A case of human anthrax in Victoria.

A human case of anthrax was identified through surveillance of knackery workers who had been exposed to infected cattle. The outbreak in cattle has affected 38 herds in the Stanhope/Tatura area of central northern Victoria. The human case, a 39 year old male, was treated in hospital and is recovering. Surveillance of other knackery workers has now been completed, and no other cases were found. Public health measures are in place to prevent further human cases.

The complete cDNA and deduced amino acid sequence of equine IgE.

The cDNA from a transcript encoding the complete heavy chain of the equine immunoglobulin IgE has been cloned and sequenced. A fragment of the equine epsilon gene was amplified from cDNA using PCR and this fragment was then used to probe a horse cDNA library prepared from peripheral blood lymphocytes. A recombinant clone containing the cDNA encoding the complete horse epsilon chain and its associated V-D-J and leader, was subsequently isolated and sequenced. Comparison of the deduced amino acid sequence of equine IgE with the C epsilon heavy chains of other species indicates it to be most similar to human C epsilon (54%), followed by sheep C epsilon (52%); the greatest sequence similarity was found in the C epsilon 3 and C epsilon 4 domains among the species compared. The associated V-region had greatest similarity in FR1 to those reported for sheep and the cow (> 80%) but less than 60% to the VH sequences of other species in FR3.

Variation in expression of MHC class II antigens on horse lymphocytes determined by MHC haplotype.

A panel of monoclonal antibodies was used to characterize the expression of equine Major Histocompatibility Complex (MHC) class II antigens on lymphocytes of horses of different MHC types. MHC class II antigen expression was compared between adult horses and foals, and the level of expression of MHC class II antigens on horse T cell subpopulations was also determined. Peripheral blood lymphocytes (PBL) from young and adult healthy horses of different MHC haplotypes were labeled with the antibodies and assayed by single- and two-color immunofluorescence flow cytometry. A variation in the expression of equine MHC class II antigens on PBL was identified that was determined by MHC haplotype. Both young and old animals carrying the MHC class II haplotype D3 expressed lower levels of MHC class II antigens on their PBL than did horses of other MHC class II types. D3 heterozygotes had an intermediate level of expression. Neonates and foals of any MHC haplotype had lower levels of expression of MHC class II antigens than did adults. MHC class II antigens were identified on surface immunoglobulin positive (sIg+) B cells as well as on T cells, with the B cells staining brightest. Dual staining for MHC class II antigens and markers for equine T cell subsets demonstrated that both EqCD4+ and EqCD8+ T cells expressed MHC class II molecules. These results suggest that the relatively low levels of MHC class II antigen expression on foal and neonate lymphocytes may be developmentally regulated, and not determined by environmental factors. The cause of the low expression of MHC class II antigens linked to the MHC class II haplotype D3 is not known, but it appears to reflect a decreased expression by all lymphocyte subpopulations.

Report of the First International Workshop on Equine Leucocyte Antigens, Cambridge, UK, July 1991.

The First International Workshop on Equine Leucocyte Antigens was organized and convened for the purposes of identifying immunologically relevant cell surface molecules of equine leucocytes and establishing a system of nomenclature for those molecules. Participating members of the workshop represented the majority of laboratories world-wide engaged in the tasks of production and characterization of equine leucocyte and lymphocyte markers using monoclonal antibodies. The workshop confirmed the identification of several equine CD molecules described previously by individual laboratories, and in addition recognized antibodies identifying new CD molecules. The workshop also succeeded in fostering co-operation between laboratories around the world which study equine immunobiology. Equine CD molecules identified by the current battery of monoclonal antibodies include EqCD2, EqCD4, EqCD5, EqCD8, EqCD11a/18, EqCD13 and EqCD44. Other antibodies are markers for MHC class I and class II molecules, for B cells, granulocytes, macrophages, T cell subsets distinct from those defined by CD4 and CD8, and other sub-populations of horse leucocytes that do not have obvious counterparts in humans, rodents, or other species. Despite the progress made in the first workshop, there are still substantial gaps in the armory of reagents available to study equine leucocyte biology, and further definition of the structure, function, and genetics of the antigens identified by the workshop clusters (WC1, WC2 etc.) and other molecules of immunological importance will be a goal of future workshops. The study of equine immunobiology and resistance to disease also urgently requires the development of tools to study equine immunoglobulins and cytokines, and these needs will provide ample scope for future studies.

