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Biomedical subjects

H P Hughes

Publications and source records attributed to H P Hughes.

At least 19 recordsLinked to original sources
PubMed1998

Cytokine adjuvants: lessons from the past--guidelines for the future?

Formulation of vaccines has for the most part relied on simple adjuvants which are able to enhance the immune response to the immunogen. Cytokines are an attractive alternative to conventional preparations, and have been tested in a number of different systems. However, experience has indicated that there are a number of guidelines that must be followed. The dose of cytokine administered is critical for optimal effect. Too little will have no effect, and too much will have undesirable side effects. For instance, at high doses IL-2 can induce autoimmune disease and interferon gamma can have a suppressive effect. Cytokines may also have to be administered at the same site or even to the same cell as the antigen for optimal effect. Conjugation or molecular chimerization of antigens and cytokines can achieve this effect efficiently. Formulation of cytokine with antigen may overcome any detrimental effect that the antigen may have. Should the antigen have any suppressive epitopes or have a direct effect on essential intracellular mechanisms, cytokines may be used to overcome these effects. In some cases, Th1 or Th2 cytokines have been used to enhance a protective Th1 or Th2 response. However, the paradigm does not always hold, and Th1 cytokines can enhance Th2 responses, or have no overall effect on phenotype. Further, in some host species, there is evidence that there may be no Th1/Th2 dichotomy. The most important aspect of using cytokines as adjuvants is in ensuring that there is a balanced response.

Adjuvants, Immunologic↗
PubMed1996

Effect of recombinant bovine interleukin-1 beta in normal calves and in calves infected with bovine herpesvirus type 1.

Bovine herpesvirus-1 (BHV-1) is an important pathogen of respiratory infections in cattle. Its continuing importance lies in its ability to predispose infected hosts to bacterial infections. In this present study, we determined whether the immunoregulatory effects induced by interleukin-1 (IL-1) could stimulate appropriate host defense mechanisms to influence the course of BHV-1 infection in cattle. We first evaluated the effect of different doses (10-1000 ng/kg) of IL-1 in normal cattle. A single administration of IL-1 was able to induce a dose-dependent increase in polymorphonuclear (PMN) cells as well as monocytes in peripheral blood. The number of CD3+ lymphocytes and gamma/delta T cells in peripheral circulation decreased transiently in a dose-dependent manner. In the disease model, the effect of IL-1 administration (300 ng/kg) 24 h before, at the time of, and 24 h after the BHV-1 challenge was assessed. As a single therapeutic modality, IL-1 did not significantly reduce the establishment or progression of BHV-1-induced disease. Nevertheless, our results demonstrated that the significant modulation of diverse immune parameters did not exacerbate disease. Thus, the use of IL-1 as an adjunct therapy or as a vaccine adjuvant in cattle can be safely considered in situations where BHV-1 infection is likely to occur.

Adjuvants, Immunologic↗
PubMed1995

Effect of recombinant bovine interleukin-1 beta on viral/bacterial pneumonia in cattle.

Bovine herpesvirus-1 (BHV-1) is an important pathogen of respiratory infections in cattle. Its continuing importance lies in its ability to predispose infected hosts to bacterial infections (e.g., Pasteurella haemolytica). In this study we determined whether the immunoregulatory effects induced by recombinant bovine interleukin-1 (rbIL-1) could stimulate appropriate host defense mechanisms to influence the course of BHV-1 and P. haemolytica infection in cattle. We first evaluated the effect of multiple doses (5 doses of 300 ng/kg) of rbIL-1 in normal cattle. An increase in polymorphonuclear (PMN) cells, as well as monocytes, in peripheral blood was observed during the course of IL-1 administration. In addition, the phagocytic activity of monocytes was increased. Although the phagocytic and oxidative burst activities in PMN decreased during the course of rbIL-1 treatment, no changes were observed in the bactericidal capacity of these cells. Lymphocyte numbers in peripheral blood remained unchanged; however, the functional activity of these cells, as measured by IFN-gamma production upon in vitro stimulation, was decreased. In the bovine respiratory disease model, multiple administration of IL-1 did not influence significantly the progression of BHV-1/P. haemolytica infection in cattle. Thus, our results demonstrated that IL-1, although not therapeutically effective, could be administered safely as an adjuvant, even during the course of BHV-1/P. haemolytica infection.

Animals↗
PubMed1995

Regulation of bovine acute phase responses by recombinant interleukin-1 beta.

