Host-specificity of myxoma virus: Pathogenesis of South American and North American strains of myxoma virus in two North American lagomorph species.

The pathogenesis of South American and North American myxoma viruses was examined in two species of North American lagomorphs, Sylvilagus nuttallii (mountain cottontail) and Sylvilagus audubonii (desert cottontail) both of which have been shown to have the potential to transmit the South American type of myxoma virus. Following infection with the South American strain (Lausanne, Lu), S. nuttallii developed both a local lesion and secondary lesions on the skin. They did not develop the classical myxomatosis seen in European rabbits (Oryctolagus cuniculus). The infection at the inoculation site did not resolve during the 20-day time course of the trial and contained transmissible virus titres at all times. In contrast, S. audubonii infected with Lu had very few signs of disseminated infection and partially controlled virus replication at the inoculation site. The prototype Californian strain of myxoma virus (MSW) was able to replicate at the inoculation site of both species but did not induce clinical signs of a disseminated infection. In S. audubonii, there was a rapid response to MSW characterised by a massive T lymphocyte infiltration of the inoculation site by day 5. MSW did not reach transmissible titres at the inoculation site in either species. This might explain why the Californian myxoma virus has not expanded its host-range in North America.

Virulence and pathogenesis of the MSW and MSD strains of Californian myxoma virus in European rabbits with genetic resistance to myxomatosis compared to rabbits with no genetic resistance.

The pathogenesis of two Californian strains of myxoma virus (MSW and MSD) was examined in European rabbits (Oryctolagus cuniculus) that were either susceptible to myxomatosis (laboratory rabbits) or had undergone natural selection for genetic resistance to myxomatosis (Australian wild rabbits). MSW was highly lethal for both types of rabbits with average survival times of 7.3 and 9.4 days, respectively, and 100% mortality. Classical clinical signs of myxomatosis were not present except in one rabbit that survived for 13 days following infection. Previously described clinical signs of trembling and shaking were observed in laboratory but not wild rabbits. Despite the high resistance of wild rabbits to myxomatosis caused by South American strains of myxoma virus, the MSW strain was of such high virulence that it was able to overcome resistance. The acute nature of the infection, relatively low viral titers in the tissues and destruction of lymphoid tissues, suggested that death was probably due to an acute and overwhelming immunopathological response to the virus. No virus was found in the brain. The MSD strain was attenuated compared to previously published descriptions and therefore was only characterized in laboratory rabbits. It is concluded that Californian MSW strain of myxoma virus is at the extreme end of a continuum of myxoma virus virulence but that the basic pathophysiology of the disease induced is not broadly different to other strains of myxoma virus.

Immunocontraceptive effects on female rabbits infected with recombinant myxoma virus expressing rabbit ZP2 or ZP3.

Recombinant myxoma viruses expressing rabbit zona pellucida 2 (rZP2) or rabbit zona pellucida 3 (rZP3) glycoproteins were constructed and tested in domestic rabbits to assess their potential to induce autoimmune infertility. The recombinant virus expressing rZP2 had no effect on fertility or ovarian histology, despite all animals developing antibodies against the rZP2 antigen. However, recombinant viruses expressing rZP3 induced infertility in 70% of animals at the first breeding. Serum antibodies were relatively short-lived, but antibody was bound to zona pellucida of all rabbits from Day 10 onward. There was no obvious correlation between infertility and rZP3 antibody titer. There was a transient inflammatory response in the ovaries of rZP3-immunized rabbits at Day 15 but no T-cell response to rZP3 could be detected at any time. Dysfunctional follicular formation was present in ovaries from rabbits infected with rZP3-expressing viruses 15-40 days postinfection but this had disappeared at later time points. A recombinant myxoma virus expressing a modified rZP3 antigen with the C-terminal hydrophobic putative anchor sequence deleted was also tested. This virus did not induce either infertility or an antibody response against the zona pellucida. Thus, the context of antigen presentation was crucial for an autoimmune response.

Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis.