Correlation between monoclonal antibody reactivity and expression of CD4 and CD8 alpha genes in the horse.

Equine peripheral blood lymphocytes (PBL) were enriched by positive selection using panning with a mixture of monoclonal antibodies against putative equine CD4 (Equine Leucocyte Antigen Workshop antibodies WS 1 and WS 72), or CD8 molecules (Workshop antibodies WS 12, WS 49, and WS 74). RNA was extracted from CD4 enriched cells (99% purity), from CD8 enriched cells (69% purity), from peripheral blood lymphocytes, and from neonatal equine thymus. RNA extracted from equine granulocytes and from equine kidney served as negative control. The RNA was electrophoresed in agarose and transferred to nylon membranes. Northern blots were hybridized with human and mouse cDNA probes for CD4 and CD8 alpha. The human CD4 probe detected a 2.9 kb RNA transcript in equine PBL, CD4 enriched lymphocytes, and thymocytes. The human CD8 alpha probe detected a 2.0 kb transcript in RNA from equine CD8 alpha enriched lymphocytes and thymocytes, but not from PBL or CD4 enriched lymphocytes. Mouse cDNA probes for CD4 and CD8 did not react with equine RNA. Results of hybridizations using the human probes support the assignment of the CD4 and CD8 specificities to the antibodies listed above. The results also suggest that the equine CD4 and CD8 alpha genes are more closely related to the human than to the murine counterparts.

Characterization of canine parvovirus (CPV) interactions with 3201 T cells: involvement of GPI-anchored protein(s) in binding and infection.

Binding of canine parvovirus (CPV) to the susceptible feline T cell line 3201 was quantitated by fluorescence-activated cell sorter (FACS) analysis. CPV bound to the cells in a dose-dependent manner, while no binding to the non-permissive MSB-1 avian lymphoma cell line was detected. Binding could be competitively inhibited by addition of excess unlabeled empty capsids, or by pre-incubation of virus with a CPV-specific monoclonal antibody. To characterize the biochemical nature of this binding, live cells were treated with a variety of enzymes prior to use in the binding assay. Treatment with neuraminidase removed a significant proportion of the wild-type virus binding activity, while both proteinase K and phosphatidylinositol-specific phospholipase C (PI-PLC) prevented binding of a non-hemagglutinating (non-HA), non-sialic acid binding mutant to 3201 cells. This suggests that CPV binds to sialic acid expressed on host cells as well as erythrocyte membranes, and that it also binds a protein moiety which is glycosylphosphatidylinositol (GPI)-anchored. The role of these components in CPV infection was also examined by pretreating cells with neuraminidase or PI-PLC prior to inoculating them with either wild-type CPV or the non-hemagglutinating mutant. Neuraminidase treatment had no effect on the ability of CPV to infect the cells, while infectivity was severely compromised by pretreating the cells with either proteinase K or PI-PLC. GPI-anchored proteins on 3201 cells were further characterized by Triton X-114 extraction and reactivity to anti-CRD after PI-PLC treatment.

Mutations adjacent to the dimple of the canine parvovirus capsid structure affect sialic acid binding.

The erythrocyte receptor on rhesus macaque erythrocytes used by canine parvovirus (CPV) for binding in hemagglutination (HA) was examined. Erythrocyte membrane proteins were electrophoresed and blotted to nitrocellulose and probed with [125I]-labeled CPV capsids, showing seven virus-binding proteins. Treatment of erythrocytes or isolated membranes with Clostridium perfringens neuraminidase virtually abolished virus binding. Binding was also affected by treatment with potassium periodate and inhibited by wheat germ agglutinin, but was not significantly affected by concanavalin A, peanut agglutinin, or soluble N-acetyl-neuraminlactose. A non-HA mutant of CPV failed to bind to erythrocytes or to blotted erythrocyte membrane proteins. The mutation was a single Arg-Lys difference of VP2 amino acid residue 377. The pH dependence of binding of the closely related feline panleukopenia virus was shown to result from a decreased binding in buffers with pH values of 6.8 or greater. The VP2 residues responsible for that difference have been shown to be 323 and 375. The sequences affecting binding were all adjacent to the dimple in the capsid, implicating that region of the capsid as the sialic acid binding site. The role of sialic acid in virus-host cell interactions was not defined, but the plaque sizes of the non-HA mutant and wild type CPV were indistinguishable.

