African swine fever virus specific porcine cytotoxic T cell activity.

African swine fever virus (ASFV) specific, cytotoxic T lymphocyte (CTL) activity has been studied in a protection model in which SLA inbred miniature swine are experimentally inoculated with a naturally occurring, non-fatal ASFV isolate (NHV). Peripheral blood mononuclear cells (PBMC) from such infected swine show significant activity in CTL assays, using cultured ASFV-infected porcine blood derived macrophages as target cells. This CTL activity is elicited from PBMC by in vitro restimulation of effector cells with low doses (multiplicity of infection = 0.1) of the homologous virus isolate for 48 to 72 h. For SLAc/c effectors, this CTL activity appears to be SLA class I restricted because (1) blocking target cell antigens with monoclonal antibodies (mAb) against SLA class I antigens causes a major reduction in CTL activity; (2) there is preferential lysis of SLA class I matched, ASFV infected targets; and (3) depletion of effector cells with CD8 specific mAb and complement causes a reduction in CTL activity. The CTL activity is ASFV specific for all pigs tested in that infected macrophages are preferentially lysed as compared to normal (non-infected) cultured macrophages or macrophages infected with hog cholera virus (HCV). Lysis of macrophages infected with different ASFV isolates revealed that there is marked lysis of macrophages infected with the virulent L60 isolate but less lysis of macrophages infected with the DR-II and Tengani isolates. In summary, our data show that ASFV specific CTL activity is triggered in swine infected with the NHV ASFV isolate.

Ex vivo expansion and differentiation of hematopoietic stem cells.

Hematopoietic stem cells are phenotypically very heterogeneous, probably reflecting the degree of activation and/or differentiation. This cell population is capable of high-level proliferative activity and multilineage differentiation. Despite its potential for self-renewal, the hematopoietic stem cell exists in a quiescent state for prolonged periods of time. The mechanism(s) involved in triggering these cells to enter the cell cycle is/are not totally clear; however, cytokines (both positive and negative regulators) are implicated. Most, if not all known cytokines that interact at the stem cell level do so not only by inducing proliferation but also differentiation. The ability to maintain a population of truly primitive stem cells for extended periods of time in vitro is currently under investigation by many research groups.

The interaction between bovine herpesvirus type 1 and activated bovine T lymphocytes.

The interaction between activated bovine T lymphocytes (BTLs) and bovine herpesvirus type 1 (BHV-1) was investigated. BHV-1 infection of BTLs reduced the amplitude of recombinant bovine interleukin 2-induced proliferative responses. This decreased proliferation was caused by a virus-induced lymphocytolysis which was dependent on viable virus and was not inhibited by recombinant bovine interferon-alpha I1. Furthermore, lymphocytolysis was not associated with virus replication or with the synthesis of detectable levels of viral proteins. Electron microscopic examination of virus-infected cells revealed that lymphocytolysis was characterized by early nuclear disintegration resembling apoptosis. These observations suggest that activated T cells, localized at the site of BHV-1 infection, may be susceptible to virus-induced cytolysis.

Expression of tumor necrosis factor-alpha receptors on bovine macrophages, lymphocytes and polymorphonuclear leukocytes, internalization of receptor-bound ligand, and some functional effects.

Tumor necrosis factor-alpha (TNF) is a pluripotent protein produced by cells of the monocyte-macrophage lineage. It has important pro-inflammatory functions and is thought to be involved in the pathogenesis of viral-bacterial lung infections of cattle. Binding and internalization of gold-labeled TNF (TNF-G10) by bovine alveolar macrophages as well as by peripheral blood mononuclear leukocytes (PBML) and polymorphonuclear granulocytes (PMN) have therefore been investigated in conjunction with studies of the effect of TNF on some leukocyte functions. TNF-G10 bound to all leukocyte types to varying extents, which however, did not correlate with the influence of TNF on cell functions. Thus, PMN appeared to possess the lowest number of TNF-receptors, but were nevertheless the most sensitive to functional modulation by TNF. The expression of TNF-receptors appeared to be regulated by other cytokines such as IFN-gamma and IL-2, with IFN-gamma down-regulating receptor expression on all cell types, and IL-2 up-regulating receptor expression on lymphocytes. In macrophages internalization of TNF-G10 occurred via clathrin-coated structures, whereas in lymphocytes and PMN the endosomes appeared to lack distinct coating.