Rabbit IL-4 was expressed in the virulent standard laboratory strain (SLS) and the attenuated Uriarra (Ur) strain of myxoma virus with the aim of creating a Th2 cytokine environment and inhibiting the development of an antiviral cell-mediated response to myxomatosis in infected rabbits. This allowed testing of a model for genetic resistance to myxomatosis in wild rabbits that have undergone 50 years of natural selection for resistance to myxomatosis. Expression of IL-4 significantly enhanced virulence of both virulent and attenuated virus strains in susceptible (laboratory) and resistant (wild) rabbits. SLS-IL-4 completely overcame genetic resistance in wild rabbits. The pathogenesis of SLS-IL-4 was compared in susceptible and resistant rabbits. The results support a model for resistance to myxomatosis of an enhanced innate immune response controlling virus replication and allowing an effective antiviral cell-mediated immune response to develop in resistant rabbits. Expression of IL-4 did not overcome immunity to myxomatosis induced by immunization.

The complete cDNA sequences of IL-2, IL-4, IL-6 AND IL-10 from the European rabbit (Oryctolagus cuniculus).

The cDNAs for four rabbit cytokine genes [interleukin 2 (IL-2), IL-4, IL-6 and IL-10] have been cloned from primary lymphocytes by polymerase chain reaction (PCR) methods. IL-2 and IL-10 are both highly conserved between rabbit and other species. IL-4 and IL-6 are less strongly conserved, at both nucleotide and amino acid levels, and exhibit structural differences. An extension of the coding region of rabbit IL-6 relative to all other reported IL-6 genes results from a mutation in the usual stop codon which allows translation to continue for a further 27 amino acids. Analysis of IL-6 from four other lagomorph species suggests that this mutation is specific to the European rabbit. Sequence and structural differences of IL-4 and IL-6, while presumably not altering function, may render them highly species-specific. Several alternatively spliced variants of IL-2 and IL-4 are also reported.

Localization of the inducible enhancer in the mouse interleukin-5 gene that is responsive to T-cell receptor stimulation.

Transcriptional regulation of the interleukin-5 (IL-5) gene in T lymphocytes appears to be of central importance in the control of the eosinophilia characteristic of allergic responses and certain parasite infections. Previous studies of IL-5 gene regulation have been hampered by the lack of a transfection assay, which detects the antigen-responsive enhancer in the IL-5 promoter. Here we show that stable transfection of the Th2 clone D10.G4.1 and the T lymphoma EL4.23 with chloramphenicol acetyltransferase reporter gene constructs carrying the region to -3859 gives inducible expression with the known regulatory characteristics of the endogenous IL-5 gene. To facilitate detailed analysis of the promoter region, 3.9 kb of DNA sequence immediately up stream of the start of transcription was determined and the minimum upstream region required for inducible expression was further localized, by stable transfection studies in EL4.23 cells, to the region up to -1016. A CTF/NF1 site in the upstream enhancer at -940 to -928 was shown to be required for regulated inducible expression. Mutation of this sequence motif abolished inducibility and also prevented binding of the sequence to a nuclear protein(s). A TCATTT-containing element in the proximal promoter region was also demonstrated to be essential for inducible expression of the IL-5 gene, similar to the role of this conserved element in the transcriptional regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 genes.

Functional role and signal-induced modulation of proteins recognizing the conserved TCATTT-containing promoter elements in the murine IL-5 and GM-CSF genes in T lymphocytes.

The TCATTT-containing element extending from -61 to -41 of the mouse IL-5 gene is highly conserved in the corresponding region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and has been previously shown to be involved in regulating inducible GM-CSF gene expression. By using stable transfection assays in the mouse Th2 clone D10.G4.1, we show that the TCATTT-containing element is also involved in the regulation of inducible IL-5 gene expression. The mouse IL-5 and GM-CSF homologues of this element were found by gel shift analysis to form DNA-nuclear protein complexes of similar electrophoretic mobility under conditions in which expression of these genes is induced. However, comparative studies using extracts of D10.G4.1 cells treated with the cellular activators Con A and PMA and the inhibitors cycloheximide and cyclosporin A indicated that the binding activities to the conserved elements in the IL-5 and GM-CSF genes (designated NF-IL-5A and NF-GM-CSFA, respectively) are regulated by different signaling pathways. In addition, NF-IL-5A is not induced in the Th1 clone HDK-1 which does not express the IL-5 gene. The strong correlation between the signal-dependent and cell-specific modulation of IL-5 and GM-CSF gene expression patterns and the binding activities of NF-IL-5A and NF-GM-CSFA suggests that these nuclear proteins are involved in the transduction of T cell activation signals to the transcriptional machinery of these genes through their interactions with their respective TCATTT-containing elements.

Differential expression of inhibin alpha and beta A subunit genes in rat and mouse ovarian follicles during pregnancy.

