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Purpose: To evaluate the clinical feasibility and tolerability of large volume subcutaneous delivery at different injection depths for lean and non-lean subjects., Methods: A single-center, randomized, subject-blinded, crossover study in 62 healthy subjects was conducted to evaluate delivery of a 10-cP solution containing hyaluronic acid. Subjects were separated into lean and non-lean cohort by SC thickness. A syringe pump was used to study the effect of different volumes (5, 12, 25 mL) of a viscous placebo solution and needle lengths (6, 9 and 12 mm) delivered at 0.5 mL/min., Results: Across all treatments, injection sites were observed to have negligible leakage, ~34 kPa of back pressure, and VAS of mild pain with higher pain from needle insertion than during injection. While mild to moderate erythema was the most frequently reported ISR and edema was most prominent for 25 mL injections, all ISRs were resolved within 4 hours post injection. Subjects were unbothered by ISRs across all treatments and rated them as low distress scores (average 1.0-1.5 out of 6)., Conclusion: SC injection of 25 mL is feasible and tolerable using a low-pain formulation for abdomen injection irrespective of subcutaneous thickness and injection depths at a delivery rate of 0.5 mL/min., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ocean health is faltering, its capability for regeneration and renewal being eroded by a steady pulse of anthropomorphic impacts. Plastic waste has infiltrated all ocean biomes, climate change threatens coral reefs with extinction, and eutrophication has unleashed vast algal blooms. In the face of these challenges, synthetic biology approaches may hold untapped solutions to mitigate adverse effects, repair ecosystems, and put us on a path towards sustainable stewardship of our planet. Leveraging synthetic biology tools would enable innovative engineering approaches to augment the natural adaptive capacity of ocean biological systems to cope with the swiftness of human-induced change. Here, we present a framework for developing synthetic biology solutions for the challenges of plastic pollution, coral bleaching, and harmful algal blooms., Competing Interests: Declaration of interests J.J.C. is a cofounder and director of Sherlock Biosciences. The other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In recent years, significant progress has been made in the studies of the spring-driven autoinjector, leading to an improved understanding of this device and its interactions with tissue and therapeutic proteins. The development of simulation tools that have been validated against experiments has also enhanced the prediction of the performance of spring-driven autoinjectors. This paper aims to address critical hydrodynamic considerations that impact the design of spring-driven autoinjectors, with a specific emphasis on sloshing and cavitation. Additionally, we present a framework that integrates simulation tools to predict the performance of spring-driven autoinjectors and optimize their design. This work is valuable to the pharmaceutic industry, as it provides crucial insights into the development of spring-driven autoinjectors and therapeutic proteins. This work can also enhance the efficacy and safety of the delivery of therapeutic proteins, ultimately improving patient outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The collapse of cavitation bubbles induced by abrupt acceleration of the syringe in an autoinjector device can lead to protein aggregation. The details of bubble dynamics are investigated using an axisymmetric, three-dimensional simulation with passive tracers to illustrate the transport of protein molecules. When a bubble near the syringe wall collapses, protein molecules are concentrated in the re-entrant jet, pushed towards the syringe wall, and then spread across the wall, potentially leading to protein adsorption on the syringe wall and aggregation. This phenomenon is more prominent for bubbles positioned closer to the bottom wall, growing to a larger maximum radius. The bubble's maximum radius decreases with the bubble's distance from the syringe wall and air gap pressure, and increases with an increase in liquid column height and nucleus size. The strain rate induced by the bubble collapse is not large enough to unfold the proteins. When the re-entrant jet impacts the bubble surface or syringe wall, the bubble breaks up, generating smaller bubbles with high surface concentration of protein molecules, potentially inducing aggregation in the bulk. The bubble dynamics are influenced by dimensionless distance of the nucleus from the wall, normalized by maximum bubble radius (γ). The re-entrant jet velocity increases with γ, while the maximum liquid pressure, typically 100∼1000 bar, first decreases and then increases with γ. For a cloud of cavitation bubbles, i.e., closely clustered bubbles, coalescence of bubbles can occur, leading to a higher peak pressure at collapse., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: The objective of this work was to systematically evaluate the effects of formulation composition on subcutaneous injection site pain (ISP) using matrices comprising of common pharmaceutical excipients., Methods: Two randomized, blinded, crossover studies in healthy subjects were conducted at a single site, where subjects received 1 mL SC injections of the buffer matrices. ISP intensity was measured using a 100 mm visual analogue scale (VAS), which was then analyzed via heatmap, categorical grouping, subgroup analysis, and paired delta analysis., Results: Buffer type, buffer concentration and tonicity agent showed a substantial impact on ISP. Citrate buffer demonstrated a higher ISP than acetate buffer or saline). The 20 mM citrate buffer was more painful than 10 or 5 mM citrate buffers. NaCl and propylene glycol were significantly more painful than sugar alcohols (mannitol, sucrose, trehalose or glycerol). Histidine buffers exhibited ISP in the descending order of 150 mM > 75 mM > 25 mM > 0 mM NaCl, while histidine buffers containing Arginine-HCl at 0, 50, or 150 mM all showed very low ISP. Histidine buffer at pH 6.5 showed a lower ISP than pH 5.7., Conclusions: This systematic study via orthogonal analyses demonstrated that subcutaneous ISP is significantly influenced by solution composition.

