Exploring opportunities for tuning phenyltris(pyrazol-1-yl)borate donation by varying the extent of phenyl substituent fluorination.

The importance of electron deficient Tp ligands motivates the introduction of electron-withdrawing substituents into the scorpionate framework. Since perfluorophenyltris(pyrazol-1-yl)borate affects significant anodic shifts in half-cell potentials in their metal complexes relative those of phenyltris(pyrazol-1-yl)borate analogues, the tuning opportunities achieved using 3,4,5-trifluorophenyl- and 3,5-bis(trifluoromethyl)phenyl(pyrazol-1-yl)borates were explored. Bis(amino)boranes ((3,4,5-F)C6H2)B(NMe2)2 and ((3,5-CF3)C6H3)B(NMe2)2 are precursors to fluorinated tris(pyrazol-1-yl)phenylborates. Thallium salts of these scorpionates exhibit bridging asymmetric κ3-N,N,N coordination modes consistent with the reduced π-basicity of the fluorinated phenyl substituents relative those of other structurally characterized tris(pyrazol-1-yl)phenylborates. While a comparative analysis of the spectral and X-ray crystallographic data for classical Mo(0), Mo(II), Mn(I), Fe(II) and Cu(II) complexes of [((3,4,5-F)C6H2)Bpz3]- and [((3,5-CF3)C6H3)Bpz3]- could not differentiate these ligands with respect to their metal-based electronic impacts, cyclic voltammetry suggests that 3,4,5-trifluorophenyl- and 3,5-bis(trifluoromethyl)phenyl(pyrazol-1-yl)borates affect similar anodic shifts within their metal complexes, with coordination of [((3,5-CF3)C6H3)Bpz3]- rendering metal centers more difficult to oxidize, and sometimes even more difficult to oxidize than their [C6F5Bpz3]- analogues. These data suggest that the extent of phenyl substituent fluorination necessary to minimize metal center electron-richness in phenyltris(pyrazol-1-yl)borate complexes cannot be confidently predicted.

Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.

The identification and characterization of stem cells is introducing a paradigm shift in the field of orthopaedic surgery. Whereas in the past, diseased tissue was replaced with allograft material, current trends in research revolve around regenerating damaged tissue. Muscle-derived stem cells have an application in regeneration of articular cartilage, bone, and skeletal muscle. These postnatal (ie, adult) stem cells can be readily isolated via muscle biopsy. They can display long-term proliferation, high self-renewal, and multipotent differentiation. They also can be genetically modified to secrete growth factors important to tissue healing, thereby functioning as implantable, long-lasting reservoirs for these molecules. Taken together, this evidence suggests that muscle-derived stem cells are well suited for gene therapy and tissue engineering applications for the musculoskeletal system. Effective implementation of even just a few applications of muscle-derived stem cell-based tissue engineering has the potential to revolutionize the way certain musculoskeletal diseases are managed.

Efficacy of particle-based DNA delivery for vaccination of sheep against FMDV.

As an alternative strategy to classical inactivated viral vaccine against FMDV, naked DNA vaccine is attractive because of safety, flexibility and low cost. However DNA vaccination is usually poorly efficient in target species. Indeed we found that naked DNA plasmids encoding for P1-2A3C3D and GM-CSF proteins did not induce any detectable immunity against FMDV in sheep. Interestingly, we demonstrate herein that formulations of DNA on poly(D,L-lactide-co-glycolide) (PLG) or in lipofectin triggered divergent types of immune responses: PLG stimulated a T cell response and could elicit significant neutralising antibody titers, whereas lipofectin generated even higher antibody titers but no significant T cell response. The DNA/PLG regimen used in five sheep protected against clinical symptoms and viraemia and prevented the carrier state in four of them. Thus formulated DNA can be remarkably efficient against FMDV in a ruminant species that is usually refractory to DNA vaccination.

Type I IFN modulates the immune response induced by DNA vaccination to pseudorabies virus glycoprotein C.