Relative levels of rat ovarian alpha inhibin (alpha I) and beta A inhibin (beta AI) mRNAs were measured during pregnancy by dot-blot hybridization of ovarian poly(A+) RNA. Follicular patterns of alpha I and beta AI expression in contralateral ovaries from the same rats were also studied by hybridization histochemistry. Oligodeoxynucleotide probes specific for porcine alpha I and beta AI were synthesized, 32P end-labelled and used as hybridization probes on dot-blots of ovarian RNA and frozen sections of ovarian tissue from pregnant rats. During pregnancy, levels of alpha I and beta AI mRNAs remained fairly constant from day 7 after mating until parturition and then fell within 16 h post partum. In all ovaries observed, expression of inhibin genes was located in granulosa cells of healthy antral follicles. In general, the strongest signals for alpha I and beta AI mRNAs were obtained in large follicles, with weaker signals in smaller follicles. Follicular patterns of alpha I and beta AI expression during pregnancy were often dissimilar when alpha I and beta AI were compared over a range of follicles. Considerable alpha I mRNA was detectable in some follicles in which beta AI was reduced or undetectable, despite strong signals for both alpha I and beta AI in an adjacent follicle. Essentially, alpha I mRNA levels were relatively consistent between groups of follicles, whereas beta AI levels varied considerably. beta AI mRNA was never observed in a follicle in the absence of alpha I mRNA, indicating that activin production in any follicle occurs in the presence of alpha I mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-3 is significantly more effective than other colony-stimulating factors in long-term maintenance of human bone marrow-derived colony-forming cells in vitro.

Human bone marrow cells cultured for 21 days in the presence of recombinant human interleukin-3 (IL-3) produced up to 28 times more colony-forming cells (CFC) than could be obtained from cultures stimulated with granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF). IL-3-cultured cells retained a multipotent response to IL-3 in colony assays but were restricted to formation of granulocyte colonies in G-CSF and granulocyte or macrophage colonies in GM-CSF. Culture of bone marrow cells in IL-3 also led to accumulation of large numbers of eosinophils and basophils. These data contrast with the effects of G-CSF, GM-CSF, and IL-3 in seven-day cultures. Here both GM-CSF and IL-3 amplified total CFC that had similar multipotential colony-forming capability in either factor. G-CSF, on the other hand, depleted IL-3-responsive colony-forming cells dramatically, apparently by causing these cells to mature into granulocytes. The data suggest that a large proportion of IL-3-responsive cells in human bone marrow express receptors for G-CSF and can respond to this factor, the majority becoming neutrophils. Furthermore, the CFC maintained for 21 days in IL-3 may be a functionally distinct population from that produced after seven days culture of bone marrow cells in either IL-3 or GM-CSF.

Molecular organization of the cytokine gene cluster, involving the human IL-3, IL-4, IL-5, and GM-CSF genes, on human chromosome 5.

The human genes for the hematopoietic growth factors interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been mapped to 5q23-31. We present in situ hybridization evidence that the human IL-4 gene is located at 5q23.3-31.2, suggesting that the four cytokine genes may be closely linked. We used pulsed-field gel electrophoresis to prepare subchromosomal restriction maps surrounding these genes to define this possible linkage more precisely. The IL-4 and IL-5 genes are tightly linked, being 90 to 240 kilobases (kb) apart, as has been shown for the IL-3 and GM-CSF genes, which are only 9 kb apart. Possible overlap of the map containing the IL-4 and IL-5 genes with restriction sites 5' to the IL-3 gene suggests that the four cytokine genes may be localized within 500 kb of each other. The endothelial cell growth factor gene (ECGF), which has also been localized to the 5q31 region, did not appear to be close to the cytokine genes. Linkage of the IL-3, IL-4, IL-5, and GM-CSF genes has important implications in the evolutionary origin and regulation of expression of these genes. The four cytokine genes are located in the region of the long arm of chromosome 5, which is deleted in the 5q- anomaly. The present study provides a basis for further investigations of this disorder.

Cellular basis for the differential response of mouse kallikrein genes to hormonal induction.

