Molecular and crystal properties of Bos d 2, an allergenic protein of the lipocalin family.

The relationship between the molecular structure of allergenic proteins and the allergenic determinants is one of the central issues in allergology. We report here that the natural preparation of Bos d 2, a mammalian lipocalin allergen, comprises three molecular variant proteins of 17,829, 17,781, and 17,800 Da. When cDNA of Bos d 2 (Genome Sequence Data Base No. L42867) was recloned and expressed in Pichia pastoris, two proteins were produced. One of the proteins (17,831 Da) and the proteins in the natural preparation had pyroglutamate as the N-terminal residue; in the other (17,849 Da) the N-terminal residue was glutamine. Recombinant Bos d 2 protein was crystallized and the native data set was collected at 1.8 A resolution.

Flow cytometric quantitation of C-myc and P53 proteins in bovine papillomavirus type 1-transformed primary mouse fibroblasts.

Bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblast cell lines were analyzed via flow cytometry (FCM) for expression of p53 and c-myc proteins along with their DNA content. In comparison to the nontransformed control cell line, significantly elevated levels of both the p53 and the c-myc protein were present in some but not all of the transformed cell lines. Quantitation of p53 and c-myc proteins in cell lines containing BPV-1 DNA revealed that the tumorigenic cell lines expressed higher levels of both the p53 (P = 0.0034; Mann-Whitney U test) and the c-myc protein (P = 0.0039; Mann-Whitney U test) as compared to the nontumorigenic cell lines. On average, at least 9,000-10,000 p53 or c-myc protein molecules per cell were detected in the transformed tumorigenic cell lines. These results show that quantitative FCM can be reliably used to detect very low levels (3,000 molecules per cell) of specific protein, and FCM is a useful tool to study the virus-induced changes in the levels of nuclear proteins within a cell population and in tumorigenesis.

Effect of gamma-interferon or cycloheximide treatment on viral and c-myc transcripts in bovine-papillomavirus-type-1-transformed primary mouse fibroblasts.

We have studied the effect of gamma-interferon (IFN-gamma) treatment on the transcription of viral and c-myc oncogenes in bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblast cell lines. Upon IFN-gamma treatment, viral transcripts always decreased in cell lines containing episomal BPV-1 DNA, while the effect was variable in cell lines containing integrated BPV-1 DNA. Two series of tumour cell lines established by in vivo passage of B6B71 (episomal) and B6B31-J (integrated) cells via nude mice (NuTu A) into immunocompetent C57BL/6 mice (NuTuA B6Tul) also showed a decrease and an increase, respectively, in viral transcripts upon IFN-gamma treatment. IFN-gamma also reduced c-myc transcription in all cell lines derived from tumours, but increased it in the transformed cell cultures. There was selection of c-myc oncogene transcripts in all the tumour-derived cell lines as compared to their transformed cell cultures. Cycloheximide treatment increased both viral and c-myc gene transcripts 3- to 20-fold in all cell lines. These results imply that IFN-gamma produced locally by immunological mechanisms may influence the expression of Papillomavirus oncogenes, and the response to IFN-gamma treatment may change when the viral DNA is integrated into the host genome.

Bovine papillomavirus type 1-transformed primary mouse fibroblasts show no correlation between tumorigenicity and viral gene expression, but c-myc gene expression is elevated in tumorigenic cell lines.

Bovine papillomavirus type 1 (BPV-1)-transformed primary mouse fibroblasts containing episomal or integrated BPV-1 sequences were analysed for virus-specific transcripts and c-myc gene expression. Total BPV-1-specific expression was high in cell lines containing episomal BPV-1 DNA in comparison to lines containing integrated BPV-1 sequences, mainly due to higher expression of the E6/E7 sequences. No correlation was found between the viral transcription and tumorigenicity, although BPV-1 gene expression occurred in all cell lines. High levels of c-myc expression were found in all cell lines exhibiting a tumorigenic phenotype as compared to the nontumorigenic lines. These data suggest that expression of BPV-1 genes may be essential for transformation but not tumorigenicity, whereas high levels of expression of cellular oncogenes like c-myc may be associated with tumorigenicity.

Integration of bovine papillomavirus type 1 DNA and analysis of the amplified virus-cell junctions in transformed primary mouse fibroblasts.