DNA vaccines have the capacity to induce strong Th1-biased immune responses that are of major importance to providing protection against intracellular pathogens. In the present study we have focused on the role played by type I IFN in immune responses induced after DNA vaccination. Mice lacking the IFNAR1 chain of the type I IFN receptor (IFNAR K/O mice) were immunized with a plasmid encoding glycoprotein C of pseudorabies virus (PRV-gC). After DNA vaccination, wild-type (WT) mice showed features characteristic of Th1 immune responses, such as high IgG2a:IgG1 anti-PRV Ab ratio and antigen-specific IFN-gamma production by spleen cells. In contrast, IFNAR K/O mice showed a significantly lower IgG2a:IgG1 Ab ratio and IFN-gamma production. In addition, the percentage of CD8(+) and B lymph-node cells expressing CD69 after PRV-gC DNA vaccination was lower in IFNAR K/O than in WT mice. These results support a major role played by type I IFN in shaping Th1 immune responses after DNA vaccination. Codelivery of plasmids encoding IL-12 and IL-18 along with the plasmid encoding PRV-gC restored Th1 responses in IFNAR K/O mice.

Interferon-alpha-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets.

Transmissible gastroenteritis virus (TGEV) infection of piglets results in a very rapid and massive release of IFN-alpha in serum and secretions. The objective of this work was to characterize the IFN-alpha-producing cells (IPC) in tissues of TGEV-infected piglets. Caesarean-derived colostrum-deprived piglets were infected orally with the TGEV virulent Miller strain and IPC were characterized in situ by immunohistochemistry, using a rabbit anti-pig IFN-alpha antiserum. IPC were almost exclusively detected in intestinal tissues and mesenteric lymph nodes (MLN), as early as 6 h post inoculation (p.i.), with a peak at 12-18 h. They disappeared by 24 h. IPC were localized between enterocytes in the small intestine epithelial layer, in the lamina propria, around the Peyer's patches and, at highest frequency, in MLN. Very few IPC were present in the spleen and popliteal lymph nodes of infected piglets. Double immunohistochemical staining for IFN-alpha and leukocyte markers on MLN cryosections showed that IPC were mainly Swine Leukocyte Antigen (SLA) class II positive, and were not stained by an anti-macrophage (SWC3a) MAb. In addition, double staining with anti-TGEV and anti-IFN-alpha MAbs showed that viral antigens were present in MLN, close to IPC. These results show for the first time the presence of IPC in gut mucosa and gut-associated lymphoid tissues in response to an enteropathogenic virus. Moreover, this work shows that IFN-alpha released in serum is likely to originate almost exclusively from gut IPC triggered locally by viral antigens to produce IFN-alpha, since there were very few IPC in spleen or peripheral lymph nodes. MHC class II molecule expression by gut-associated IPC suggests that these cells may be the in vivo mucosal counterparts of the dendritic cells recently shown to produce IFN-alpha after in vitro viral induction.

Antibody-dependent induction of type I interferons by poliovirus in human mononuclear blood cells requires the type II fcgamma receptor (CD32).

The induction of type I interferons (IFNs) in peripheral blood mononuclear cells (PBMCs) can be triggered by viral infection or exposure to viral glycoproteins. Here we show that the IFN-alpha-inducing capacity of attenuated poliovirus vaccine strains is dramatically enhanced in the presence of human polyvalent immunoglobulin G (IgG). The transcription of both IFN-alpha and IFN-beta genes was detected by RT-PCR in stimulated cells. This antibody-dependent activation of type I IFNs genes was also observed with Formalin-inactivated or UV-inactivated poliovirus, but not with empty poliovirus capsids. The ability of poliovirus-antibody complexes to induce IFN-alpha was specifically inhibited when PBMCs were preincubated with an excess of the Fc fragment of IgG. Monoclonal antibodies directed to FcgammaRII (CD32) were also inhibitory, whereas antibodies to the two other classes of Fcgamma receptors, CD16 and CD64, were not. Also, aggregation of FcgammaRII by anti-CD32 antibodies alone failed to induce IFN-alpha production. Our results suggest that induction of type I interferons by poliovirus-antibody complexes depends on CD32-mediated phagocytosis of RNA-containing viral particles. As suggested by the results of an ELISPOT analysis, only a fraction of the IFN-alpha-producing cells are able to synthesize IFN-alpha in response to poliovirus-IgG complexes.

Transient IFN-gamma synthesis in the lymph node draining a dermal site loaded with UV-irradiated herpes simplex virus type 1: an NK- and CD3-dependent process regulated by IL-12 but not by IFN-alpha/beta.

