SK&F 86002, a dual cytokine and eicosanoid inhibitor, attenuates endotoxin-induced cardiopulmonary dysfunction in the pig.

Cytokines and eicosanoids are well documented important mediators of endotoxemia. Bicyclic imidazoles are a novel class of nonsteroidal anti-inflammatory compounds that display unique pharmacological profiles by reducing cytokine production and arachidonic acid metabolism. In this study, we evaluated the ability of the bicyclic imidazole, SK&F 86002, to attenuate endotoxin-induced cardiopulmonary dysfunction. Pigs were randomly assigned to one of four groups: LPS (n = 5), given .5 microgram/kg/h 055:B5 Escherichia coli lipopolysaccharide (LPS) intravenously (i.v.) for 6 h; saline (n = 5); SK&F 86002 (n = 3), given 50 mg/kg SK&F 86002 orally 30 min prior to anesthesia; and SK&F 86002 + LPS (n = 5). Administration of LPS resulted in cardiopulmonary dysfunction characterized by decreased stroke volume and arterial oxygen tension, and increased room air alveolar-arterial oxygen gradient, pulmonary arterial pressure, pulmonary vascular resistance, and peak intratracheal pressure. Additionally, LPS administration was associated with leukopenia and increased pulmonary myeloperoxidase activity. Pretreatment with SK&F 86002 attenuated LPS induced hypotension, hypoxemia and bronchoconstriction and blocked the pulmonary hypertension. SK&F 86002 blocked the LPS-induced increase in myeloperoxidase activity, indicating a reduction in pulmonary neutrophil infiltration, but had no effect on systemic leukopenia. Pretreatment with SK&F 86002 significantly attenuated LPS-induced increases in plasma thromboxane B2 and tumor necrosis factor-alpha. We hypothesize that ameliorating effects of SK&F 86002 in this endotoxin model of cardiopulmonary dysfunction are related to inhibition of cytokine and eicosanoid biosynthesis.

Positive selection of EqCD8+ precursors increases equine lymphokine-activated killing.

Lymphokine activated killing (LAK) is an example of natural cytotoxicity, and as such is a critical means of defense against diseases such as viral infection and neoplasia. Despite this important role, the specific molecular interactions involved in LAK or other forms of natural cytotoxicity are only partially understood. In some species, cells capable of mediating natural cytotoxicity express the CD8 molecule, although no specific role has been demonstrated for CD8 in non-MHC restricted cytotoxicity. In this study the role of the EqCD8 equine homolog of CD8 in LAK cell activity was examined. A series of LAK assays were performed using equine lymphocyte populations enriched or depleted for EqCD8 expression by positive or negative selection. The results indicate that positive selection of LAK precursors using an anti-EqCD8 (CVS8) antibody greatly increases LAK cytotoxicity. The implications for the role of the EqCD8 molecules in LAK are discussed.

Report of the Second Equine Leucocyte Antigen Workshop, Squaw valley, California, July 1995.

The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaborations were fostered among the participating laboratories and observers. Overall, enormous advances have been made in the past decade since mAbs specific for equine leucocyte antigens and immunoglobulins were first reported. There remains enormous scope and need for further studies of equine leucocyte antigens and immunoglobulins, both for the purposes of comparative immunology and for the good of the horse. In the future novel techniques will be required to develop reagents for specific target antigens such as the orthologues of the CD25 or CD45 isoforms. In studies of equine immunoglobulins the functional role of the IgG isotypes must be better established, reagents for IgE must be developed, and cloning of the immunoglobulin heavy chain genes will be essential if the complexities of the IgG sub-isotypes are to be elucidated. The tasks still facing the currently small group of equine immunologists throughout the world remain formidable, and will only be tackled successfully in a spirit of collaboration.

Effects of dexamethasone on development of immunoglobulin G subclass responses following vaccination of horses.

OBJECTIVE: To determine the effects of dexamethasone on development of IgG subclass responses following vaccination of healthy horses. ANIMALS: 11 mature Thoroughbreds. PROCEDURE: Horses received 2 IM injections at 2-week intervals of a vaccine containing inactivated infectious bovine rhinotracheitis, bovine viral diarrhea, and parainfluenza-3 viral antigens and were then randomly assigned to 2 groups. Six horses received dexamethasone (0.2 mg/kg of body weight, IM) twice weekly for 8 weeks starting the day of the first vaccination. Five control horses received an equivalent volume of saline (0.9% NaCl) solution. Antigen-specific serum IgG subclass titers were determined weekly after vaccination by use of an ELISA. RESULTS: Vaccination resulted in similar antigen-specific serum IgG(T) titers in dexamethasone-treated and control horses. In contrast, although control horses developed IgGa and IgGb responses after vaccination, corticosteroid administration completely inhibited these responses in treated horses. CONCLUSIONS AND CLINICAL RELEVANCE: Cortico steroids can have profound effects on primary immune responses in horses and can significantly affect IgG responses to inactivated vaccines. Corticosteroid treatment regimens commonly used to treat diseases in horses may result induction of a nonprotective IgG subclass response, leaving treated horses susceptible to disease. Additionally, mechanisms regulating IgGa and IgGb responses appear to differ from those regulating IgG(T) responses. Further defining these mechanisms is a critical step in designing effective vaccines, and corticosteroid-induced immunomodulation may be a valuable tool for studying immune responses in horses.

Effect of Metal Cations on the Viscosity of a Pectin-Like Capsular Polysaccharide from the Cyanobacterium Microcystis flos-aquae C3-40.

The properties of purified capsular polysaccharide from the cyanobacterium Microcystis flos-aquae C3-40 were examined by capillary viscometry. Capsule suspensions exhibited similar viscosities between pH 6 and 10 but were more viscous at pH <=4 than at pH 6 to 11. At pH 7, a biphasic effect of metal ion concentration on capsule viscosity was observed: (i) capsule viscosity increased with increasing metal ion concentration until a maximal viscosity occurred at a specific concentration that was a reproducible characteristic of each metal ion, and (ii) the viscosity decreased with further addition of that ion. Because the latter part of the biphasic curve was complicated by additional factors (especially the precipitation or gelation of capsule by divalent metal ions), the effects of various metal chlorides were compared for the former phase in which capsule viscosity increased in the presence of metal ions. Equivalent increases in capsule viscosity were observed with micromolar concentrations of divalent metal ions but only with 10 to 20 times greater concentrations of Na(sup+). The relative abilities of various metal salts to increase capsule viscosity were as follows: CdCl(inf2), Pb(NO(inf3))(inf2), FeCl(inf2) > MnCl(inf2) > CuCl(inf2), CaCl(inf2) >> NaCl. This pattern of metal efficacy resembles known cation influences on the structural integrity of capsule in naturally occurring and cultured M. flos-aquae colonies. The data are the first direct demonstration of an interaction between metal ions and purified M. flos-aquae capsule, which has previously been proposed to play a role in the environmental cycling of certain multivalent metals, especially manganese. The M. flos-aquae capsule and the plant polysaccharide pectin have similar sugar compositions but differ in their relative responses to various metals, suggesting that capsular polysaccharide could be a preferable alternative to pectin for certain biotechnological applications.

Susceptibility of equine chorionic girdle cells to lymphokine-activated killer cell activity.

PROBLEM: Equine chorionic girdle cells are a subpopulation of highly invasive trophoblast cells that attach and invade the uterine epithelium on Day 35 (Day 0 = day of ovulation). These invading chorionic girdle cells form endometrial cups that are associated with a marked local maternal leukocytic response that may result in the demise of the cups at Day 120 of pregnancy. Once endometrial cups become established in the uterine wall they do not express MHC antigens, and therefore may only be susceptible to non-MHC restricted cytotoxic cells. The susceptibility of cultured chorionic girdle cells to LAK cell cytotoxicity was tested in order to evaluate the role of this type of cytotoxicity in the life-cycle of endometrial cup tissue. METHODS: Chorionic girdle cells from ten Day 34 conceptuses were collected, cultured, and used as a target cell in a lymphokine activated killer assay to determine if these cells were susceptible to lymphokine activated killing. RESULTS: Cultured chorionic girdle cells demonstrated similar in vitro morphological features and patterns of antigen expression to those seen in vivo in endometrial cups. Cultured chorionic girdle cells were susceptible to lymphokine activated killing. CONCLUSIONS: Lymphokine activated killer cells may play a role in the degeneration of endometrial cup tissue. The potential mechanisms of regulation of uterine LAK cell activity in the horse are discussed.

Okadaic acid induces dephosphorylation of histone H1 in metaphase-arrested HeLa cells.

It is shown here that treatment of metaphase-arrested HeLa cells with okadaic acid (0.15-2.5 microM) leads to dephosphorylation of histone H1. This effect is presumably due to the specific ability of okadaic acid to inhibit protein phosphatases 1 and/or 2A, because okadaic acid tetraacetate, which is not a phosphatase inhibitor, has no effect. Dephosphorylation of H1 does not occur if okadaic acid-treated cells are simultaneously treated with 20 nM calyculin A, or if the okadaic acid concentration is 5.0 microM or greater. The mechanism behind this phenomenon is not known. However, the results suggest that the chain of events leading to histone dephosphorylation may be negatively controlled by a protein phosphatase 2A, while the phosphatase which actually dephosphorylates H1 could be a protein phosphatase 1. It remains to be determined whether the phosphatase involved here is the same enzyme as that which dephosphorylates H1 at the end of normal mitosis.

Local and systemic isotype-specific antibody responses to equine influenza virus infection versus conventional vaccination.

Inactivated alum-adjuvanted conventional equine influenza virus vaccines are of poor efficacy and offer limited short-term protection against infection. In sharp contrast, natural infection with equine influenza virus confers long-term protective immunity. In order to identify the protective immune responses to equine influenza virus, the influenza virus-specific IgA, IgGa, IgGb, IgGc and IgG(T) antibody responses in nasal secretions and serum induced by natural infection and a commercial vaccine were studied by ELISA. Two groups of four influenza-naive ponies were established. In the natural infection group, ponies received 10(8.5) EID50 of A/equine/Ky/1/81 by intranasal instillation, were allowed to recover, and then were rechallenged 100 days later. All four ponies exhibited clinical signs of influenza virus infection and viral shedding following primary infection, but were completely protected from challenge infection. Antibody responses to primary infection were characterized by nasal IgA and serum IgGa and IgGb responses. Ponies in the conventional vaccine group received a commercially available vaccine by intramuscular injection followed by a booster injection 3 weeks later. Challenge infection 100 days after vaccination resulted in clinical signs of infection and viral shedding. Antibody responses to vaccination were restricted to serum IgG(T) responses only. These results demonstrate that the protective immunity generated by natural equine influenza virus infection is associated with a mucosal IgA immune response and humoral IgGa and IgGb sub-isotype responses, and that this pattern of response is not generated by conventional vaccines.

Receptor-specific regulation of B-cell susceptibility to Fas-mediated apoptosis and a novel Fas apoptosis inhibitory molecule.

The susceptibility of primary B cells to Fas (APO-1, CD95)-mediated apoptosis is modulated by signals derived from additional surface receptors: CD40 engagement produces upregulation of Fas expression and marked sensitivity to Fas-induced cell death, whereas antigen receptor engagement, or interleukin-4 receptor (IL-4R) engagement, inhibits Fas killing and thereby produces Fas resistance, even in otherwise susceptible, CD40-stimulated targets. Surface immunoglobulin (sIg) and IL-4R utilize distinct signaling pathways to produce Fas resistance that rely on protein kinase C and signal transducer and activator of transcription 6, respectively sIg signaling for inducible Fas resistance requires nuclear factor-kappaB and depends on new macromolecular synthesis. Proximate mediators for Fas resistance include the known anti-apoptotic gene products Bcl-xL and FLIP (but not Btk), and a novel anti-apoptotic gene that encodes Fas apoptosis inhibitory molecule (FAIM). FAIM was identified by differential display and was cloned as two alternatively spliced forms: FAIM-S is broadly expressed, whereas faim-L expression is tissue specific. faim is highly evolutionarily conserved, suggesting an important function throughout phylogeny. Inducible resistance to Fas-mediated apoptosis is speculated to protect antigen-specific B cells during potentially dangerous interactions with FasL-bearing T cells; the elevated sIg-signaling threshold for inducible Fas resistance in autoreactive, tolerant B cells would insure against autoimmunity. However, aberrant acquisition of Fas resistance may allow autoreactive B cells to escape Fas deletion and malignant lymphocytes to thwart antitumor immunity.

c-Rel is required for the protection of B cells from antigen receptor-mediated, but not Fas-mediated, apoptosis.

The NF-kappaB/Rel transcription factor family has been shown to protect many cell types from apoptotic signals. However, it is not known whether NF-kappaB is required for all survival pathways and whether each NF-kappaB member plays a unique or a redundant role. Here we describe the results of studies on the role of c-Rel in survival. Mature B cells from c-Rel(-/-) mice exhibit defects in survival, including sensitivity to Ag receptor-mediated apoptosis as well as increased sensitivity to ionizing radiation and glucocorticoids. Transgene expression of Bcl-x(L), a c-Rel target gene, rescues c-Rel(-/-) B cells from their survival defects. Thus, c-Rel-dependent survival pathways are crucial for protection from apoptotic signals that target the mitochondrial pathway. Despite a lack of Bcl-x(L), c-Rel(-/-) B cells can still be rescued from Fas-mediated apoptosis via B cell receptor signaling. The Fas apoptosis inhibitor molecule and FLICE inhibitory protein (c-FLIP) proteins are up-regulated normally in c-Rel(-/-) B cells, and these two molecules may play a more physiological role in the Fas pathway. Furthermore, unlike the TNF sensitivity of RelA(-/-) fibroblasts, c-Rel-deficient fibroblasts are refractory to TNF-mediated cell death. Thus, c-Rel is dispensable for protection against death receptor-mediated apoptosis. Taken together, our data suggest that distinct NF-kappaB/Rel members are required for protecting cells from different types of apoptotic signals.

Immunogenicity and efficacy of baculovirus-expressed and DNA-based equine influenza virus hemagglutinin vaccines in mice.

Two fundamentally different approaches to vaccination of BALB/c mice with the hemagglutinin (HA) of A/Equine/Kentucky/1/81 (H3N8) (Eq/KY) were evaluated, that is, administration of HA protein vs administration of HA-encoding DNA. Each vaccine was tested for its immunogenicity and ability to provide protection from homologous virus challenge. HA protein was synthesized in vitro by infection of Sf21 insect cells with a recombinant baculovirus. Intranasal administration of this vaccine induced virus-specific antibodies, as measured by enzyme-linked immunosorbent assay (ELISA), but did not induce virus neutralizing (VN) antibodies. This route of administration provided partial protection from virus challenge, but interestingly, this protection was completely abrogated, rather than enhanced, by co-administration of 10 micrograms of cholera holotoxin. As a second approach, mice were directly vaccinated in vivo by Accell gene gun delivery of plasmid DNA encoding the Eq/KY HA gene. This approach induced VN antibodies as well as virus-specific ELISA antibodies. When two doses of DNA vaccine were administered 3 weeks apart, mice were not protected from challenge, although they cleared the infection more rapidly than control mice. However, when the second DNA vaccination was delayed until 9 weeks after the first, 9 out of 10 vaccinated mice were completely protected. These results indicate that the time between initial and booster DNA vaccinations may be an important variable in determining DNA vaccination efficacy.

Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene.

Equine influenza virus infection remains one of the most important infectious diseases of the horse, yet current vaccines offer only limited protection. The equine immune response to natural influenza virus infection results in long-term protective immunity, and is characterized by mucosal IgA and serum IgGa and IgGb antibody responses. DNA vaccination offers a radical alternative to conventional vaccines, with the potential to generate the same protective immune responses seen following viral infection. Antigen-specific antibody isotype responses in serum and mucosal secretions were studied in ponies following particle-mediated delivery of hemagglutinin (HA)-DNA vaccination on three occasions at approximately 63-day intervals. One group of four ponies were vaccinated at skin and mucosal sites and the another group were vaccinated at skin sites only. All ponies were subjected to a challenge infection 30 days after the third vaccination. Skin and mucosal vaccination provided complete protection from clinical signs of infection, while skin vaccination provided partial protection; DNA vaccination provided partial protection from viral shedding. DNA vaccination generated only IgGa and IgGb antibody responses, which occurred with a higher frequency in the skin and mucosa vaccinated ponies. No mucosal IgA response was generated prior to challenge infection and IgA responses were only detected in those ponies which shed virus postchallenge. These results demonstrate that HA-DNA vaccination induces IgG(a) and IgG(b) antibody responses which are associated with protection in the absence of mucosal IgA responses. In addition, additional DNA vaccinations of mucosal sites increased protection and the frequency of seroconversion in ponies.