The expression of many mouse kallikrein genes in the salivary gland is sexually dimorphic and inducible in females by administration of testosterone or thyroxine. Induction is slow (3-7 days) and is accompanied by the non-uniform differentiation of the cell type expressing these genes from striated duct (SD) cells (female) to granular convoluted tubule (GCT) cells (male). One kallikrein gene, mGK-6, is expressed at an apparently constant total level in male and female and is not induced by either hormone. In situ hybridization histochemistry shows that all kallikrein genes analyzed exhibit uniform cellular distribution of expression in the SD cells of the female. The hormonally mediated differentiation of some, but not all, of these cells has different effects on kallikrein gene expression--mGK-6 is repressed while other kallikreins are induced--leading to non-uniform distribution of expression.

Mouse glandular kallikrein genes. Identification, structure, and expression of the renal kallikrein gene.

Glandular kallikreins are a highly homologous subfamily of serine proteases encoded by 25 genes in the mouse. Several of the gene products have been implicated as having specific roles in growth factor biosynthesis, suggesting a general role for members of this gene family in the generation of biologically active peptides (Mason, A.J., Evans, B.A., Cox, D.R., Shine, J., and Richards, R.I. (1983) Nature 303, 300-307). Here we describe the identification, structure, and expression of the renal kallikrein gene, the major kinin-generating member of the family. Kinins are potent vasodilators implicated in regulation of local blood flow and ion transport. A gene-specific oligodeoxyribonucleotide probe was designed and chemically synthesized from the renal kallikrein gene sequence to distinguish this gene's transcripts from those of other members of the gene family. Differential expression phenotypes were found between the renal kallikrein gene and other members of the kallikrein gene family.

Tissue-specific control of glandular kallikreins.

Glandular kallikreins are kinin-generating serine proteases found in the salivary glands, pancreas, reproductive organs, and kidney and in their secretions. They are physico-chemically, enzymatically, and immunologically identical but it is not known whether they perform similar functions at these separate anatomical sites. A specific radioimmunoassay was used to measure immunoreactive kallikrein in these tissues of the rat after various stimuli. Salt depletion significantly increased kallikrein in the kidney from 2.35 +/- 0.17 to 4.11 +/- 0.27 microgram/g tissue (P less than 0.01, 16 DF) and in the salivary glands from 16.8 +/- 2.1 to 24.2 +/- 0.9 mg/g tissue (P less than 0.01, 16 DF), but did not change pancreatic kallikrein. Deoxycorticosterone (2.4 mg s.c. over 8 days) led to a doubling in kidney kallikrein, from 2.35 +/- 0.17 to 5.27 +/- 0.56 microgram/g tissue (P less than 0.01, 18 DF), without changing salivary gland or pancreatic kallikrein, whereas testosterone (240 micrograms/day s.c. over 15 days) significantly increased salivary gland kallikrein from 26.6 +/- 2.1 to 42.2 +/- 1.8 mg/g tissue (P less than 0.01, 18 DF) with no increase in kidney or pancreatic kallikrein. Accordingly, the glandular kallikreins in these tissues vary independently, which suggests they are under different electrolyte and endocrine control and may perform different physiological functions at each anatomical site.

Release of active and inactive kallikrein from the isolated perfused rat kidney.

The release of kallikrein into the perfusate and urine of the isolated perfused rat kidney was studied. Comparison between enzymic and immunological assays for kallikrein demonstrated the presence of an enzymically inactive form of kallikrein. Of kallikrein found in normal rat urine 77 +/- 4% is active and 23% is in an inactive form. In the isolated perfused rat kidney a similar proportion of active kallikrein (84%) was excreted into the urine but very little enzymically active kallikrein (2%) could be detected in the perfusate. However, significant amounts of enzymically inactive but immunologically reactive kallikrein could be found in the kidney perfusate. The rate of release of kallikrein into the perfusate was approximately one-fifth of the rate of release into the urine. Renin showed a similar pattern of release into the perfusate and urine but the lysosomal enzyme marker acid phosphatase was not detectable. These results show that kallikrein is secreted from the kidney into the circulation as well as being excreted in the urine. However, in urine the enzyme is predominantly in an enzymically active form whereas it is secreted into the circulation in an inactive form.

Effect of ACTH administration on urinary kallikrein excretion in man.

The effect of ACTH administration on urinary kallikrein excretion and its relationship to changes in plasma and urine electrolytes, renin concentration and steroids was examined in normotensive and mildly hypertensive subjects. ACTH administration produced hypokalaemia, initial urinary sodium retention, a fall in active plasma renin concentration, a transient rise in plasma aldosterone concentration and sustained rises in plasma deoxycorticosterone concentration and urinary kallikrein activity. Changes in patients with mild hypertension were similar in pattern to normotensives, but urinary kallikrein concentrations were lower. The effects of ACTH on urinary kallikrein excretion appeared to be independent of aldosterone and correlated most closely with deoxycorticosterone concentrations.