This study focuses on developing a predictive dynamic model for spring-driven autoinjectors. The values of unknown physical parameters, such as the heat convection coefficient and the friction force between the plunger and the syringe barrel, are obtained by fitting the experimentally measured displacements of the plunger and the syringe barrel. The predicted kinematics of the components, such as the displacement and velocity of the syringe barrel, agree well with the experiments with a l 2 -norm error smaller than 10%. The predictions of the needle displacement at the start of drug delivery agree with the experimental measurements with a l 2 -norm error of 20%. The maximum air gap pressure and temperature decrease with the initial air gap height but increase with the elasticity and viscosity of the plunger and the mechanical stop. The proposed experimentally validated dynamic model can be effectively used for device design optimization as it is not computationally demanding., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Interface motion and hydrodynamic shear of the liquid slosh during the insertion of syringes upon autoinjector activation may damage the protein drug molecules. Experimentally validated computational fluid dynamics simulations are used in this study to investigate the interfacial motion and hydrodynamic shear due to acceleration and deceleration of syringes. The goal is to explore the role of fluid viscosity, air gap size, syringe acceleration, syringe tilt angle, liquid-wall contact angle, surface tension and fill volume on the interface dynamics caused by autoinjector activation., Methods: A simplified autoinjector platform submerged in water is built to record the syringe and liquid motion without obstruction of view. The syringe kinematics is imported to the simulations based on OpenFOAM InterIsoFoam solver, which is used to study the effects of various physical parameters., Results: The simulations agree with experiments on the air-liquid interface profile and interface area. The interfacial area and the volume of fluid subject to high strain rate decrease with the solution viscosity, increase with the air gap height, syringe velocity, tilt angle and syringe wall hydrophobicity, and hardly change with the surface tension and liquid column height. The hydrodynamic shear mainly occurs near the syringe wall and entrained bubbles., Conclusion: For a given dose of drug solution, the syringe with smaller radius and larger length will generate less liquid slosh. Reducing the air volume and syringe wall hydrophobicity are also helpful to reduce interface area and effective shear. The interface motion is reduced when the syringe axis is aligned with the gravitational direction.

Recombinant human hyaluronidase PH20 (rHuPH20) facilitates subcutaneous (SC) delivery of co-administered therapeutic agents by locally and transiently degrading hyaluronan in the SC space, and can be administered with therapeutics using a variety of devices. Two SC delivery studies were carried out to assess auto-injector (AI) performance, each in 18 Yucatan miniature pigs. Abdominal injections were administered using three auto-injectors of 1 mL (AI1) and 2 mL (AI2 and sAI2) with different injection speeds and depths (5.5-7.5 mm) and two pre-filled syringe (PFS) devices of 1 and 2 mL. The injection included a placebo buffer with and without rHuPH20 to evaluate the effect of rHuPH20 on SC injection performance. The feasibility of using similar devices to deliver a placebo buffer in humans was investigated. rHuPH20 was not studied in humans. In miniature pigs, postinjection swelling was evident for most PFS/AI injections, particularly 2 mL. Swelling heights and back leakage were typically lower with rHuPH20 co-administration versus placebo for most device configurations (1 or 2 mL PFS or AI). Auto-injections with versus without rHuPH20 also resulted in reduced swelling firmness and faster swelling resolution over time. Slow injections with rHuPH20 had shorter and more consistent injection time versus placebo. In humans, minimal injection site swelling and negligible back leakage were observed for 2-mL injections of placebo, while more erythema was observed in humans versus miniature pigs. Even at high delivery rates with PFS or AI, the addition of rHuPH20 resulted in improved SC injection performance versus placebo in miniature pigs.

