. Upon binding of ferrichrome-iron, conformational changes are transduced to the periplasmic pocket of FhuA, signaling the ligand-loaded status of the receptor. Sequence homologies and mutagenesis data are used to propose a structural mechanism for TonB-dependent siderophore-mediated transport across the outer membrane.

A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins.

BACKGROUND: Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. RESULTS: The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. CONCLUSIONS: These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

Active transport of an antibiotic rifamycin derivative by the outer-membrane protein FhuA.

BACKGROUND: FhuA, an integral membrane protein of Escherichia coli, actively transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane. The transport is coupled to the proton motive force, which energizes FhuA through the inner-membrane protein TonB. FhuA also transports the semisynthetic rifamycin derivative CGP 4832, although the chemical structure of this antibiotic differs markedly from that of ferric hydroxamates. RESULTS: X-ray crystallography revealed that rifamycin CGP 4832 occupies the same ligand binding site as ferrichrome and albomycin, thus demonstrating a surprising lack of selectivity. However, the binding of rifamycin CGP 4832 is deviant from the complexes of FhuA with hydroxamate-type ligands in that it does not result in the unwinding of the switch helix but only in its destabilization, as reflected by increased B factors. Unwinding of the switch helix is proposed to be required for efficient binding of TonB to FhuA and for coupling the proton motive force of the cytoplasmic membrane with energy-dependent ligand transport. The transport data from cells expressing mutant FhuA proteins indicated conserved structural and mechanistic requirements for the transport of both types of compounds. CONCLUSIONS: We conclude that the binding of rifamycin CGP 4832 destabilizes the switch helix and promotes the formation of a transport-competent FhuA-TonB complex, albeit with lower efficiency than ferrichrome. Active transport of this rifamycin derivative explains the 200-fold increase in potency as compared to rifamycin, which is not a FhuA-specific ligand and permeates across the cell envelope by passive diffusion only.

Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA.

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of gram-negative bacteria. These "Trojan Horse" antibiotics may prove useful as an efficient means to combat multi-drug-resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Identification of the high-risk asthmatic patient. Experience with 39 patients undergoing ventilation for status asthmaticus.

Thirty-nine asthmatic patients required mechanical ventilation (IPPV) for status asthamticus over a seven and a half year period. We reviewed their clinical records with particular emphasis on the events leading to intermittent positive pressure ventilation (IPPV) and the long-term courses of those patients who survived IPPV. Long delays by patients before seeking medical attention, incomplete assessment of acute attacks, underuse of corticosteroids prior to admission and overuse of sedation were important factors often influencing the necessity for IPPV. Four patients died during IPPV. Of the 35 who survived, 32 were regularly followed in our Respiratory Clinic. Nine patients subsequently died, eight undoubtedly from asthma. Serial measurements of forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1) were retrospectively analyzed to determine patterns of asthma. Of the 23 patients still alive, 14 have well controlled asthma, five have a pattern of persistent airflow obstruction, two have markedly labile asthma, and two have gradually deteriorating airflow obstruction. By contrast, seven patients who died sufficiently long after IPPV to enable categorization of their patterns showed either markedly labile asthma or gradually deteriorating airflow obstruction. None had relatively constant ventilatory function at either normal or suboptimal levels. We suggest that patterns of asthma are useful guides in detecting patients at high-risk. Patterns characterized by markedly labile asthma or gradually deteriorating airflow obstruction appear to be associated with an increased risk of sudden death from asthma.

The clinical course and management of thoracic empyema.

We report a prospective multi-centre study of the clinical course and hospital management of thoracic empyema in 119 patients (mean age 54.8). The commonest presenting symptom was malaise (75%), 55% were febrile; 31% were previously well with no predisposing condition. Initial treatments were antibiotics alone (5), needle aspirations (46), intercostal tube drainage (61), rib resection (3) and decortication (4). Overall, intercostal drainage was used in 77 patients (16 failed aspirations), surgical rib resection in 24 (1 failed aspirations, 20 failed drainage), and surgical decortication in 28 (6 failed aspirations, 17 failed drainage). Only 4 patients received intrapleural fibrinolytic agents. Aspiration and drainage were likely to fail if the empyema was > 40% of the hemithorax. Median time from treatment start to discharge was: aspirations, 26 days; drainage, 23 days; resection 11 days; decortication, 12 days. Overall 21 patients died (12 with empyema as the major cause); two had been surgically treated. Mortality correlated with age, diabetes, heart failure, and low serum albumin at admission. Infecting organisms, identified in 109 patients (92%) included anaerobes (37), Str. melleri (36), and Str. pneumoniae (28). Six months after discharge, all but six survivors had regained their previous health.