Role of prostaglandins in the renal response to angiotensin-converting enzyme inhibition and renal artery occlusion.

We investigated the role of prostaglandins in the renal effects of endogenous kinins in anaesthetized dogs. Endogenous kinin levels, as indicated by urinary kinin excretion, were increased by angiotensin-converting enzyme inhibition or by 10-min renal artery occlusion. Angiotensin-converting enzyme inhibition with captopril resulted in increased renal blood flow that was not prevented by prior inhibition of prostaglandin synthesis with indomethacin. The renal vasodilation after temporary renal artery occlusion was also not altered by indomethacin treatment. Thus both angiotensin-converting enzyme inhibition and renal artery occlusion caused increased kinin levels and renal vasodilation that was not dependent on prostaglandins. These results also suggest that intrarenal kinins have a role in the control of renal blood flow.

Radioimmunoassay of blood bradykinin: purification of blood extracts to prevent cross-reaction with endogenous kininogen.

Alcoholic extracts of blood collected for measurement of circulating bradykinin were analysed for co-extracted kininogen which would artificially elevate measured bradykinin levels. The blood extract contained a glycoprotein with identical chromatographic properties to kininogen. Bradykinin immunoreactivity in the extract eluted as a single peak from an immunoaffinity column of anti-bradykinin IgG bound to Sepharose identically to both bradykinin and kininogen showing immunoidentity of these substances. Affinity chromatography on Concanavalin A-Sepharose or ion-exchange chromatography on CM-Sephadex C25 separated bradykinin and the glycoprotein which again eluted identically to kininogen. The decrease in measured values of blood bradykinin after purification of the extract on CM-Sephadex C25 was a similar amount to that calculated as cross-reactivity with the amount of co-extracted kininogen. Hence radioimmunoassay of bradykinin in ethanolic extracts of blood is inaccurate owing to the presence of co-extracted kininogen, and additional purification steps such as chromatography on CM-Sephadex C25 are mandatory for accurate assay by ligand binding techniques.

The consequences of predation in the population biology of the monocarpic species Cirsium palustre and Cirsium vulgare.

Predation by rabbits, Cheilosia grossa and especially Epiblema scutulana considerably reduces achene production in Cirsium palustre and Cirsium vulgare. In both species larger plants in a population are more frequently attacked, however the effect of predation is greater in medium sized plants. As a result the contribution of larger plants to the total achene production of the population is increased.In Cirsium vulgare larger populations are more susceptible to attack. The pattern and consequences of predation are discussed in relation to the occurrence in temporary habitats and to selectional processes. Predation intensifies existing developments in the population biology of these species. To compare the effect of predation for both species, detailed information of their population biology is necessary. At the moment the outcome of the comparison can only be indicated.

Influence of micro-organisms on the germination of the monocarpic Cirsium vulgare in relation to disturbance.

Cirsium vulgare is a plant species of disturbed sites. To understand this distribution the effect of micro-organisms on the germination in this species was investigated as the microflora of disturbed and non-disturbed sites shows differences. It was found that micro-organisms from fruitcoat and environment do influence germination. In an environment containing humus, germination is stimulated.Mortality of seedlings is higher in undisturbed sites, resulting in the observed distribution. It is hypothesized that this mortality is the result of germination, forced by microbial activity (break-down of the fruitcoat, exudation of growth-regulating substances) under conditions in which the survival chances of the seedling are low.

The role of pollination in the population biology of the monocarpic species Cirsium palustre and Cirsium vulgare.

The absence of cross-pollination in Cirsium palustre and Cirsium vulgare resulted in reduced achene production while the achenes produced were heavier than those produced after cross-pollimation. Establishment of plants from non-cross-pollinated achenes is comparatively higher, facilitating the founding of a population from isolated individuals in these wind-dispersed species.If achene weight is the result of a balance between cross-pollination and self-pollination (and/or apomixis), the first causing more and thereby lighter achenes to develop, increasing density of flowering individuals in a population may lower achene weight and consequently seedling survival. This may account for the frequently observed decline of populations of fugitive monocarpic perennials.