Objective: Autoinjectors are a convenient and efficient way to self-administer subcutaneous injections of biopharmaceuticals. Differences in device mechanical design can affect the autoinjector functionality and performance. This study investigates the performance differences of two single-spring-actuated autoinjectors. Methods: We compare the performance between Emgality (120 mg/mL) and Aimovig (140 mg/mL) autoinjector devices from an engineering point of view at two test conditions: room (25 C[Formula: see text]) and storage (5 C[Formula: see text]) temperatures. We employ a novel experimental procedure to simultaneously acquire the force and acoustic signals during operation, and high-speed imaging during the needle insertion and drug injection. Results: We perform 18 quantitative comparisons between Emgality and Aimovig, and we observe that 14 of these have statistically significant differences. For both test conditions, Emgality requires an 8 N activation force while Aimovig requires 14 N activation force, and the needle of Emgality has an insertion depth of 5 mm while Aimovig has an insertion depth of 7 mm. The injection speeds are significantly affected by temperature. Emgality has an injection speed of 0.40 mL/s and 0.28 mL/s at room and storage temperature condition, respectively; while Aimovig has an injection speed of 0.24 mL/s and 0.16 mL/s at those conditions. Lastly, confirmation "click" sound of Emgality occurs 0.75-1.53 s after dose completion, while in Aimovig, the confirmation "click" sound occurs 0.26-0.46 s before dose completion. Conclusions: This study revealed performance differences between Emgality and Aimovig autoinjector devices, despite the fact that the delivery principle of these single-spring-actuated autoinjectors are the same. These differences may result in different risk of intramuscular injection and premature device removal, both of which need to be further verified in clinical trials.

Subcutaneous (SC) delivery of biotherapeutics is well established as a route of administration across many therapeutic areas and has been shown to be effective and well-tolerated. It can offer several advantages over intravenous administration. This notwithstanding, there remain critical development issues and knowledge gaps in SC drug delivery. To articulate and address these issues, the SC Drug Delivery and Development Consortium was convened in 2018 as a pre-competitive collaboration of industry experts in drug delivery, device development, and commercialization. In this review, we outline the Consortium's vision and mission in advancing the development of patient-centered biotherapeutics and establishing a collaborative organization that facilitates open sharing of information and gives voice to diverse viewpoints from SC experts across industries and disciplines. Additionally, we describe the current landscape and challenges associated with SC administration of therapeutic proteins (specifically monoclonal antibodies) and offer insights into potential solutions to these challenges within the context of 8 problem statements developed by the Consortium to highlight key gaps, unmet needs, and actionable issues. Current and future opportunities to accelerate progress in the field through technological advances and the development of drug delivery tools are also discussed., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumor microenvironment is a significant physical barrier to the effective delivery of chemotherapy into solid tumors. To overcome this challenge, tumors are pretreated with an agent that reduces cellular and extracellular matrix densities prior to chemotherapy. However, it also comes with a concern that metastasis may increase due to the loss of protective containment. We hypothesize that timely priming at the early stage of primary tumors will help control metastasis. To test this, we primed orthotopic 4T1 breast tumors with a paclitaxel (PTX)-loaded iron-oxide-decorated poly(lactic- co-glycolic acid) nanoparticle (NP) composite (PTX@PINC), which can be quickly concentrated in target tissues with the aid of an external magnet, and monitored its effect on the delivery of subsequently administered NPs. Magnetic resonance imaging and optical whole-body imaging confirmed that PTX@PINC was efficiently delivered to tumors by the external magnet and help loosen the tumors to accommodate subsequently delivered NPs. Consistently, the primed tumors responded to Doxil better than nonprimed tumors. In addition, lung metastasis was significantly reduced in the animals PINC-primed prior to Doxil administration. These results support that PINC combined with magnetophoresis can facilitate the timely management of primary tumors with a favorable secondary effect on metastasis.