Production of cytokines by mouse B cells: B lymphomas and normal B cells produce interleukin 10.

We have examined a panel of murine Ly-1+ B lymphomas and purified normal murine peritoneal B cells separated into subsets on the basis of expression of the Ly-1 surface antigen, for their ability to produce cytokines. Where possible, we have used a combination of cytokine detection methods in order to compensate for differences in sensitivity and specificity, and the possibility of inhibitors masking an activity. All the lymphomas tested were shown to constitutively express TGF-beta and CSIF/IL-10. In addition, varying levels of IL-6, TNF-alpha and TNF-beta, and G-CSF, were demonstrable in most of the lymphomas, and variants of one lymphoma (CH12) additionally produced varying levels of IL-3, IL-4, and GM-CSF. FACS purified normal Ly-1+ and Ly-1- peritoneal B cells, were also shown to express RNA encoding CSIF/IL-10, IL-6, TNF-alpha and TNF-beta, and very low levels of G-CSF, following stimulation with LPS. These data were supported by the detection of IL-6 and CSIF/IL-10 in supernatants from LPS-stimulated Ly-1+ and Ly-1- B cells using specific immunoassays. None of the lymphomas or B cell preparations produced IL-1 alpha, IL-2, IL-5, IL-7, or IFN-gamma. The purity of our normal B cell populations was assessed by phenotypic analysis on the FACS and also by the disappearance of certain mRNA transcripts after purification, e.g. CD4, c-fms, GM-CSF, and IFN-gamma, most of which could be detected in LPS-stimulated total peritoneal cell populations. This suggested that our B cell purification method had reduced, to a level undetectable in our assays, contaminating T cells (CD4), macrophages (c-fms, GM-CSF), and NK cells (IFN-gamma). Absence of IL-3, IL-4, IL-5, and GM-CSF expression by LPS-stimulated Ly-1+ and Ly-1- B cells reduced the concern that contaminating peritoneal mast cells could account for the observed cytokine production. We therefore believe our data provide strong support for production of a subset of cytokines by LPS-stimulated normal B cells. Both the Ly-1+ B lymphomas and normal Ly-1+ and Ly-1- B cells appear capable of expressing IL-6, TNF-alpha, TNF-beta, and CSIF/IL-10.(ABSTRACT TRUNCATED AT 400 WORDS)

The BCL1 B lymphoma responds to IL-4, IL-5, and GM-CSF.

Proliferation in vitro of the in vivo passaged murine B cell tumor line BCL1 has been used as a standard assay for mouse interleukin-5 (IL-5) for a number of years. We demonstrate that this line will also respond to human IL-5. The response to murine IL-5 is abrogated by transforming growth factor-beta and to a lesser extent by interferon-gamma. This suggests a possible regulatory role for these lymphokines in the proliferation of B cells induced by IL-5. Other purified recombinant lymphokines were also tested for their ability to induce BCL1 proliferation. The lymphokines IL-1, IL-2, IL-3, and IL-6 had no effect on the growth of BCL1. In contrast, IL-4 and more surprisingly granulocyte-macrophage colony-stimulating factor (GM-CSF) also induced proliferation of this cell. These effects could be inhibited by specific antibodies directed against the respective lymphokines. These data suggest that GM-CSF, as well as IL-4 and IL-5, may be yet another regulator of neoplastic and possibly even normal B-cell growth and differentiation.

Constitutive production of lymphokines by cloned murine B-cell lymphomas--CH12 B lymphoma produces interleukin-4.