We have analysed the site of bovine papillomavirus type 1 (BPV-1) DNA integration in clones originating from a transformed primary mouse fibroblast cell line established by transfection of linear BPV-1 DNA. Viral DNA was integrated at a single site in the host genome with an intact early region and an almost complete long control region. Sequence analysis showed that the BPV-1 DNA was integrated at the HindIII site (the enzyme used to linearize the BPV-1 DNA for transfection) with short deletions at both ends. These deletions correspond to a 534 bp segment spanning the 3' end of the L1 open reading frame and the replication enhancer element in the BPV-1 genome. The cellular sequences 5' to the viral integration site exhibited 85 to 97% identity to several sequences belonging to the mouse L1 family of long interspersed repetitive sequences. Cellular sequences 3' to the viral DNA exhibited no significant similarity to any known sequence. The BPV-1 sequences and the cellular flanking sequences were found to be amplified 45- to 50-fold. All the cell clones shared an identical integration site but one of the clones had an additional population of amplified and integrated BPV-1 DNA molecules with an internal deletion of 1136 bp in the late region. The significance of viral DNA integration at a murine long interspersed repetitive sequence containing an amplification-promoting sequence is discussed.

Class-specific antibodies during follow up of patients with farmer's lung.

Sequential serum samples of 13 patients with acute farmer's lung (FL) taken during a follow-up of 18-36 months, were tested for antibodies of immunoglobulin G (IgG), IgA, IgM and IgE classes against Thermoactinomyces vulgaris and Micropolyspora faeni, and compared with contemporary lung function parameters. In the acute phase, antibodies of several Ig classes were present, those of IgG and IgA being most common. At the end of the follow-up, the mean values of all antibody titres were lower than in the acute phase, and antibodies were now mostly of one or two Ig classes only. The reduction in antibody levels was most often detectable in IgG and IgA antibodies against T. vulgaris. Antibody titres correlated inversely with tested lung function parameters, especially IgA antibodies with pulmonary diffusing capacity. Our results show that a follow-up of levels of class-specific antibodies, especially of IgG and IgA gives valuable information on causative microbes and on temporal changes of the exposure.

Comparative immunochemical analysis of five Thermoactinomyces strains.

The antigen structures in mycelial extracts of 4 strains of Thermoactinomyces vulgaris and 1 strain of Thermoactinomyces candidus were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. For immunological characterization, antigens were transferred electrophoretically to nitrocellulose membranes and blotted with T. vulgaris strain-specific antisera and with sera of farmer's lung patients. Cross-reactions between the strains were also studied using immunodiffusion and crossed immunoelectrophoresis. Protein components between 50 and 60 kilodaltons were found to be the most immunogenic. Patient sera showed heterogeneous responses, but all reacted with an antigen at 55 kilodaltons that was also common to all strains studied.

Lymphocyte activation with release of soluble mediators induced by Thermoactinomyces vulgaris in vitro.

Murine spleen lymphocytes stimulated in vitro with a hypersensitivity pneumonitis-associated bacterium, Thermoactinomyces vulgaris, were found to secrete interleukin-2 up to 7 days after mitomycin C blockade. They exerted helper effect in secondary mitogen or antigen-induced lymphocyte proliferation. Cyclosporin A, an inhibitor of interleukin-2 synthesis, caused a complete abrogation of the helper effect, suggesting that the effect was mainly due to interleukin-2. Indomethacin, an inhibitor of prostaglandin synthesis, enhanced the helper effect in some inbred strains of mice, indicating prostaglandin-dependent downregulation. The strain variation in the prostaglandin-induced downregulation was not H-2 linked.

Tumour-associated antigens in primary mouse fibroblasts induced by transformation with bovine papillomavirus type 1.

Bovine papillomavirus type 1 (BPV 1) DNA was used to transform primary fibroblasts of C57BL/6J mice. Transformation frequency in these cells was much lower than in C127 cells and not associated with the appearance of morphologically distinct foci. However, continuous lines of transformed C57BL/6J cells were developed by serial subculturing of transfected cells. These transformed cell lines showed phenotypic properties associated with transformation including abnormal karyotypes. They contained variable amounts of viral DNA, but the copy number was in the same range as in six C127 transformants tested for comparison. In two cell lines monomeric viral DNA in an episomal form was detected. Slowly migrating viral sequences in these and in the third line were probably episomal concatamers, but the possibility of integration could not be excluded. There was some variation in immunogenicity, but all cell lines induced a cell-mediated immune response in syngeneic mice detected by the chromium release assay. In addition to BPV 1-transformed cell lines, the effector cells also reacted against an unidentified antigen shared by 2 cell lines transformed by SV40 and UV irradiation, respectively.

Circulating IgG antibodies against fungal and actinomycete antigens in the sera of farmer's lung patients from different countries.