Our previous studies have shown that UV-inactivated, non-replicating herpes simplex virus type 1 (UV-HSV-1) triggers early and transient synthesis of IFN-alpha/beta in the mouse regional lymph node when delivered upstream (i.e. in the ear dermis). In this study, it is demonstrated, by use of a quantitative RT-PCR readout assay, that IFN-gamma mRNA expression was rapidly and transiently upregulated in draining lymph nodes when UV-HSV-1 was delivered in the ear dermis of C57Bl/6 mice. An increased number of IFN-gamma-producing cells was also detected in the lymph node by flow cytometric analysis. Two different subsets of cells, namely DX5(+) NK cells and CD3epsilon(+) T cells, accounted for this early IFN-gamma synthesis. Prompt upregulation of IFN-alpha and IL-12p40 mRNA was also recorded. We took advantage of IFN-alpha/beta-receptor knockout and wild-type 129 mice to study a potential role of IFN-alpha/beta in the signalling pathway leading to IFN-gamma transcription/translation. IFN-gamma mRNA upregulation still occurred in IFN-alpha/beta-receptor(-/-) mice, showing that IFN-alpha/beta was dispensable. The use of IL-12-neutralizing antibodies, prior to UV-HSV-1 delivery, confirmed the major role played by IL-12 in the early/transient IFN-gamma burst.

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus.

This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky's disease virus) DNA vaccine. Aujeszky's disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-gamma. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-gamma). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-gamma had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky's disease.

Absence of porcine interferon alpha secreting cells in severe combined immunodeficiency (SCID) mice inoculated with porcine leukocytes.

A low frequency leukocyte subpopulation, referred to as natural interferon producing cells (NIPC) is able to produce high amounts of interferon alpha (IFN-alpha) following contact with noninfectious viral structures. In order to examine the possible leukocytic nature and bone marrow origin of NIPC, severe combined immunodeficiency (SCID) mice were reconstituted with porcine leukocyte populations, including bone marrow cells. At different times after reconstitution, enriched CD4 and CD45 positive porcine cells were isolated from various mouse organs and tested for the presence of porcine NIPC by porcine IFN-alpha specific ELISPOT assay, after in vitro stimulation by UV inactivated transmissible gastroenteritis virus (TGEV). Although engraftment of porcine cells in SCID mice was shown by flow cytometry and by the production of pig immunoglobulins, no IFN-alpha secreting cells could be detected. This result suggests that NIPC do not derive from bone marrow precursor cells, or that growth factors needed for in vivo expansion of porcine NIPC were absent in mice.

Interferon alpha inducing property of coronavirus particles and pseudoparticles.

Previous work in our laboratory have provided evidence that the membrane glycoprotein M of TGEV is centrally involved in efficient induction of alpha interferon (IFN-alpha) synthesis by non-immune peripheral blood mononuclear cells incubated with fixed, TGEV-infected cells or inactivated virions. Here we report recent completion of studies initiated to get a better understanding of the nature of the interferogenic determinant(s). Transfected cells expressing TGEV M together with the minor structural component E (formerly called sM) were found to trigger IFN-alpha synthesis. Co-expression of these two proteins was shown to be necessary and sufficient for assembly and release of pseudoparticles resembling TGEV virions. Purified pseudoparticles exhibited an interferogenic activity close to that of authentic virions. Chimeric recombinant particles expressing BCV M ectodomain also induced IFN. Examination of cell cultures infected by viruses representative of the three Nidovirales genera revealed that the capacity to act as an efficient IFN-alpha inducer is a common feature of viral particles of the coronavirus genus. Altogether these data bring new insights regarding the putative nature of the viral structure involved in IFN-alpha induction.

Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes.

Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-alpha synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-alpha-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-alpha almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-alpha is more likely to involve a specific, multimeric structure than a definite sequence motif.

Interferon-alpha response to swine arterivirus (PoAV), the porcine reproductive and respiratory syndrome virus.

We studied the interferon-alpha (IFN-alpha) system in relation to the porcine arterivirus (PoAV), porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant porcine IFN-alpha inhibited the growth of this virus in alveolar macrophage cultures. When pigs were challenged intranasally with PoAV, their serum contained IFN-alpha in relatively low concentrations on the second day after challenge and up to 5 days at the latest. Most animals had no IFN-alpha in their lung secretions, even though PoAV replicates in the respiratory tract. In vitro, PoAV replicates in alveolar macrophages, but neither these nor peripheral blood mononuclear cells (PBMC) produced IFN-alpha in response to infection. This may be because PoAV suppresses IFN-alpha production. When macrophages treated with PoAV were superinfected with swine transmissible gastroenteritis virus (TGEV), a known good inducer of IFN, no IFN-alpha was detected. This suppressive effect was lost when the virus was inactivated by UV light. Our results suggest that downregulation of IFN-alpha production may play an important part in enabling PoAV to replicate in cell cultures and in pigs.

In vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero TGEV coronavirus injection.

Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFN alpha) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFN alpha secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFN alpha-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFN alpha was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFN alpha in utero inducibility by mutant virus as compared with wild virus. Flow cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFN alpha secretion. The present data provided in vivo evidence of IFN alpha secretion at the cell level in foetal lymphohaematopoietic organs. Such IFN alpha-secreting cells in lymphohaematopoietic tissues may be the source of IFN alpha detected during foetal infections.

Reconstituted coronavirus TGEV virosomes lose the virus ability to induce porcine interferon-alpha production.

The transmissible gastroenteritis virus (TGEV) is a coronavirus which induces a strong interferon-alpha (IFN-alpha) production in vivo and in vitro. Previous studies have shown that the TGEV external protein M plays a major role in IFN-alpha induction by a non-infectious virus, whereas protein S is not involved. The present study extended these results by showing that monoclonal antibodies (MAbs) directed at the external viral protein sM could not block IFN-alpha induction, which argues against a direct role for this protein. In the same type of blocking experiment, MAbs to the TGEV receptor aminopeptidase N did not inhibit IFN-alpha induction, which strongly indicates that viral replication or entry through the receptor is not needed for TGEV induction of IFN-alpha in leukocytes. In an attempt to isolate functional envelope proteins, TGEV virions were detergent-solubilized and reconstituted in virosomes. Although BIAcore antigenic analysis revealed that the three external viral proteins were present on the virosomes, these proteins were unable to induce IFN-alpha in porcine leukocytes, and seemed to compete with the native virus for IFN-alpha induction. These data indicated that IFN-alpha inducing interactions between TGEV external proteins and leukocytes required a complex native envelope protein structure which has been lost in the virosomes.

Reconstituted coronavirus TGEV virosomes lose the virus ability to induce porcine interferon-alpha production.

The transmissible gastroenteritis virus (TGEV) is a coronavirus which induces a strong interferon-alpha (IFN-alpha) production in vivo and in vitro. Previous studies have shown that the TGEV external protein M plays a major role in IFN-alpha induction by a non-infectious virus, whereas protein S is not involved. The present study extended these results by showing that monoclonal antibodies (MAbs) directed at the external viral protein sM could not block IFN-alpha induction, which argues against a direct role for this protein. In the same type of blocking experiment, MAbs to the TGEV receptor aminopeptidase N did not inhibit IFN-alpha induction, which strongly indicates that viral replication or entry through the receptor is not needed for TGEV induction of IFN-alpha in leukocytes. In an attempt to isolate functional envelope proteins, TGEV virions were detergent-solubilized and reconstituted in virosomes. Although BIAcore antigenic analysis revealed that the three external viral proteins were present on the virosomes, these proteins were unable to induce IFN-alpha in porcine leukocytes, and seemed to compete with the native virus for IFN-alpha induction. These data indicated that IFN-alpha inducing interactions between TGEV external proteins and leukocytes required a complex native envelope protein structure which has been lost in the virosomes.

In vivo induction of interferon-alpha in pig by non-infectious coronavirus: tissue localization and in situ phenotypic characterization of interferon-alpha-producing cells.

A low frequency peripheral blood mononuclear cell (PBMC) subpopulation, referred to as natural interferon-producing (NIP) cells, is described as producing interferon-alpha (IFN-alpha) following contact with non-infectious viral structures, namely viral glycoproteins. These cells are characterized in vitro as non-T, non-B, MHC class II+ and CD4+ cells. In this study, NIP cells were analysed in vivo after an intravenous injection of UV-inactivated transmissible gastroenteritis virus in newborn piglets, which resulted in strong serum IFN-alpha production. Splenocytes, but not PBMC, were the IFN-alpha producers in vivo. Using double immunohistochemical labelling for both IFN-alpha and leukocyte markers, we established that splenic NIP cells were not T or B cells. The majority were MHC class II+ and only a minority expressed a macrophage marker. NIP cells were localized in contact with MHC class II-expressing cells and T cells, which suggested that NIP cells might modulate the antiviral immune response.

