Bent Aasted

Analysis of the immunological cross reactivities of 213 well characterized monoclonal antibodies with specificities against various leucocyte surface antigens of human and 11 animal species.

213 Monoclonal antibodies (mAbs) raised against leucocyte surface antigens from human and 11 animal species were analyzed for reactivities against leucocytes from human and 15 different animal species. We found 77 mAbs (36%) to cross-react. Altogether, 217 cross reactions were registered out of 3195 possible combinations (7%). Most of the cross reacting mAbs had integrin or MHC class II specificities. This study defined cross reactions on the following markers: CD1a, 1c, 2, 4, 5, 8, 9, 11a, 11b, 14, 18, 20, 21, 23, 29, 31, 41, 43, 44, 45, 45R, 46, 49, 61, 62L, TCR gamma/delta, BCR, Thy-1, MHC class I and MHC class II, Swine-WC7 and Cattle-WC1. In order to characterize the molecular weight (MW) of the corresponding cross reacting antigens, selected mAbs were used to immunoprecipitate the antigens. The MWs of the analyzed precipitated antigens were in good agreement with the MWs of the homologous antigens. The followed strategy was found to be efficient and economical in defining new leucocyte antigen reactive mAbs.

ANALYSES OF MONOCLONAL ANTIBODIES REACTING WITH PORCINE WCD6 : RESULTS FROM THE SECOND INTERNATIONAL SWINE CD WORKSHOP

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.

Analyses of mAb reactive with porcine CD8

Among all mAb submitted to the first porcine CD workshop, based on FCM analyses six mAb could be identified to recognize the porcine CD8 analogue (workshop Nos. 004, 051, 052, 053, 108 and 109). In immunoprecipitation studies three mAb (Nos. 004, 108 and 109) recognized an antigen with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. The molecular masses of the antigens recognized by the three other mAb (Nos. 051, 052 and 053) are still unknown. Epitope analyses performed by blocking experiments led to the determination of two CD8 epitopes: CD8a and CD8b. CD8a is recognized by mAb Nos. 004, 051 and 052, and CD8b by Nos. 053, 108 and 109.

Cytokine Profiles in Peripheral Blood Mononuclear Cells and Lymph Node Cells from Piglets Infected In Utero with Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT The aim of the present study was to investigate at 2, 4, and 6 weeks after birth cytokine expression by peripheral blood mononuclear cells and bronchial lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). Technically, by flow cytometry we were able to measure gamma interferon (γ-IFN), tumor necrosis factor alpha (TNF-α), interleukin-4 (IL-4), and IL-8 levels. In general, we found increases in the percentages of IL-4-, γ-IFN-, and TNF-α-producing lymphocytes in the infected piglets compared to the percentages in the uninfected control animals, while there was a decrease in the percentage of IL-8-producing monocytes. We believe that these findings reflect a general lymphocyte activation stage that is created due to the infection and that occurs in combination with impairment of the monocyte function, possibly due to the ongoing viral replication in these cells. Single-cell bronchial lymph node preparations exhibited very much the same cytokine profiles as peripheral blood mononuclear cells except for a lack of IL-8 production. When the levels of the individual cytokines in the three groups of PRRSV-infected piglets were compared, the levels of cytokine expression at 4 weeks diverged from those at 2 and 6 weeks, in that there was a significant decrease in the numbers of lymphocytes producing γ-IFN and TNF-α. This tendency was also observed among blood monocytes and lymph node macrophages. Possible reasons for this temporary immunosuppression in the piglets at 4 weeks are discussed.

Sequence comparison of the non-structural genes of four different types of Aleutian mink disease parvovirus indicates an unusual degree of variability.