For polymeric nanoparticles (NPs) to deliver more drugs to tumors than free drug solution, it is critical that the NPs establish interactions with tumor cells and avoid removal from the tumors. Since traditional polyethylene glycol (PEG) surface layer interferes with the cell-NP interaction in tumors, we used a water-soluble and blood-compatible chitosan derivative called zwitterionic chitosan (ZWC) as an alternative surface coating for poly(lactic-co-glycolic acid) (PLGA) NPs. The ZWC-coated PLGA NPs showed pH-dependent surface charge profiles and differential cellular interactions according to the pH of the medium. The in vivo delivery of ZWC-coated NPs was evaluated in mice bearing LS174T-xenografts using magnetic resonance (MR) imaging and fluorescence whole body imaging, which respectively tracked iron oxide particles and indocyanine green (ICG) encapsulated in the NPs as tracers. MR imaging showed that ZWC-coated NPs were more persistent in tumors than PEG-coated NPs, in agreement with the in vitro results. However, the fluorescence imaging indicated that the increased NP retention in tumors by the ZWC coating did not significantly affect the ICG distribution in tumors due to the rapid release of the dye. This study shows that stable drug retention in NPs during circulation is a critical prerequisite to successful translation of the potential benefits of surface-engineered NPs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The subcutaneous route offers myriad benefits for the administration of biotherapeutics in both acute and chronic diseases, including convenience, cost effectiveness and the potential for automation through closed-loop systems. Recent advances in parenteral administration devices and the use of additives which enhance drug dispersion have generated substantial additional interest in IV to SQ switching studies. Designing pre-clinical and clinical studies using SQ mediated delivery however requires deep understanding of complex inter-related physiologies and transport pathways governing the interstitial matrix, vascular system and lymphatic channels. This expert review will highlight key structural features which contribute to transport and biodistribution in the subcutaneous space and also assess the impact of drug formulations. Based on the rapidly growing interest in the SQ delivery route, a number of potential areas for future development are highlighted, which are likely to allow continued evolution and innovation in this important area.

Recent advances in subcutaneous drug delivery and device design are transforming the biopharmaceutical sector and improving patient care., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Modern mangroves are among the most carbon-rich biomes on Earth, but their long-term (≥10 6 years) impact on the global carbon cycle is unknown. The extent, productivity and preservation of mangroves are controlled by the interplay of tectonics, global sea level and sedimentation, including tide, wave and fluvial processes. The impact of these processes on mangrove-bearing successions in the Oligo-Miocene of the South China Sea (SCS) is evaluated herein. Palaeogeographic reconstructions, palaeotidal modelling and facies analysis suggest that elevated tidal range and bed shear stress optimized mangrove development along tide-influenced tropical coastlines. Preservation of mangrove organic carbon (OC) was promoted by high tectonic subsidence and fluvial sediment supply. Lithospheric storage of OC in peripheral SCS basins potentially exceeded 4,000 Gt (equivalent to 2,000 p.p.m. of atmospheric CO 2 ). These results highlight the crucial impact of tectonic and oceanographic processes on mangrove OC sequestration within the global carbon cycle on geological timescales.

Nanoparticle (NP)-based approaches to cancer drug delivery are challenged by the heterogeneity of the enhanced permeability and retention (EPR) effect in tumors and the premature attrition of payload from drug carriers during circulation. Here we show that such challenges can be overcome by a magnetophoretic approach to accelerate NP delivery to tumors. Payload-bearing poly(lactic-co-glycolic acid) NPs were converted into polymer-iron-oxide nanocomposites (PINCs) by attaching colloidal Fe3O4 onto the surface, via a simple surface modification method using dopamine polymerization. PINCs formed stable dispersions in serum-supplemented medium and responded quickly to magnetic field gradients above 1 kG/cm. Under the field gradients, PINCs were rapidly transported across physical barriers and into cells and captured under flow conditions similar to those encountered in postcapillary venules, increasing the local concentration by nearly three orders of magnitude. In vivo magnetophoretic delivery enabled PINCs to accumulate in poorly vascularized subcutaneous SKOV3 xenografts that did not support the EPR effect. In vivo magnetic resonance imaging, ex vivo fluorescence imaging, and tissue histology all confirmed that the uptake of PINCs was higher in tumors exposed to magnetic field gradients, relative to negative controls., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cholecystoenteric fistulas are a complication of biliary disease, having an estimated incidence in the range of 0.1 - 0.5 percent. Roughly 8-27 percent of these are of the cholecystocolonic type,1 occurring most commonly at the hepatic flexure. An 82-year-old male presented to our tertiary hospital emergency department and was diagnosed with a cholecystocolonic fistula (CCF) by CT imaging. Surgical evaluation confirmed the CT diagnosis with subsequent removal of the fistula and gallbladder, which was curative.