Herpes simplex virus induces appearance of interferon-alpha/beta-producing cells and partially interferon-alpha/beta-dependent accumulation of leukocytes in murine regional lymph nodes.

As in vivo experimental system involving the local induction of interferon-alpha/beta (IFN-alpha/beta) responses was established in mice by injecting s.c. ultraviolet (UV)-inactivated herpes simplex virus (HSV) in the right ears, the left ears receiving phosphate-buffered saline (PBS) as a control. Circulating IFN-alpha/beta was present in blood as early as 6 h postinjection, and little or none was found 24 h postinjection. Identification of IFN-alpha/beta-producing cells, carried out by immunohistochemistry and in situ hybridization, demonstrated that the IFN response occurred mainly in the lymph node draining the HSV-injected ear and not in the contralateral lymph node. Occasionally, IFN-alpha/beta-producing cells were found in the spleen and in the skin. The injected HSV caused an inflammatory reaction in the skin and an almost threefold enlargement of the draining lymph node within 6 h. The latter was characterized by a general accumulation of all major lymphocyte subsets and a striking infiltration of neutrophils. Injection s.c. of neutralizing anti-IFN-alpha/beta antibodies before HSV injection reduced the increase in size of the draining lymph node by approximately 50% at 6 h, and no significant effects were seen at 24 h. The localization of cells producing IFN-alpha/beta in the lymph node and the capacity of such IFN-alpha/beta to at least partially mediate an early accumulation of cells suggest that the local IFN-alpha/beta response may have an important role in the initiation of early antiviral immune responses.

The immunology of domestic animals: its present and future.

Veterinary immunology, defined as the immunology of domestic and wild animals having economical or sentimental value to man, provides both practical knowledge that is useful to animal husbandry, and new insights into fundamental immunology. Defining new immunologically based diagnostic tools and immunotherapeutic approaches, including vaccine strategies, obviously still represents important applied objectives. Resistance of domestic animals to disease might also be improved through genetic selection of animals for immunological traits. New contributions to fundamental immunology will arise from so-called 'Immunological opportunities in farm animals' (M. Parkhouse) such as: the ileal Peyer's patch of large animals as an accessible model for studying B cell differentiation; the significance of a high number of gamma delta T cells in ungulates and of double positive CD4+ CD8+ T cells; the possible manipulation of the immune system at a regional level in the living animal by cannulating lymphatic vessels; the use of spontaneous immunodeficiency or autoimmune diseases of animals as models for human clinical research. The fulfillment of these objectives benefits from the flow of information and communications among immunologists working on domestic animals. In addition to national specialized groups, the Veterinary Immunology Committee of the International Union of Immunological Societies (VIC-IUIS) was created to facilitate communication on veterinary immunology; to establish 'official' links between veterinary immunology and the rest of the immunologists' community; and to support the organization of international workshops on domestic animal immunological reagents.

Early ontogeny of immune cells and their functions in the fetal pig.

The origin of immune cells and their products have been studied in the prenatal period in miniature pigs. Macrophages were first detected on day 25, and myelocytes and lymphoid cells by day 28. Membrane antigens SLA-DR and CD45 were found by day 22, membrane molecules MG-7, 8/1, CD1, CD2 and 74-22 by day 28, Gamma/delta T cells were found initially in extrathymic sites (in the liver). The first gamma/delta T cells were detected as early as 40 days of gestation. The expression of fibronectin, Thy-1 and its message, Ig isotypes and the first induction of IFN alpha were described.

Frequency of interferon-alpha-secreting blood leukocytes in irradiated and bone-marrow-grafted pigs.

The effects of irradiation were studied on porcine interferon-alpha (IFN-alpha) secreting cells (IFN-alpha SC). IFN-alpha SC were characterized by an ELISPOT assay on non-adherent PBMC following incubation with the transmissible gastroenteritis coronavirus. In vitro irradiation of PBMC was followed by a decrease in the number of IFN-alpha SC while IFN-gamma production and cell viability were not affected. These data indicate that porcine IFN-alpha SC are relatively radiosensitive. Indeed, the frequency of blood IFN-alpha SC decreased markedly and rapidly after in vivo whole body or partial lymphoid irradiation. In addition, within several days of compatible bone-marrow engraftment in the irradiated animals, the number of blood IFN-alpha SC returned to normal values. These data demonstrate that circulating porcine IFN-alpha SC are derived from bone-marrow progenitors.