Effect of chronic administration of recombinant bovine tumor necrosis factor to cattle.

Cachectin/tumor necrosis factor-alpha (TNF), a protein produced by macrophages upon stimulation, has been implicated as an important mediator of inflammatory processes and of clinical manifestations in chronic infectious diseases. In order to study further the potential role of TNF in infectious diseases, a homologous system was employed in which recombinant Escherichia coli (E. coli) derived bovine TNF (rBoTNF) was injected in cattle, either as a single bolus or in a repetitive treatment-regime. No clinical signs were observed, although changes occurred in hematologic and immunologic parameters when less than 0.5 mg of TNF/100 kg body weight was administered twice daily for 18 days. Prolonged treatment with 0.05-0.5 mg/100 kg induced histologic but no gross changes in the kidneys and liver. When doses were increased above 0.5 mg/100 kg, depression, anorexia, cachexia, and diarrhea appeared rapidly. Pathologic changes were apparent in various tissues including liver, kidneys, and lymphoid organs; body fat depots were depleted. Most of these changes appeared to be reversible; return to normal tissue-morphology occurred within 3 weeks of withdrawal of rBoTNF. The clinical and pathologic changes induced by prolonged rBoTNF administration resembled those observed in some chronic parasitic and viral infections of cattle in which macrophage-activation characteristically occur. Our finding may be relevant to the elucidation of the pathogenesis of these and other chronic infections.

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres.

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.

Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor.

A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.

Role of interferon-gamma in inducing cytotoxicity of peripheral blood mononuclear leukocytes to bovine herpesvirus type 1 (BHV-1)-infected cells.

In the present study, the possible role of interferon (IFN)-gamma on the induction of cytotoxic activity of peripheral blood mononuclear leukocytes (PBML) from BHV-1-immune cattle was investigated. Supernatants obtained from BHV-1-immune PBML, stimulated under conditions similar to those required to demonstrate cytotoxicity, contained an antiviral substance capable of inducing 2'-5'-oligoadenylate synthetase activity in MDBK cells and MHC class II antigen expression on epithelial cells. These supernatants also contained IFN-alpha, but were devoid of tumor necrosis factor and interleukin-2 biological activities. Further studies during primary infection and hyperimmunization with BHV-1 showed that IFN-gamma production and non-MHC-restricted cytotoxicity against BHV-1-infected targets always occurred concomitantly, suggesting that they represent an important part of the detectable CMI responses mounted against this virus. Furthermore, it was also demonstrated that cytotoxicity of PBML against BHV-1-infected cells was reduced with the addition of antibodies to bovine IFN-gamma to the cytotoxic assay. Bovine recombinant IFN-gamma was able to enhance the in vitro cytotoxic activity of PBML from immune cattle, but not from their nonimmune counterparts. This suggests that other factors, in addition to IFN-gamma, may be essential in the development of non-MHC-restricted cytotoxic responses during BHV-1 infection.

Bovine peripheral blood leukocyte population dynamics following treatment with recombinant bovine interferon-alpha I1.

The in vitro functional activity of bovine peripheral blood lymphocytes and the population dynamics of the major peripheral blood leukocyte subpopulations were investigated following the administration of recombinant bovine interferon-alpha I1 (rBoIFN-alpha I1). The intramuscular injection of rBoIFN-alpha I1 induced a 24-h lymphopenia characterized by a decrease in both circulating T and non-T/non-B lymphocytes. An increased CD4/CD8 ratio indicated that there was a relatively greater depletion of the CD8 equivalent subpopulation of T lymphocytes. These changes in lymphocyte trafficking were observed in the absence of a cortisolemia. Coincidental with the lymphopenia, there was a marked decrease in the amplitude of mitogen-induced lymphocyte proliferation responses that probably was caused by a numerical deficit rather than functional deficit in the responder T cells.

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells.

Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.

Studies on the in vitro biological activities of recombinant bovine tumor necrosis factor (rBoTNF) alpha. I. Synergistic antiviral efficacy of rBoTNF alpha, recombinant bovine interferon gamma (rBoIFN gamma) and their combination.