SummaryThe present work shows that at least four different sequence types of Aleutian mink disease parvovirus (ADV) are present in ADV isolates from mink. We here report the nucleotide sequences of these four types of ADV from nucleotide 123 to 2208 (map unit 3 to 46). This part of the genome encodes three non-structural (NS) proteins of ADV. Comparison of the deduced amino acid sequences of these NS proteins showed that the ADV proteins are much less conserved than the NS proteins from other members of the autonomous group of parvoviruses. In general, we found that the middle region of the ADV NS-1 protein was relatively well conserved among the types, while both the amino-and carboxy-terminal ends of the protein had higher amino acid variability. Interestingly, the putative NS-3 protein from type 3 ADV is truncated in the carboxy-terminal end. The molecular evolutionary relationship among the four types of ADV was examined. This analysis, taken together with the unusually high degree of variability of the ADV types, indicates that the ADV infection in mink is likely to be an old infection compared to the other parvovirus infections or, alternatively, that ADV accumulates sequence changes much faster than other parvoviruses.

Acute Interstitial Pneumonia in Mink Kits Inoculated with Defined Isolates of Aleutian Mink Disease Parvovirus

The present study addressed the causal role of Aleutian mink disease parvovirus (ADV) in acute interstitial pneumonia in mink kits. All the examined isolates of ADV caused interstitial pneumonia in newborn kits, although the severity of disease and the mortality varied. These findings indicate that ADV is the direct causal agent of this disease in mink kits and that cofactors, which could have been present in the original ADV-K isolate, do not play a role. Acute interstitial pneumonia characterized by hypertrophy and hyperplasia of alveolar type II cells, intranuclear viral inclusions, interstitial edema, and hyaline membrane formation was experimentally reproduced in mink kits infected as newborns with five different isolates of ADV. Four hundred forty-nine newborn mink kits were included in the study, of which 247 were necropsied. The lesions caused by the different isolates were indistinguishable by histopathologic examination, but the incidence (50–100%) and severity (mortality of 30–100%. n = 218) of disease among the mink kits varied. Also, the content of ADV antigens in the lungs of infected kits varied among the groups. According to these features, the examined isolates could be placed in groups of high and low virulence. ADV-K, ADV-Utah I, and ADV-DK were in a highly virulent group producing a mortality of 90–100% (n = 110) in mink inoculated as newborns. ADV-GL and ADV-Pullman belonged to a group of low virulence, with an incidence of clinical disease of 50–70% and a mortality of approximately 30–50% (n = 118) in kits inoculated as newborns. The mortality in the control group receiving a mock inoculum was around 12% (n = 34). The period from infection to development of fatal disease varied from approximately 12 days for the highly virulent isolates up to around 20 days for the isolates of low virulence. The 107 mink kits that survived inoculation with ADV as newborns developed lesions typical of classical Aleutian disease irrespective of the ADV isolate used. The lesions consisted of chronic immune complexmediated glomerulonephritis and infiltrations with mononuclear cells, including plasma cells in lung, liver, spleen, kidney, mesenteric lymph node, and intestine. Surviving kits also had hypertrophy of the bronchusassociated lymphoid tissue and focal subpleural, intraalveolar accumulations of large cells with foamy cytoplasm, so-called lipid pneumonia.

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink.

Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop

After initial evaluation of the 176 new and 19 control monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cell/activation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyers patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number.

Vaccination with Aleutian mink disease parvovirus (AMDV) capsid proteins enhances disease, while vaccination with the major non-structural AMDV protein causes partial protection from disease.

Vaccination studies were performed with partially purified recombinant AMDV VP1/2 capsids as well as with the major AMDV non-structural protein (NS1). All vaccine constructs induced an antibody response, but did not prevent infection upon challenge with AMDV. The severity of Aleutian disease (AD) was judged by the serum gammaglobulin level, the quantity of peripheral blood CD8 lymphocytes, antibody titers to VP1/2 and NS1 proteins and mink death rates. The VP1/2 vaccine constructs enhanced the disease process with drastic death rates for the vaccinated mink. On the contrary, the NS1 vaccine constructs resulted in milder AD than seen in the non-vaccinated mink.

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop.

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8+ cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8+ lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8low and CD8high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8low and CD8high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4-/CD8high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8+ lymphocyte subsets in health and disease.