Sixty-nine farmer's lung patients and 28 normal controls from four countries (Finland, Switzerland, Canada and the United States) were investigated for antibody levels against 13 antigens commonly used for the screening panel for hypersensitivity pneumonitis. Of these antigens, eight were from the Medical College of Wisconsin (United States) and five were from the University of Kuopio (Finland). IgG antibodies against these antigens were studied in 97 sera using a sensitive biotin-avidin-linked enzyme immunoassay. The results indicate that the mean antibody titer against Micropolyspora faeni was highest in the United States (U.S.) followed by Finland. Both Finnish and U.S. antigens reacted almost identically against various groups of patients, although the degree of reactivity varied considerably. Higher antibody levels against Thermoactinomyces vulgaris were detected in Finnish patients than patients from other countries while patients from all four countries showed elevated levels of antibodies against T. candidus. This study demonstrates that antigens from identical species, irrespective of geographic origin, reacted similarly. However, variability between antigens of the same species was still considerably significant. Since the microbiological flora of moldy hay varies widely in different regions, the microbial species associated with the disease at a given geographical area has to be determined before selecting antigens for serological studies. The antigens currently used in various laboratories are crude preparations and need to be purified and standardized for dependable results. Until such antigens are available, all antigenic preparations used in the immunological evaluation of patients should be immunochemically characterized for their reproducibility and reliability although the ultimate goal should be to obtain standardized pure antigens for dependable immunodiagnosis of farmer's lung.

Precipitins against microbes in mouldy hay in the sera of farmers with farmer's lung or chronic bronchitis and of healthy farmers.

The aim of this study was to determine which precipitins against four antigens in 2,440 farmers are associated with the occurrence of farmer's lung (FL). The antigen panel consisted of mycelial antigens of Micropolyspora faeni, Thermoactinomyces vulgaris, Aspergillus fumigatus and Aspergillus umbrosus. As reference groups we used healthy farmers and those with chronic bronchitis. For the occurrence of precipitins against the four antigens there was a statistically significant difference between farmers with FL and healthy farmers but not between farmers with chronic bronchitis and healthy farmers. In a stepwise logistic linear regression analysis, farmers with FL and chronic bronchitis were compared to healthy farmers with respect to precipitins to the four antigens. Precipitins against Thermoactinomyces vulgaris differentiated farmers with FL (p less than 0.05) but not farmers with chronic bronchitis from healthy farmers. In Finland the occurrence of FL seems to be associated mainly with precipitins against Thermoactinomyces vulgaris, not with precipitins against Micropolyspora faeni as in Great Britain, and not with precipitins of Aspergillus umbrosus, which occurred most frequently in the sera of Finnish farmers. This association is in accordance with the exposure to spores of airborne moulds in farmers' work environment, where spore concentrations of Thermoactinomyces vulgaris have been measured to be about six times higher than those of Micropolyspora faeni.

Prognostic value of precipitins for working ability in dairy farmers.

In dairy farmers exposed to the microbes present in hay, precipitating antibodies against these microbes are frequently found regardless of the state of health of the farmer. The prognostic value of these antibodies for the future health and working ability of farmers was studied in a six-year follow-up survey of 292 farmers. During these six years, of the farmers aged 45-59 years in the primary survey, 14 men (22%) and 15 women (22%) had retired or changed occupation because of illness. Among the men, the presence of precipitins was negatively correlated with their working ability reported in the follow-up study. The risk of occupationally disabling respiratory disease was three times higher in men with precipitins against microbes present in mouldy hay than in precipitin-negative farmers of the same age. No similar correlation was found for women.

Serum precipitins against microbes in mouldy hay with respect to age, sex, atopy, and smoking of farmers.

This study was based on a sample of 3,065 farmers from a larger survey population of 12,056 Finnish farmers. Data were collected in a postal survey conducted by the Social Insurance Institution of Finland. Serum samples for determination of precipitating antibodies were taken at local health centres. Precipitins were determined by the method of microplate immune diffusion. The antigen panel consisted of mycelial antigens of Micropolyspora faeni, Thermoactinomyces vulgaris, Aspergillus fumigatus, and Aspergillus umbrosus. Precipitins to any one of the four microbes were detected in 8.6% of the sera. The most common causes of positive precipitin tests were Aspergillus umbrosus and Thermoactinomyces vulgaris, which agrees with previous findings reported from Finland. In general, the precipitins were more prevalent among women, which corresponds to local cultural traditions, and in older farmers. The prevalence of precipitins did not differ between non-atopic and atopic (defined as past or present co-existence of atopic dermatitis including infantile eczema and/or hay fever or other allergic rhinitis) subjects. In contrast, the prevalence of precipitins was about 1.5-2 times larger among non-smokers than among smokers, which confirms the findings in previous reports. In future studies on occurrence of precipitins, the data should be controlled with respect to age, sex, and smoking.

Interactions of Thermoactinomyces vulgaris and mouse alveolar macrophages. Loss of mitogenic activity and release of suppressive prostaglandins and interleukin 1.