B cells can be activated by T-independent antigens or mitogens such as lipopolysaccharide (LPS) which will induce proliferation and differentiation of the B cells into Ig-secreting cells, without the intervention of T cells. The precise mechanism of T-independent proliferation and differentiation of B cells is still unclear. It is possible however that antigen-stimulated B cells may produce some factors which play a role in T-independent B-cell responses. In addition, since it has now been established that B cells can function as antigen-presenting cells, it is possible that they too secrete a molecule which is involved in the activation of T cells, analogous to IL-1 production by antigen-presenting macrophages. A number of human B-cell lines, as well as human normal B cells activated appropriately, have been shown to produce various cytokines, and similar studies are now being undertaken in the mouse. In the present study, six cloned murine B-cell lymphomas of different origin were analyzed for the presence of mRNA encoding a number of lymphokines by hybridization of specific cDNA probes to poly-A RNA, followed by the sensitive S1 nuclease digestion technique. The lymphokines included (IL-) 1, 2, 3, 4, 5, and neuroleukin. Whereas none of the lines expressed detectable levels of IL-2, IL-3, or IL-5 mRNA, all the lines expressed high levels of neuroleukin mRNA. Three of the lymphomas (CH12, CH31, and NBL) expressed low levels of IL-1 mRNA. The most striking finding was that one lymphoma, CH12, constitutively expressed IL-4 mRNA. This mRNA appeared to be functional, as IL-4 activity measured by the HT-2 T cell proliferation assay could be detected in supernatants collected from CH12 cells. The growth-inducing activity of CH12 supernatant on HT-2 cells could be completely blocked by an anti-IL-4 monoclonal (11B11), but not by an anti-IL-2 antibody (S4B6), consistent with our observations that CH12 cells produce IL-4 but not IL-2. CH12 cells were also found to express high affinity receptors for IL-4. Proliferation of CH12 cells was not affected by the addition of exogenous IL-4. Addition of anti-IL-4 antibodies to CH12 cells in culture caused a slight but reproducible increase in their proliferation at low cell numbers, which is probably not highly significant. These findings open the possibilities that murine B lymphocytes are capable of lymphokine production or alternatively that aberrant lymphokine production underlies B-lymphocyte transformation.

Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum.

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.

Molecular approaches to the study of nucleotide excision repair in eukaryotes.

Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes. Studies on human cells have been stimulated by the availability of excision repair-defective cell lines from patients suffering from the autosomal recessive disease xeroderma pigmentosum (XP). Such studies have contributed significantly to an understanding of the genetic complexity of excision repair in human cells. However, to date, no human excision repair genes or gene products known to complement the repair defect in XP cells have been isolated. The yeast Saccharomyces cerevisiae is an interesting model for exploring the molecular mechanism of nucleotide excision repair in eukaryotic cells. As is true in human cells, multiple yeast genes are involved and at least five genes are required for the specific incision of UV-irradiated DNA in vivo. These five genes have been isolated by molecular cloning and the nucleotide sequences of four of them have been determined. Each of these cloned genes is being used for overexpression of protein.

Studies on gene transfer and reversion to UV resistance in xeroderma pigmentosum cells.

We have examined several parameters which address the feasibility of complementing the UV-sensitive phenotype of xeroderma pigmentosum (XP) fibroblasts by gene transfer. We present a comparative study which demonstrates that, relative to immortalized cells, human diploid cells are poor recipients for gene transfer. As measured by both transient and stable expression assays, diploid fibroblasts were completely refractory to DNA transfer by calcium phosphate coprecipitation and exhibited substantially reduced levels of expression following gene transfer by fusion with E. coli protoplasts. We also examined the significance of reversion of the phenotype of UV sensitivity in SV40-immortalized XP-A cell lines. In addition to confirming a previous report of reversion to wild-type levels of UV resistance at a frequency of approximately 10(-7), we have attempted to facilitate the identification of XP-A cells complemented with genomic DNA by employing less stringent selection schemes and cotransfection of a selectable marker. Under these conditions, we observed an increased frequency of reversion and were unable to identify true transfectants.