Background: Segmental antigen challenge in allergic volunteer subjects leads to the recruitment of inflammatory cells, including eosinophils, to the lung and to lung injury as shown by albumin influx into the alveolar air space. The goal of this study was to determine whether eosinophil-active cytokines, including IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor, are released into the lung 24 hours after segmental antigen challenge of ragweed allergic subjects with allergic rhinitis and to determine whether the presence of the cytokine or cytokines is correlated with markers of lung inflammation and lung injury., Methods: Volunteers underwent challenge with a wide variety of antigen doses, which resulted in the recruitment of inflammatory cell mixtures both with and without eosinophils., Results: Eosinophil survival activity (ESA), the ability of the cytokine to prolong blood eosinophil survival in culture, was found in 5 of 17 ragweed allergic subjects and only in subjects challenged with relatively high doses of ragweed antigen (0.2 ragweed antigen units/ml or more). No ESA was found in bronchoalveolar lavage (BAL) fluid in any of eight nonragweed allergic subjects. This activity could be almost completely neutralized by preincubating BAL fluid with specific antibody to IL-5, although a small contribution by granulocyte-macrophage colony-stimulating factor may also have been present. ESA correlated with eosinophil recruitment (r = 0.72, p < 0.001) and degranulation in the lung (r = 0.63 to 0.81, p < 0.01, for eosinophil granule constituents in BAL fluid) and lung injury as shown by albumin influx into the alveolar air spaces (r = 0.83, p < 0.001). ESA was unrelated to the presence of other inflammatory cells in the lung. Subjects who had IL-5 in BAL fluid appeared to undergo more severe initial reactions to antigen challenge., Conclusions: We conclude that IL-5 is the most important constituent in ESA in the lung 24 hours after antigen challenge and that it correlates with eosinophil recruitment, degranulation, and lung injury.

An increase in bronchovascular permeability is thought to play an important role in the pathogenesis of allergic asthma. We sought to determine whether the increase in permeability observed 24 h after segmental antigen challenge in ragweed-allergic human volunteers was associated with the infiltration and degranulation of a specific cell type. A 20,000-fold range of antigen concentrations was used to alter the number and type of inflammatory cells recruited to the lung by challenge. Although large numbers of inflammatory cells were recruited to lung air spaces over a large range of antigen concentrations, significant numbers of eosinophils (731.3 +/- 232.9 x 10(3)/ml) were recruited only when the concentration of antigen used for segmental challenge was > or = 100-fold higher than the concentration needed to produce an 8 to 10 mm wheal 20 min after intradermal skin testing. In addition, large increases in bronchoalveolar lavage (BAL) albumin concentration (636.3 +/- 170.5 micrograms/ml) were observed only in this same group of subjects. The correlation coefficient between the logarithms of the BAL eosinophil concentration and albumin concentration was +0.82 (p < 0.001), and between eosinophil-derived neurotoxin and albumin it was +0.88 (p < 0.001). In a stepwise, multiple regression analysis, eosinophils accounted for 67% of the variance in BAL albumin concentration, whereas no other cell type was a significant predictor of albumin flux into BAL fluid. We conclude that eosinophil recruitment and degranulation are associated with large increases in bronchovascular permeability after segmental antigen challenge in humans.

Acid-sensitive liposomes have been developed for cytosolic delivery of encapsulated substances. We now demonstrate delivery of liposome-encapsulated Ag into the class I MHC Ag processing pathway in peritoneal macrophages in vitro using several types of acid-sensitive liposomes, including those composed of dioleoylphosphatidylethanolamine (DOPE)/palmitoylhomocysteine, DOPE/cholesterol hemisuccinate, DOPE/dioleoylsuccinylglycerol, and DOPE/dipalmitoylsuccinylglycerol. Our previous studies showed that acid-resistant liposomes (dioleoylphosphatidylcholine/dioleoylphosphatidylserine) did not engender class I-mediated presentation in vitro. However, in vivo immunization with OVA encapsulated in acid-resistant as well as acid-sensitive liposomes generated class I MHC-restricted T cell responses, as determined by subsequent in vitro cytotoxicity assays using OVA-transfected target cells. Target lysis by these cells was OVA- and class I MHC (Kb)-specific. This response was not generated by immunization with equivalent amounts of soluble OVA. Thus, a pathway for in vivo class I processing of Ag encapsulated in acid-resistant liposomes has been missed in vitro, perhaps because it is dependent on specific populations of APC or interactions between cells that have not been reconstituted in vitro. This pathway may explain the ability of many exogenous particulate Ag (liposomes, bacteria, parasites, and mammalian cells) to generate class I MHC-restricted T cell responses.