The recent demonstration of the antiviral activity of recombinant human TNF has launched an interest in the use of TNF alpha for antiviral therapy in veterinary medicine. In the precent report, we demonstrate that pretreatment of bovine cells with rBoTNF alpha reduces the yield of bovine herpesvirus type-1 (BHV-1) from infected cells. Reduction in yield was similar to that observed in the presence of rBoIFN gamma. Similarly, rBoTNF alpha was able to protect bovine cells from virus-induced cytopathology. Enhanced antiviral activity was demonstrated when rBoTNF alpha was administered in combination with rBoIFN gamma. Studies on the induction of 2',5'-oligoadenylate synthetase (2'-5' AS) production by cytokine-treated cells showed that although rBoTNF alpha by itself did not induce 2'-5' AS activity it was capable of enhancing the production of 2'-5' AS by rBoIFN gamma-treated cells. Combination of these two cytokines was also evident in the inhibition of proliferation of treated cells. In contrast, the cytotoxic effect of rBoTNF alpha towards actinomycin D-treated cells was not affected by the combination of rBoTNF alpha with rBoIFN gamma.

Induction of MHC class II antigens on bovine cells of nonlymphoid origin by recombinant bovine interferon-gamma and tumor necrosis factor-alpha.

To test a hypothesis that immune mechanisms may be involved in the disease process in cattle persistently infected with and immunotolerant to bovine viral diarrhea virus, the effect of interferon-alpha I 1 (IFN-alpha I 1) and -gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) on bovine endothelial, epithelial, and fibroblast cell class II major histocompatibility complex (MHC) gene product expression was investigated. Untreated control cultures did not express MHC class II antigens. However, following exposure to either recombinant bovine IFN-gamma or TNF-alpha (rBoIFN-gamma, rBoTNF-alpha) MHC class II (Ia) antigen expression was induced on these nonlymphoid cell types. rBoIFN-alpha I 1 did not induce class II antigens, but suppressed their induction by rBoIFN-gamma and TNF-alpha. Induction of Ia-antigen expression, which was dependent on de novo protein synthesis, showed dose- and time-dependency, but once induced, Ia-antigen expression appeared to be fairly stable. Bovine viral diarrhea virus, which, at least in vivo, can induce IFN-alpha, neither induced Ia-antigen expression in the nonlymphoid cell types, nor did the virus interfere with cytokine induction of Ia-antigen expression.

A monoclonal antibody detects macrophage maturation antigen which appears independently of class II antigen expression. Reactivity of monoclonal EBM11 with bovine macrophages.

A mAb EBM11, raised against human macrophages [M phi] was found to detect a bovine M phi diameter-subpopulation. The Ag was strictly intracellular and was expressed in M phi only at a certain state of maturation. Its expression was regulated independently of the activation state of the cells, as revealed by treating M phi in vitro with bovine rIFN-alpha I1, IFN-gamma, or TNF-alpha, all potent M luminal diameter activators. Such treatment had no apparent effect on Ag expression. The Ag was present in 1 to 5% of peripheral blood leukocytes, i.e., up to 20% of circulating blood monocytes, in both normal noninfected cattle and in cattle persistently infected with bovine viral diarrhoea virus. Blood monocytes of the latter group were activated in vivo, but apparently did not reach a more mature state than found in noninfected animals. After an acute infection with bovine herpes virus type 1, the frequency and total number of EBM11+ cells decreased dramatically in inverse relationship to an equally significant increase in frequency and total number of circulating monocytes. In cryostat sections of normal tissues, the EBM11 mAb reacted with sinus M phi and M phi in germinal centers of lymphoid tissues, with alveolar M phi and liver Kupffer cells. In skin it reacted with few scattered M phi in the dermis, but not with epidermal Langerhans cells. This latter feature distinguishes the bovine system from the human. In virus-induced inflammatory processes in skin and keratinized epithelia EBM11+ cells constituted a subpopulation of the infiltrating M phi. The data obtained suggest that EBM11 mAb could be useful both for the elucidation of differentiation/maturation pathways of cells of the monocyte-macrophage lineage as well as for studies of M phi-virus interactions in virus infections. In either aspect cattle could provide a useful comparative model.

Viral-bacterial synergistic interaction in respiratory disease.