Interactions of mouse alveolar macrophages from three different inbred strains of mice and Thermoactinomyces vulgaris, a microbe associated with Farmer's lung disease, were studied. Alveolar macrophages were found to abolish the mitogenic activity of T. vulgaris. A prostaglandin synthesis inhibitor indomethacin, could not restore the activity. Alveolar macrophage supernatants generated by T. vulgaris treatment exerted strong suppression in secondary concanavalin A-induced lymphocyte transformation. Indomethacin partly relieved the suppression but a histamine 2 receptor blocker, cimetidine, had no effect. Interleukin 1 activity was practically undetectable by the thymocyte co-stimulation assay unless indomethacin was used. When indomethacin was used, interleukin 1 activity could be detected in all strains of mice tested. Major differences in the abolition of the mitogenic effect, in the suppressive effect, or in the release of interleukin 1 were not detected between inbred strains of mice tested. The results indicate that alveolar macrophages exert suppressive actions in vitro after T. vulgaris treatment but in vivo activities remain to be elucidated.

Significance of IgG antibodies against environmental microbial antigens in a farming population.

A group of dairy farmers studied 6 years earlier in a field survey was re-surveyed for respiratory symptoms, occupational capability and the presence of antibodies against environmental micro-organisms. Specific IgG antibodies to Micropolyspora faeni, Thermoactinomyces vulgaris, Aspergillus umbrosus and Aspergillus fumigatus were measured by ELISA from the serum samples obtained in the two surveys. Antibody titres remained constant in 70% of both farmers and controls, but where changes took place, the titres against the actinomycete antigens tended to rise, whereas both increases and decreases were detected equally against the Aspergillus antigens. The titre of specific antibody to any of the four micro-organisms, when measured from a single serum specimen, seemed to be of little diagnostic value. Observed changes however, were more diagnostic, in that a fall in titre, especially against the Aspergillus antigens, was closely associated with a definite decrease in exposure, such as after retirement. Increased titres occurred in farmers with continued exposure, and those against the actinomycetes were associated with the appearance of symptoms in previously symptom-free individuals. In a case of farmer's lung which developed in this population during the follow-up period, significant increases were detectable against T. vulgaris and M. faeni.

Pulmonary and immune responses to a Thermoactinomyces vulgaris antigen respiratory sensitization in C57BL/6J mice.

Lung histology and immune responses were followed during a six month period of Thermoactinomyces vulgaris antigen respiratory sensitization of C57BL/6J mice. Histologically, the mice developed a picture resembling the subacute stage of human farmer's lung. At the early phase of the sensitization a T-cell response was seen in vitro, characterized by an increased spleen but no peripheral blood lymphocyte reactivity to T-cell mitogens at the same time as increased reactivity to the sensitizing antigen was detected. An increase in the percentage of T-cells in the lavage cell population was also seen. The sensitization was associated with the appearance of both IgG and IgA antibodies in lavage and in serum of which lavage IgA antibodies were found to follow the course of the sensitization most accurately. The increase in antibody titers was not, however, reflected in lavage cytology or in lymphocyte reactivity. The results suggest that different immunological reactions may be predominant at different stages of farmer's lung.

Carcinogenicity testing in nude mouse cell cultures.

Secondary cultures of newborn NMRI nu/nu (nude) mouse skin fibroblasts were used as targets for transformation by the combined administration of SV40 and 3-methylcholanthrene (MC). The long (72 h) post-treatment with MC increased virus transformation as much as 4.3-fold. In contrast, anthracene, a non-carcinogenic compound, had no effect on viral transformation frequency. Despite considerable variation within a group, the cell lines transformed by the combination treatment, as a group, were more tumourigenic than cell lines transformed by SV40 alone.

Enhancement of simian virus 40-induced transformation of nude mouse skin fibroblasts by 3-methylcholanthrene.

Secondary cultures of newborn NMRI nu/nu (nude) mouse skin fibroblasts were used as targets for transformation by the combined administration of SV40 and 3-methylcholanthrene (MC). The SV40-induced transformation was enhanced both by pre- and post-treatment with MC at concentrations which themselves were nontransforming. The short pretreatment caused a 2-fold enhancement in the transformation frequency at a low concentration of MC that allowed greater than 50% of the cells to survive. A short post-treatment also caused a concentration dependent enhancement in SV40 transformation of the same magnitude. The long (72 h) post-treatment with MC increased virus transformation as much as 4.3-fold. In contrast, anthracene, a non-carcinogenic compound, had no effect on the viral transformation frequency. An analysis of the morphology of the foci revealed that long post-treatment with MC induced a high number of type I foci. Combination dishes lacked these foci almost completely, and the enhancement was due to the increased number of type II and type III foci.