Bronchoscopic antigen challenge of atopic volunteers results in an immediate release of inflammatory mediators and, after a number of hours, the recruitment of inflammatory cells to the lung. The purpose of this work was to investigate the effect of antigen dose on the subsequent recruitment of inflammatory cells to the lung. Twenty-two volunteers without asthma, eight nonatopic control subjects, and 14 ragweed-allergic subjects underwent 25 local antigen-challenge procedures that consisted of a baseline lavage of a control segment, antigen challenge of another segment in the contralateral lung, and lavage of the challenged segment 24 hours later. A 25,000-fold range of antigen doses was used from 0.004 to 100 PNU/ml (0.02 to 500 ng/ml of ragweed antigen E [Amb a I]). Challenge of nonatopic control subjects resulted in the recruitment of only a small number of inflammatory cells, less than a twofold increase in comparison with the cells of control lavage; this increase was primarily due to an increase in neutrophils. Challenge of atopic subjects, in contrast, resulted in approximately a threefold to ninefold increase in inflammatory cells with more cells recruited at larger doses of antigen. Only subjects challenged with a "high" dose of antigen (greater than or equal to 1 PNU/ml) recruited significant quantities of eosinophils to the lung. In these subjects, a twofold increase in macrophages, a fourfold increase in lymphocytes, a 90-fold increase in neutrophils, and an 800-fold increase in eosinophils were observed; the number of neutrophils and eosinophils recruited averaged between 30 and 60 million.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2-macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells.

Liposome-encapsulated protein Ag were used to dissect the roles of various subcellular compartments in Ag processing for class I and class II MHC-restricted presentation. Macrophages exhibited efficient processing of Ag encapsulated in acid-resistant dioleoylphosphatidylcholine/dioleoylphosphatidylserine liposomes, which sequester their contents from potential endosomal processing events and release them only after delivery to lysosomes. Lysosomal processing was demonstrated for all four Ag studied (OVA, murine hemoglobin, bovine ribonuclease A, and hen egg lysozyme), establishing the recycling of immunogenic peptides from lysosomes after Ag processing. These acid-resistant liposomes did not engender class I processing. Ag encapsulated within acid-sensitive dioleoylphosphatidylethanolamine/palmitoylhomocysteine liposomes were also processed via the class II pathway. Of the four Ag encapsulated in liposomes, one, OVA, was tested for ability to stimulate a class I-specific response. OVA in acid-resistant liposomes did not engender a class I-specific response. In contrast, OVA encapsulated in acid-sensitive liposomes was presented by class I molecules, albeit less efficiently than it was presented by class II molecules. We interpret this to be the result of the release of a minor portion of the encapsulated Ag into the cytosol.

In this randomized, crossover study comparing the bioavailability of a film-coated (Ansaid) with a sugar-coated (Froben) 100 mg tablets of racemic flurbiprofen in 23 healthy young men, no significant differences were found for Cmax, tmax or AUC, using a nonstereoisomeric assay for flurbiprofen. Minor differences in the appearance of flurbiprofen in serum during the first 30-min post-dosing period were noted, with Ansaid appearing earlier than Froben. These differences likely reflect dissolution rate dissimilarity between the two products. Stereospecific determinations demonstrate a small (7.8 per cent) but significant difference in AUC of the active S-configuration (Froben greater than Ansaid). No significant differences between Ansaid and Froben were found for tmax or Cmax for the S-flurbiprofen. In bioequivalency studies of chiral drugs, stereospecific approaches provide a more accurate assessment of products.