The acute phase response is a collection of physiologic changes initiated early in the inflammatory process. This response is comprised of both localized changes at the site of infection or injury and the initiation of systemic responses, such as the increase in production of acute phase proteins. Cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) play key roles in the regulation of acute phase response in the species studied to date. To better characterize the acute phase response of cattle, recombinant bovine (rBo). IL-1 beta was administered to cattle. A single administration of rBoIL-1 beta was able to induce a dose dependent increase in body temperature, circulating leukocytes, and serum haptoglobin and fibrinogen concentrations, as well as a decrease in plasma zinc concentration. Five daily administrations of rBoIL-1 beta resulted in heightened and prolonged elevations of haptoglobin and fibrinogen. In addition, alpha 1-acid glycoprotein levels were increased, a response not seen after a single administration of rBoIL-1 beta. These results indicate that IL-1 is an important regulator of the acute phase response in cattle.

Acute-Phase Reaction↗
PubMed1994

A slow release formulation for recombinant bovine interferon alpha I-1.

Recombinant bovine interferon-alpha I1 (rBoIFN-alpha) has known antiviral and immunomodulatory effects which have been exploited to reduce clinical disease in a number of clinical situations including bovine respiratory diseases. A slow release rBoIFN-alpha formulation may be of value to reduce bovine respiratory disease under field conditions by extending the period of protection, and hence improving the prophylactic benefits of rBoIFN-alpha. In this report, we describe a formulation of rBoIFN-alpha in sesame oil containing calcium stearate which can successfully sustain the release of rBoIFN-alpha over an 8-day period. Recombinant bovine IFN-alpha could be measured in serum for 8 days following treatment with an initial burst of release 6 h after injection. After a single subcutaneous depot injection of 50 mg and 100 mg of rBoIFN-alpha, initial serum levels reached 12-15 ng/ml and 25 ng/ml respectively. Correlating with this burst of release, there was a decrease in the number of circulating CD4-CD8- gamma delta+ T lymphocytes, and a slight neutropenia. No alterations in other cell phenotypes tested (CD4, CD8, CD2, CD6, B cells, monocytes or MHC class II) were observed, nor were there changes in lymphokine activated killer (LAK), natural killer (NK) cell activity, or oxygen radical formation (assessed by reduction of nitroblue tetrazolium). However, despite the rapid and short-lived burst of rBoIFN-alpha, levels of 2-5 oligoadenylate (2-5 A) synthetase remained elevated for 8 days. The sustained increase of 2-5 A synthetase was not due to the high initial dose released during the burst 6-12 h after injection, since injection of a bioavailable equivalent dose of interferon induced a significant rise in 2-5 A synthetase activity for 4 days only. As 2-5 A synthetase is known to be a correlate of antiviral activity, we propose that this formulation of rBoIFN-alpha may be one approach to increase the window of protection, leading to more effective prevention of bovine respiratory disease.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Regulation of major histocompatibility complex class II expression by Pasteurella haemolytica leukotoxin.

Many properties have been associated with Pasteurella haemolytica leukotoxin and other repeat-in-toxin toxins, including their cytotoxic activity on various cells of the lymphoid and nonlymphoid systems as well as their ability to modulate the immunological activity of lymphocytes and monocytes. In this study, we assessed the ability of P. haemolytica leukotoxin to affect the expression major histocompatibility complex (MHC) class II molecules on bovine peripheral monocytes. Peripheral blood mononuclear cells were isolated from P. haemolytica leukotoxin-seronegative calves and incubated with various concentrations of authentic leukotoxin as well as the recombinant lktA gene product (LktA). Expression of MHC class II antigen on cells was evaluated by flow cytometric methods. The results indicated that both a crude, authentic leukotoxin preparation and LktA were able to affect MHC class II expression by inducing a marked downregulation of MHC class II expression on bovine monocytes. However, when cells were activated with gamma interferon (IFN-gamma), LktA and Lkt had little or no detectable effect. By using a cell line which expresses MHC class II only after activation by INF-gamma, we were able to confirm the observation that LktA had no effect on the expression of MHC class II after IFN-gamma treatment. Leukotoxin affected the functional capacity of monocytes to present antigen, as demonstrated by the ability of LktA or authentic leukotoxin to totally inhibit a mixed lymphocyte culture from MHC-mismatched calves. Thus, leukotoxin was able to downregulate constitutive expression of MHC class II expression, and we propose that this is a novel way in which this molecule can affect the immune function of monocytes, playing an important role in bacterial pathogenesis and survival of organisms at the infection site.