Chronic obstructive airways disease following treated pulmonary tuberculosis.

Seventy-one subjects who had previously been treated for tuberculosis up to 16 years before underwent pulmonary function assessment. Evidence of airways obstruction was found in 48 (68%). There was an inverse relationship between the extent of the disease on the original chest radiograph and the forced expired volume in one second (FEV1). A similar inverse relationship between the amount of sputum produced and the FEV1 and the original chest radiograph was also found. Treated pulmonary tuberculosis is a cause of significant chronic obstructive airways disease.

Effects of maternally administered dexamethasone and acute hypoxemia at 0.7 gestation on blood pressure and placental perfusion in sheep.

Glucocorticoid administration to women in premature labor significantly decreases preterm infant morbidity and mortality. Fetal exposure to maternally administered glucocorticoids in late gestation causes fetal hypertension. We determined the effects of a single course (4 injections at 12-hr intervals) of dexamethasone (DM; 2 mg, a weight-adjusted dose equivalent to one-third the dose administered to pregnant women) or saline (S) in sheep at 103-104 days of gestation (dGA; term 149 dGA) on maternal and fetal blood pressure (BP). We also determined the BP and placental perfusion effects of acute maternal hypoxemia. Venous and arterial catheters were placed in 10 ewes and fetuses (DM = 6, S = 4) at 96 +/- 1 dGA. Maternal and fetal placental perfusion was determined with fluorescent microspheres. Dexamethasone increased fetal but not maternal BP; maternal and fetal placental blood flow and vascular resistance (VR) were unchanged. At 105 dGA, hypoxemia was induced for 1 hr by maternal nitrogen gas inhalation to decrease fetal PaO2 by 40%. Hypoxemia increased BP in DM but not S fetuses or mothers in either group. Hypoxemia decreased maternal placental blood flow by 39 +/- 7% and 51 +/- 9% and increased maternal placental VR by 65 +/- 7% and 69 +/- 6% in S and DM mothers, respectively. Hypoxemia did not alter fetal placental blood flow or VR in either treatment group. In summary, at 0.7 gestation, DM induces a hypertensive response to fetal hypoxemia that is characteristic of older fetuses but does not alter hypoxemia-induced reductions in maternal placental blood flow.

Epidemiological features of drug resistant tuberculosis in Harare, 1994 to 1996.

OBJECTIVES: To characterise the prevalence, clinical and radiological features of drug resistant tuberculosis in selected patients with pulmonary tuberculosis in Harare between 1994 and 1996. DESIGN: A retrospective review of medical and microbiological records. SETTING: Beatrice Road Infectious Diseases Hospital, Harare, Zimbabwe. SUBJECTS: 381 smear-positive tuberculosis patients who had samples submitted to the National Tuberculosis Reference Laboratory for culture and susceptibility testing. MAIN OUTCOME MEASURES: Prevalence of resistance of isolated cultures of Mycobacterium tuberculosis to anti-tuberculosis drugs; clinical, radiological and microbiological response to treatment with recommended anti-tuberculosis regimens. RESULTS: Resistance to one or more drugs was detected in 16 isolates (16/165, 9.7%), single drug resistance in five (3.0%) and resistance to two or more drugs in 11 (6.7%). There were no distinctive clinical or radiological features of drug-resistant tuberculosis, although a higher percent of drug resistant cases had evidence of pleural disease (25% vs 2.5%, p = 0.005). Neither past history of tuberculosis or known or suspected HIV infection was associated with the presence of drug resistance. CONCLUSIONS: In spite of the resurgence of tuberculosis and the high prevalence of HIV infection in Zimbabwe, the rates of drug resistance have remained relatively low, even among a selected population at high risk of resistance. A significant proportion of cases of drug-resistant tuberculosis appear to be due to new transmission of drug resistant strains, which reinforces the importance of maintaining a surveillance system for the monitoring of drug susceptibility. Ongoing prospective studies should provide more reliable estimates of the prevalence and determinants of drug resistance in Zimbabwe.

Fluorescent probe for high-throughput screening of membrane protein expression.

Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture.