Wayne G. Kimpton

The kinetics of hyaluronan in normal and acutely inflamed synovial joints: Observations with experimental arthritis in sheep

The metabolic half-life of hyaluronan (HA) in synovial fluid was estimated in sheep from the rate of appearance of 3H2O in plasma after injection of highly polymerized labeled HA. This material is substituted with 3H in its acetyl group and is rapidly and almost completely degraded in sheep and other species to yield 3H2O. Previously sensitized sheep were studied before and after induction of acute monoarticular arthritis by intraarticular challenge with type II collagen. In both circumstances 3H was released from the joint in a monophasic exponential pattern and appeared in plasma only as 3H2O. Before challenge, the mean metabolic half-life of [3H]HA was 20.8 hours (range, 15.8 to 27.9 hours, n = 5); an estimate in a single unsensitized sheep (27.0 hours) fell within this range. After challenge, swelling occurred around the joint without frankly increased synovial fluid. The mean half-life fell to 11.5 hours (range, 9.0 to 16.8 hours), with a corresponding increase in mean fractional turnover from 3.5%/h to 6.3%/h; an increased amount of the label was also retained within the peripheral tissues. It is concluded that a relatively mild acute inflammation can induce major changes in the metabolic turnover of synovial HA without the development of gross effusions. In the course of this study, mean synovial fluid volume in the normal sheep hock joint was estimated to be 1.54 mL; the concentration and content of HA were 0.54 mg/mL and 0.84 mg, respectively. These data add to other evidence that the volume and HA content of normal synovial fluid vary widely in different joints and species.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokines as adjuvants for avian vaccines

The worldwide trend towards a reduced reliance on in‐feed antibiotics has increased the pressure to develop alternative strategies to manage infectious diseases in poultry. With this in mind, there is a great emphasis on vaccine use and the enhancement of existing vaccines to provide long‐term protection. Currently existing adjuvants for poultry can have deleterious side‐effects, such as inflammation, resulting in the down‐grading of meat quality and a subsequent reduction in profits. Therefore, to enhance the use of vaccination, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants in poultry is attracting considerable attention, and their potential role as such has been addressed by several studies. The recent identification of a number of chicken cytokine genes has provided the possibility to study their effectiveness in enhancing the immune response during infection and vaccination. This review focuses on the recent studies involving the assessment of cytokines as vaccine adjuvants.

Efficacy of DNA vaccination by different routes of immunisation in sheep.

DNA vaccination, delivered through various routes, has been used extensively in laboratory animals. Few studies have focused on veterinary species and while results obtained in laboratory animals can often be extrapolated to veterinary species this is not always the case. In this study we have compared the effect of the route of immunisation with DNA on the induction of immune responses and protection of sheep to challenge with live Corynebacterium pseudotuberculosis. Intramuscular injection of plasmid DNA encoding an inactivated form of the phospholipase D (PLD) antigen linked to CTLA4-Ig resulted in the induction of a strong memory response and sterile immunity following challenge in 45% of the animals. In contrast, gene gun delivery or subcutaneous (SC) injection of the DNA vaccine induced comparatively poor responses and insignificant levels of protection. Thus, DNA vaccine efficacy in sheep is strongly influenced by the route of vaccination. Amongst intramuscular vaccinates, protected sheep had significantly elevated IgG2 responses compared to unprotected animals, while both subgroups had equivalent IgG1 levels. This suggests that the presence of IgG2 antibodies and hence a Th1-like response, induced by the DNA vaccine gave rise to protective immunity against C. pseudotuberculosis.

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

Induction of lymphocyte recruitment in the absence of a detectable immune response.

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.

Interleukin-2 Directly Induces Activation and Proliferation of Chicken T Cells In Vivo

Cytokines, as immune activators, have been investigated in mammalian systems as natural adjuvants and therapeutics. In particular, interleukin-2 (IL-2) has been studied widely as a vaccine adjuvant and immuno-enhancer because of its role in activating T cell proliferation. We show here that the first nonmammalian IL-2 gene cloned, chicken IL-2 (ChIL-2), exhibits similar biologic activities to those of mammalian IL-2. To assess the activities of ChIL-2 in vivo, we injected birds with recombinant ChIL-2 (rChIL-2) protein. rChIL-2 treatment induced peripheral blood lymphocytes to express cell surface IL-2 receptors (IL-2R) within 48 h and resulted in an increase in the proportion of peripheral blood CD4+ and CD8+ T cells. Using bromodeoxyuridine (BrdU) incorporation as a measurement of cell proliferation, we showed the increase in T cell populations to be due to cell proliferation. The ability of ChIL-2 to cause both activation and proliferation of T cells in vivo indicates that it has the potential to be used as an immune activator.

Inflammation‐induced changes in the phenotype and cytokine profile of cells migrating through skin and afferent lymph

In the present study, we have localized cytokine‐secreting cells within an ectoparasite‐induced inflammatory lesion and monitored the phenotype and cytokine profile of cells migrating from the inflammatory lesion to the local draining lymph node via the afferent lymphatics. Interleukin (IL)‐8‐producing cells were first detected in skin within 6 hr of infection, with increased numbers observed at 24 and 48 hr post infection. While these cells were concentrated within the neutrophil influx, adjacent to disrupted epidermis; they were also found scattered throughout the surrounding dermis in areas where significant cellular infiltration was not apparent. IL‐1α‐ and IL‐1β‐producing cells could not be detected until 24 hr after infection and were restricted to areas of intense neutrophil accumulation. Concurrent with the onset of inflammation was a threefold increase in the total number of cells migrating through the draining afferent lymph. This increase in cellularity was due primarily to increased migration of CD4 and γδ T cells. Cytokine mRNA synthesis by migrating afferent lymph cells was examined by reverse transcription–polymerase chain reaction (RT–PCR) analysis of RNA extracted prior to, and at regular intervals during the course of the inflammatory response. IL‐1β and IL‐8, but not IL‐1α or IL‐6 mRNA, was detected in migrating afferent lymph cells. Tumour necrosis factor (TNF)‐α‐specific mRNA was present in migrating afferent lymph cells at all time points both prior to, and following infection. Soluble IL‐8 protein, but not IL‐1α, IL‐1β or TNF‐α protein, could be detected in lymph, with the amount of IL‐8 detected increasing as the infection progressed. mRNA coding for cytokines associated with T‐cell activation, such as IL‐2, IL‐4 or interferon (IFN)‐γ, was also detected in migrating cells, although the cytokine profiles of different experimental animals were extremely variable.

The in vitro and in ovo responses of chickens to TLR9 subfamily ligands

Although Toll-like receptors (TLRs) have been well characterised in mammals, less work has been carried out in non-mammalian species, such as chickens. In this study the response of chicken cells to the TLR9 subfamily of ligands was characterised in vitro and in ovo. It was found that even though chickens appear to have only one functional receptor to represent the TLR9 subfamily, stimulation of chicken splenocytes with TLR7 and TLR9 ligands induced proinflammatory cytokine production and cell proliferation, similar to that observed when the homologous mammalian receptors are stimulated. Furthermore, we demonstrated that the in ovo administration of these TLR ligands elicits a response, such as cytokine production, that can be detected post-hatch. The current knowledge of the action of TLR ligands in mammals, in conjunction with their immunomodulating ability shown in this study, draws attention to their potential use as therapeutic agents for the poultry industry.

THE SYNTHESIS OF IMMUNOREACTIVE PROLACTIN BY DECIDUA‐CHORION

Normal term human decidua‐chorion, amnion and placenta were examined for possible synthesis of prolactin (PRL) by organ culture of tissue fragments in medium M199. Before incubation, higher PRL concentrations were found in decidua‐chorion (27.7±5.7 ng/mg protein; mean±SE) and amnion (19.7±6.9 ng/mg protein) than in placenta (3.1±0.5 ng/mg protein; p <0.005). Incubated deciduachorion released significantly more PRL into culture medium than did amnion or placenta. After 72 hours culture, the PRL concentration of decidua‐chorion remained high (17.9±6.4 ng/mg protein); that in amnion (2.3±1.9 ng/mg protein) fell to approach that of placenta (1.4±1.0 ng/mg protein). In further studies, fragments of decidua‐chorion were incubated in medium RPMI 1640 without or with puromycin (0.04 mM) or cycloheximide (0.1 mM). A decrease in medium PRL concentration was observed within four hours of adding these inhibitors of protein synthesis. At four hours, the PRL content of decidua‐chorion cultured in puromycin (12.4±3 8 ng/mg protein) or cycloheximide (11.9±2.9 ng/mg protein) was lower than tissue incubated in medium alone (25.5±4.1 ng/mg protein; p <0.05). Comparable differences in tissue content and medium concentration were also seen at 24 and 72 hours. We conclude that human decidua‐chorion synthesizes a protein immunologically simjlar to PRL and that this tissue may represent the principal source of amniotic fluid PRL.

H5N1 infection causes rapid mortality and high cytokine levels in chickens compared to ducks.

Abstract Infection with H5N1 influenza virus is often fatal to poultry with death occurring in hours rather than days. However, whilst chickens may be acutely susceptible, ducks appear to be asymptomatic to H5N1. The mechanisms of disease pathogenesis are not well understood and the variation between different species requires investigation to help explain these species differences. Here we investigated the expression of several key proinflammatory cytokines of chickens and ducks following infection with 2 highly pathogenic H5N1 (A/Muscovy duck/Vietnam/453/2004 (Vt453) and A/Duck/Indramayu/BBVW/109/2006 (Ind109)) and a low-pathogenic H5N3 influenza virus (A/Duck/Victoria/1462/2008 (Vc1462)). H5N1 viruses caused fatal infections in chickens as well as high viral loads and increased production of proinflammatory molecules when compared to ducks. Cytokines, including Interleukin 6 (IL6) and the acute phase protein Serum Amyloid A (SAA), were rapidly induced at 24h post infection with H5N1. In contrast, low induction of these cytokines appeared in ducks and only at later times during the infection period. These observations support that hypercytokinemia may contribute to pathogenesis in chickens, whilst the lower cytokine response in ducks may be a factor in their apparent resistance to disease and decreased mortality.