Animals↗
PubMed1994

Flow cytometric and microscopic evaluation and effect on fertility of abnormal chromatin condensation in bovine sperm nuclei.

The techniques of Feulgen staining, acridine orange staining, and a sperm chromatin structure assay using acridine orange and flow cytometry were compared for selective examination of bovine sperm nuclei. Twenty frozen semen samples were simultaneously analysed by all three methods. The prevalence of abnormally condensed DNA and its relationship to other semen traits were determined in ejaculates from 70 bulbs presented for routine examination for breeding soundness and in frozen semen from 348 bulls evaluated over five years. A breeding trial with 118 beef heifers using semen from six bulls with different degrees of nuclear abnormalities was performed to assess the importance of the defects with respect to fertility. The results indicate that few spermatozoa with abnormal DNA condensation are found in normal semen, but the incidence increases with disturbance of spermatogenesis. However, high numbers of abnormally condensed nuclei were found in the absence of an increase in other defects. This nuclear defect might be at least partially of epididymal origin; it can lower fertility and can be compensated for by increasing the numbers of normal spermatozoa in the insemination dose. The percentage of abnormally condensed sperm nuclei as detected by Feulgen staining was significantly correlated with that detected by microscopy after acridine orange staining and by the sperm chromatin structure assay. We therefore consider the Feulgen technique to be a valuable tool for assessing the nuclear integrity of bovine spermatozoa.

Acridine Orange↗
PubMed1992

Multiple administration with interleukin-2 potentiates antigen-specific responses to subunit vaccination with bovine herpesvirus-1 glycoprotein IV.

Interleukin-2 has been described as an effective adjuvant for a number of antigens in different host species. Previously, we demonstrated the adjuvant activity of recombinant bovine IL-2 with a glycoprotein IV (gIV) subunit vaccine from bovine herpesvirus type-1 (BHV-1). In the present study, primary antibody responses were assessed in cattle immunized with either 2 or 50 micrograms of gIV, and treated with multiple doses of IL-2 or combinations of IL-2 and IFN-alpha or IL-2 and IFN-gamma. IL-2 was able to augment significantly antibody responses detected by either ELISA or virus neutralization. More significantly, IL-2 was able to enhance antibody titres in animals immunized with only 2 micrograms gIV to levels similar to those immunized with 50 micrograms gIV in the absence of IL-2. For optimal stimulation, multiple injections of IL-2 and Avridine had to be used in the formulation; other oil adjuvants or IL-2 alone could not induce a primary serum antibody response. Addition of IFN-alpha or IFN-gamma to the IL-2/gIV/Avridine formulation did not affect any of the immune parameters tested. As IFN-alpha is an effective immunoprophylactic agent for infectious bovine rhinotracheitis (IBR), combination vaccine-immunoprophylaxis may become feasible using IL-2 as a co-adjuvant. Thus, extremely low doses of antigen and only one immunization may be an effective vaccine given in combination with interferon prophylactic treatment.

Animals↗
PubMed1992

Molecular chimerization of Pasteurella haemolytica leukotoxin to interleukin-2: effects on cytokine and antigen function.

A chimeric recombinant protein composed of the lktA gene product from Pasteurella haemolytica fused to bovine interleukin-2 (IL-2) was made. The LKT-IL-2 chimera was compared with recombinant bovine IL-2 with regard to the ability to induce proliferative responses and LAK cell activity in bovine peripheral blood mononuclear cells in vitro. In both instances, chimerization had no effect on IL-2 activity. Similarly, the LKT component was unaffected in its ability to induce an effective immune response after immunization. The adjuvant properties of IL-2 have been established in a number of models, and this effect was tested by using the chimera. A multiple-injection protocol of LKT-IL-2 was compared with single-dose administration of LKT. The results obtained indicate that while there was no increase in specific antibody production, the IL-2 component of the chimera may be able to affect antigen-specific proliferation, as assessed by limiting-dilution analysis. Use of cytokine-antigen chimeras may provide a valuable antigen-adjuvant formulation that is simple to produce and purify and thus have economic advantages over conventional preparations. Furthermore, chimerization will also ensure that the adjuvant acts at the same site as the antigen, thus optimizing immunostimulatory activity.

Animals↗
PubMed1992

Clinical and immunological effects of single bolus administration of recombinant interleukin-2 in cattle.