In the present review we have identified how viruses can alter the host's susceptibility to bacterial infections by altering both environmental conditions in the lung which favor bacterial replication as well as by suppressing the host's defense mechanisms which prevent clearance of the bacteria. In many instances, these interactions are extremely complex but similar for many viruses. If the virus can overcome the initial host defense mechanisms, which include local antibody and mucus, the virus initiates tissue damage as a result of direct replication within the epithelial cells lining the mucosal surfaces of the respiratory tract. As a result of virus infection, the host cells respond by producing a variety of mediators including various types of interferons, which can alter both virus replication and host response. Replication also produces by-products of virus infection capable of initiating an inflammatory process, which in turn, through release of other mediators, can further modify lung defense mechanisms and encourage bacterial adherence and growth. The bacterium, in turn, releases chemotactic factors which encourage infiltration of specific effector cells into the lung. These effector cells can cause tissue damage and immunopathology, which encourage rapid bacterial growth and may result in death of the animal. In order to be able to control this complicated scenario, it is important either to prevent the initial infection with viruses or to reduce the degree of immunosuppression, so that bacterial clearance can occur rapidly before microcolony formation and extensive lung damage occur. Once a large amount of bacterial replication and lung damage is present, the use of antibiotics is generally of limited value. A schematic illustration of the complexity of the various interactions and counteractions occurring during virus--bacterial synergistic interactions is presented in Fig. 1.

Activity of polymorphonuclear (PMN) leukocytes during bovine herpes virus-1 induced respiratory disease: effect of recombinant bovine interferon alpha I1.

Following infection of cattle with bovine herpes virus-1 there is a state of generalized immunosuppression involving various leukocytes including polymorphonuclear (PMN) leukocytes. Since the PMN is considered to be pivotal in recovery from secondary bacterial infections during bovine respiratory disease, investigations were initiated to determine PMN activity in this disease and whether interferon could modulate PMN activity. In this study, the in vivo administration of recombinant interferon alpha-I1 was shown to increase PMN functions as measured by migration/chemotaxis and generation of reactive oxygen species. This augmented activity of PMN appeared to correlate with the reduction of overall clinical disease, that is, number of sick days, lung lesions and weight loss. In the study administration of interferon by the intranasal or intramuscular route were as effective in stimulating PMN function. Based on these studies it was concluded that the reason for improved performance of calves treated with interferon would be due to its immunomodulatory effects on leukocytes. Although interferon did not alter the initial suppression of PMN functions, these functions returned to normal and exceeded normal activities by 7-9 days post-infection, the time when maximal bacterial activity normally is present.

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo.

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.

Bovine interferon: its biology and application in veterinary medicine.

Investigations of the production and potential use of bovine interferons against viral infections have occurred since the first descriptions of interferons in other systems. The recent advent of recombinant DNA-technology has facilitated such studies and furthered our knowledge about the bovine interferon system in general. This review gives an overview of the biology, antiviral and immunomodulatory activities of bovine interferons. Areas in which the interferons are now applied or have potential application in viral diseases in cattle are described. Finally, the value of studies of the bovine interferon system with respect to comparative interferon research is discussed.

Use of recombinant bovine alpha 1 interferon in reducing respiratory disease induced by bovine herpesvirus type 1.

Intranasal or intramuscular treatment of calves with recombinant bovine alpha 1 interferon before challenge with bovine herpesvirus type 1 and Pasteurella haemolytica reduced clinical signs, number of sick days, lung lesions, and weight loss. The effective dose was determined to be relatively broad within the range of 1 to 50 mg per animal. No adverse effects were observed even at high doses of interferon (50 mg per animal). Administration before virus infection was more effective than administration at the same time as virus infection. Although interferon administration had dramatic effects on the survival of animals, it did not have much effect on virus secretion in the upper respiratory tract. Therefore, the mechanism by which interferon reduces the susceptibility of animals to viral-bacterial synergy was postulated to be via its immunomodulatory effects.

Modulation of porcine peripheral blood-derived macrophage functions by in vitro infection with African swine fever virus (ASFV) isolates of different virulence.

Macrophages play an important role in defense against invading pathogens and neoplastic diseases. Their ability to maintain their functional characteristics is also important in the survival of the host. Loss of macrophage function during infection may be important in determining resistance or susceptibility of a host species. In this communication, we describe the effect of ASFV replication, in macrophages in vitro, on the functional characteristics of these cells. Infection with ASFV did not alter the expression of Fc receptors nor their ability to mediate ADCC. However, African swine fever virus was shown to modulate antibody mediated phagocytosis, chemiluminescence and chemotaxis. Similar modulation of certain macrophage functions was observed with ASFV isolates that differed in their virulence. We were therefore, unable to correlate the virulence of ASFV with the ability to alter macrophage function.