Antigen processing requires intracellular antigen catabolism to generate immunogenic peptides that bind to class II MHC molecules (MHC-II) for presentation to T-cells. We now provide direct evidence that these peptides are produced within dense lysosomes, as opposed to earlier endocytic compartments. The protein antigen hen egg lysozyme was targeted to endosomes or lysosomes by encapsulating it in liposomes of different membrane composition. Acid-sensitive liposomes released their contents in early endosomes, whereas acid-resistant liposomes sequestered their contents from potential endosomal processing events and released their contents only after delivery to lysosomes. Antigen encapsulated in acid-resistant liposomes was processed in a chloroquine-sensitive manner and presented more efficiently than soluble antigen or antigen encapsulated in acid-sensitive liposomes. Thus, peptides may be recycled from lysosomes, transported to endosomes to bind MHC-II, and then expressed at the cell surface.

A syndrome causing depressed egg production is described. It is characterised either by a failure to attain predicted production targets or by a fall in egg numbers. The depression in production can reach 30% and it may or may not return to normal. For a short period the eggs produced are smaller, lose colour, have poor egg shell strength and many soft shelled eggs are laid. The birds remain apparently healthy and there is a marked age incidence, with most flocks starting this depression in egg production at 29-31 weeks of age. This syndrome has been recently recorded in the Netherlands, but has not been seen before in Northern Ireland. Viruses which agglutinated fowl erythrocytes to very high titres were isolated in chick embryo liver cells from six affected flocks. Three of these isolates were from the oviduct, two from the upper respiratory tract and one from the faeces. These agents are similar to adenoviruses, but were not neutralised by antisera to 11 prototype fowl adenoviruses. In addition, 17 adenoviruses were also isolated from the flocks showing the syndrome described. These isolates fell into five serological types, in addition to nine which could not be typed using antisera to 11 prototype adenoviruses. Investigations of flocks with falls in egg production not conforming to this syndrome yielded five isolates. Six adenoviruses were also isolated from birds with diarrhoea.

An outbreak of mastitis involving approximately 70 out of 140 cows over a two-month period is described. Common mastitis pathogens were not incriminated. Leptospires belonging to the Hebdomadis serogroup were isolated from the milk of three out of five cows and the blood of two of those cows.

Six isolates of rotavirus were made from turkeys and two from chickens. Three of these required trypsin treatment for isolation and serial passage in cell cultures. The remainder were isolated without trypsin treatment. Most of these viruses were isolated in chick embryo liver cell cultures from the faeces of birds aged under 1 week. In six of the eight instances, rotavirus isolation was associated with enteric disturbance, characterised by signs such as diarrhoea, poor or abnormal appetite, abnormally fluid or gaseous intestinal contents or increased mortality. Cross immunofluorescence tests showed that while avian and mammalian rotaviruses shared a common group antigen, the avian viruses were more closely related to each other than to the Nebraska calf rotavirus isolate. On the basis of serum neutralisation tests seven of the eight avian rotaviruses were grouped into three serotypes, with two turkey isolates (Ty1 and Ty3) and a chicken (Ch1) virus being the prototype strains. The remaining virus, Ty2, was intermediate in type between Ty1 and Ch1. Analysis of the RNA of these viruses by polyacrylamide gel electrophoresis showed that they could also be grouped into a number of electropherotypes. The isolates which were serologically distinct were also electrophoretically distinct. Similarly the five isolates which belonged to the Ty3 sero-type were electrophoretically identical. Analysis of the serological and electrophoretic differences suggested that RNA segment 5 may code for a type-specific antigen.

The isolation of a number of strains of infectious bursal disease (IBD) virus from fowl, turkeys and ducks is described. These isolates could be grouped into two serotypes using the neutralisation test. It is proposed that the cell culture adapted vaccine strain from fowl should be the prototype virus for serotype 1 and that the TY89 isolate from a turkey should be the prototype for serotype 2. The isolates in serotype 2 consisted of an antigenically homogeneous group of viruses from turkeys and fowl. However, within serotype 1, which represented isolates from fowl and ducks, some isolates showed only a 30% cross reaction with the vaccine strain. If cross protection mirrors cross neutralisation, then infection with viruses belonging to serotype 2 or with antigenically distant strains from serotype 1 provides one explanation for the apparent failure of the vaccine on certain sites. However, if cross protection does not mirror cross neutralisation, then a virus from serotype 2 could be used as a heterotypic vaccine for young birds with high levels of maternally derived antibody to serotype 1.