Recombinant bovine interleukin-2 (rBoIL-2) was administered as a single intramuscular bolus to healthy calves to determine the minimal dose capable of exerting a biological response. Doses ranging from 2.5 to 0.05 micrograms rBoIL-2/kg did not induce pyrexia, diarrhea, or depression, nor did they alter any blood chemistry or hematological parameters commonly associated with IL-2 toxicity. Moreover, the only significant immunological change observed was a reduction in the number of peripheral blood lymphocytes identified with the monoclonal antibodies B7A, BAQ4A (WC1+ cells), CACTB6A (WC2+ cells) and DH59B (monocytes). The decrease in cells associated with these markers did not influence non-MHC restricted cytotoxicity or in vitro lymphocyte proliferative responses to mitogens and IL-2. The treatments had no effect on delayed type hypersensitivity responses to phytohemagglutinin. These results indicate that IL-2 may be involved in the regulation of trafficking patterns of a unique subpopulation of lymphocytes in cattle.

Animals↗
PubMed1991

Rotavirus particles function as immunological carriers for the delivery of peptides from infectious agents and endogenous proteins.

A major problem in the development of useful animal subunit vaccines has been the generation of immune responses to weakly immunogenic molecules. For this purpose a new and effective delivery system has been devised. This system is based upon the inner capsid of bovine rotavirus. Under the appropriate conditions, the inner capsid protein, designated BP6, can be made to self-assemble in vitro and form spherical particles. These particles possess an inherent capacity to target to cells of the immune system. Exploitation of these properties has led to the development of technology to couple antigens to the VP6 particles such that the sphere acts as a novel immunological carrier. This is based on a "binding peptide" derived from another rotavirus peptide, VP4, as well as on more traditional techniques of chemical coupling. We have coupled peptides or proteins to this carrier via the binding peptide and have shown that every epitope tested to date gave excellent immune responses. Furthermore, using this carrier, immunity has been developed without the use of adjuvants. This has far-reaching implications for animal and human immunization.

Amino Acid Sequence↗
PubMed1991

Application of interferons in the control of infectious diseases of cattle.

Recovery from infection involves a number of complex interactions between specific cells of the immune system. Many of these interactions are mediated by cytokines, which can activate these cells to kill or reduce the replication rate of the pathogen. Availability of large quantities of recombinant cytokines has provided the opportunity to investigate the mechanism or mechanisms of action of each cytokine in vitro and in vivo. In the present review, we describe the application of interferons to reduce morbidity and mortality of cattle suffering from bovine respiratory disease and mastitis. The potential application of interferons in disease modulation as well as the impediments to their use are discussed.

Animals↗
PubMed1991

Immunopotentiation of bovine herpes virus subunit vaccination by interleukin-2.

Cattle were immunized with glycoprotein IV (gIV) from bovine herpes virus-1 (BHV-1). Groups of five animals were then given either 2, 3, 4, or 5 doses of interleukin-2 (IL-2) (0.5 microgram/kg) at 12-hr intervals. Animals that received no IL-2 exhibited specific immune responses that are typical for BHV-1 infection, i.e. enhanced specific cytotoxicity, lymphocyte proliferative responses to gIV, and increased gIV-specific (ELISA) and virus-neutralizing antibodies. Treatment of animals with five doses of IL-2 significantly augmented all of these responses except serum neutralization (P less than 0.05). Furthermore, the dose of IL-2 that was selected did not induce any non-specific responses, i.e. hypergamma-globulinaemia, changes in blood chemistry, increased lymphokine-activated killer (LAK) cell activity, changes in mitogen responsiveness or alterations in the phenotypic profile of circulating lymphocytes. Nor were there any clinical changes associated with IL-2 therapy (e.g. depression, pyrexia, diarrhea). Animals that were treated with less than five doses of IL-2 also exhibited elevated immune responses, but they were not significantly different from untreated immunized controls. Interestingly, animals given five doses of IL-2 responded to minor contaminants present in the gIV preparation. This allows speculation that this dose regimen of IL-2 is not only a potent adjuvant for conventional vaccine immunizing doses, but will also allow the use of minute quantities of antigen for immunization.

Adjuvants, Immunologic↗
PubMed1989

Susceptibility of Eimeria bovis and Toxoplasma gondii to oxygen intermediates and a new mathematical model for parasite killing.

Eimeria bovis and Toxoplasma gondii differ in their susceptibility to macrophages activated by lymphokines. Interferon-gamma can activate macrophages to totally inhibit E. bovis sporozoite development, whereas growth of T. gondii tachyzoites in macrophages is not totally affected. The susceptibility of these parasites to oxygen intermediates and their ability to evade the oxidative burst by macrophages were investigated in cell-free systems. Using a logistic model to assess growth inhibition, T. gondii growth was impaired by 50% at 10(-4.25) M (56 microM) H2O2, with 30 min as the optimum time for measuring inhibition. Preliminary results indicate that T. gondii follows mode-one and mode-two killing with relation to time after exposure to H2O2, implying a role for OH. and the induction of a DNA repair mechanism. The same model was used to assess inhibition of E. bovis growth that was more susceptible, being inhibited to 50% by 10(-5) M (10 microM) H2O2. Both parasites were susceptible to the effects of xanthine-xanthine oxidase that releases a full complement of oxygen intermediates (H2O2, OH., (1)O2, and O2-). Adding quenchers or scavengers to the system confirmed that T. gondii was susceptible to products of the interaction of O2- and H2O2 (OH. and (1)O2), and that E. bovis sporozoites were at least partially susceptible to H2O2 and O2-, but extremely susceptible to OH.. These data were supported by studies on scavenging enzymes present in the parasites. Toxoplasma gondii was rich in superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPO), and E. bovis had less catalase and SOD.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Immunity patterns during acute infection by Eimeria bovis.

Cellular and humoral responses were investigated following gavage inoculation of 6-wk-old bull calves with 35,000-40,000 oocysts of Eimeria bovis. At 3-4-day intervals for 40 days after inoculation (DAI), blood was taken and assessed for serum IgG against merozoites and sporozoites of E. bovis. Proliferative responses of peripheral blood lymphocytes were measured following stimulation with either concanavalin A (Con A) or a soluble antigen derived from E. bovis oocysts (EbAg). Serum IgG against merozoites and sporozoites reached a peak of activity between 10 and 20 DAI, coinciding with oocyst shedding on days 17 to 24. Serum antibody titers had dropped to base levels by 40 DAI, although anti-merozoite titers remained elevated for the duration of the study (i.e., from days 12 and 20 to day 40). Con A stimulation of lymphocytes was not affected by infection; there was no evidence of suppressed or augmented responsiveness. Lymphocyte responses to EbAg had reached a maximum by day 20 and remained elevated throughout the study. These results indicate (a) that sporozoites and merozoites share antigens recognized by serum IgG, (b) that there is no episode of marked immunosuppression during acute infection, and (c) that cellular immunity is probably more important in resistance against reinfection than humoral immunity.

Acute Disease↗
PubMed1988

Oxidative killing of intracellular parasites mediated by macrophages.

An important function of macrophages is to eliminate invading pathogens, and one of their main weapons involves the generation of lethal oxygen radicals. Yet some parasites and pathogens - notably Leishmania, Toxoplasma, and Listeria and Mycobacterium - make use of macrophages as their primary cellular hosts displaying a capacity to survive the oxidative killing mechanisms of these host cells. It is now clear that more than one pathway is involved in the activation of macrophages to kill intracellular pathogens. Here, Huw Hughes discusses the biochemistry of the oxidative metabolism of macrophages, and the steps taken by parasites to survive within this hostile environment.

Journal Article↗
PubMed1988

Antigen-specific lymphocyte transformation induced by oocyst antigens of Eimeria bovis.

Lymphoproliferative responses against a preparation of Eimeria bovis antigens (EBAg) were measured in E. bovis-immune and naive animals. Optimal lymphocyte responsiveness could be measured after 7 days of culture in the presence of antigen at a cell concentration of 2 X 10(5) cells per well. The specificity of the reaction was confirmed by limiting dilution analysis. Whereas immune peripheral blood mononuclear cells responded to EBAg (f = 1/18,824), naive cells did not (f = 0). The helper function of cells proliferating in response to EBAg was investigated by raising T-cell lines and a clonal population derived from a line. The T-cell line showed an enhanced reactivity to EBAg by limiting dilution analysis (f = 1/256) and was interleukin-2 dependent. Limiting dilution analyses indicated at least two populations of cells: one that was interleukin-2 restricted and antigen dependent and another that was antigen independent. Supernatants from T-cell lines and the clone were analyzed for the production of lymphokines after antigen stimulation. Minimal amounts of interleukin-2 were produced. The T-cell line produced both gamma interferon (IFN-gamma) (750 U) and IFN-alpha (1,250 U), whereas the clone produced IFN-gamma (1,250 U) only. Short-term (4-day) stimulation of immune cells by EBAg induced the production of IFN-gamma (600 U) and a non-IFN macrophage-activating lymphokine. We conclude that this macrophage-activating lymphokine is only produced after short-term culture and that further culture of T cells results in the proliferation of other clones producing other factors (such as IFN).

Animals↗