Radioimmunoassay for protein p28 of murine mammary tumor virus in organs and serum of mice and search for related antigens in human sera and breast cancer extracts.

The main protein of the core of murine mammary tumor virus, with a molecular weight of 28,000 (p28), was solubilized by deoxycholate treatment of the virus and purified by Ultrogel ACA-54 filtration and hydroxyapatite chromatography. This protein was used as labeled antigen in a highly specific and reproducible radioimmunoassay. Organ extracts of uninfected C57BL mice did not contain p28, but organ extracts of infected RIII mice did contain the antigen. Despite the high content in the mammary gland, the level of p28 in the other organs was identical in male and female mice. Sera of uninfected mice and the majority of the sera of infected mice did not contain the antigen. The investigation included 338 human sera (50, normal; 157, breast cancer; 77, polycystic disease; 32, benign mastopathy; 12, fibroadenoma; 10, at risk of developing breast cancer). None contained an antigen related to p28. Eight of 24 extracts of human breast cancer gave results that appeared weakly positive, possibly as a result of proteolysis. Extracts of healthy breast tissue and the serum from the breast arterial and venous blood of corresponding patients were negative.

Radioimmunoassay for glycoprotein gp47 of murine mammary tumor virus in organs and serum of mice and search for related antigens in human sera.

Murine mammary tumor virus main glycoprotein (gp47), prepared by diethylaminoethyl cellulose and hydroxyapatite chromatography of detergent-mercaptoethanol-KCI-disrupted virion, was used as labeled antigen in a highly specific and reproducible radioimmunoassay. Seven other (glyco) proteins of the virus were antigenically distinct from gp47. Serum and organs of unifected C57BL mice did not contain gp47, but sera of infected Swiss and RIII mice did contain the antigen. Despite the high content in the mammary gland, the level of gp47 in other organs was identical in male and female mice. The titer of gp47 in serum was high in tumor-bearing females, but it varied with the mouse strain. Anti-gp47 immunoglobulins could not be detected. The investigation included 314 human sera (107 normal, 65 benign mastopathy, 89 breast vancer, and 53 digestive cancer). None contained an antigen related to gp47. One of 20 human mammary cyst fluids was positive.

Identification, partial purification and biochemical characterization of gamma-glutamyltranspeptidase present as a membrane component in skimmed milk and milk fat-globule membranes, and in mammary-tumour virus from the milk of infected mice.

The enzyme gamma-glutamyltranspeptidase was reproducibly found to be associated with mouse milk particles; it is present in milk fat-globule membranes and mouse mammary-tumour virus of infected Swiss mice, also in particles from the milk of uninfected mice. The enzymatic activities observed range among the highest reported for mammalian tissues. The enzyme was partially purified from mouse mammary-tumour virus, and from milk fat-globule membranes. The molecule requires the presence of detergents to remain soluble, behaves as a high molecular weight component, properties characterizing integral membrane proteins. Kinetics, and the effect of competitors as well as of specific inhibitors show this enzyme to be identical to the well-known kidney gamma-glutamyltranspeptidase ((gamma-glutamyl)-peptide:amino-acid gamma-glutamyltransferase, EC 2.3.2.2). Other oncornaviruses budding from cultured cells originally expressing the enzyme in their plasma membrane also incorporate the enzyme in their structure.

Comparative study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice.

Milk fat globule membranes and mammary tumour virus particles (d=1.17 g/cm3) have been obtained from the milk of a Swiss albino mice strain. Comparative biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.

[Pseudomonas aeruginosa and surgical intensive care units]. / Pseudomonas aeruginosa et unités de soins intensifs chirurgicaux.

The prevalence of Pseudomonas aeruginosa in intensive care unit (ICU) is 19 per cent although its incidence in blood culture is only 3.5 per cent and remains the same even though the total prevalence of this bacterium increases. A review of the clinical results shows that this germ causes severe complications in only 3 per cent of patients. A study by epidemiological markers reveals the presence of 16 different IATS serotypes in the ICU, this distribution of the serotypes being similar to this observed in ambulatory patients. The serotype 12 of Ps. aeruginosa is multiresistant. In 1984, it represented 22 per cent of the isolated Ps. aeruginosa, its incidence decreases in 1985 and in 1986 (14.5%). Ps. aeruginosa seems weakly pathogenic for the surgical patients in the ICU. On the other hand, the incidence of Klebsiella pneumoniae, Serratia marcescens (014 imm. serotype resistant to tobramycin), Enterobacter aerogenes and Enterobacter cloacae increases in blood cultures proportionally to their total prevalence in ICU. These bacteria are probably responsible for hospital epidemics.

Vaccine could not have been prepared in Stanleyville.

In Stanleyville, at the time of vaccination campaigns, tissue cultures were primitive, experimental and used solely for diagnostic purposes. Production of vaccine was impossible to carry out. A few chimpanzee kidneys were minced and sent to Philadelphia as part of the hepatitis experiments of Dr Deinhardt. Vaccine was never handled in my laboratory and contamination with chimpanzee cells was not possible.

Peroral infection of suckling mice with milk-borne mouse mammary tumour virus: uptake of the main viral antigens by the gut.

Persistence of mouse mammary tumour virus (MMTV) components in the digestive tract of suckling mice was investigated by immunoperoxidase staining of the main viral antigens and micro-immunoenzyme assays of gp52 and p28; these latter assays were also performed after ingestion of milk enriched in viral antigens using Cr2O3 as a marker for the alimentary bolus migration. When compared to the ingested antigens, the amounts of gp52 and p28 decreased during transit, p28 being more rapidly digested than gp52. The antigens were, however, destroyed to a much larger extent in the gut of the adult than in that of the newborn mouse. A fraction of the marker remained for a long time in the stomach; a prolonged retention was also observed with gp52 and especially with p28. Significant amounts of viral antigens were detected in the intestinal walls: both p28 and gp52 were found in the duodenum and small intestine. Moreover, the four viral antigens gp52, gp36, p28 and p8 were clearly observed in very large supranuclear vacuoles inside the epithelial cells of the distal part of the gut. Total particles can reach the intestine; the viral material could then be either destroyed or taken up in the epithelial cells by endocytosis, so that the intestinal epithelium might serve as a portal of entry for MMTV in the suckling mouse.

Detection of virus antigens in Swiss albino mice infected by milk-borne mouse mammary tumour virus: the effect of age, sex and reproductive status. I. Localization by immunofluorescence of four antigens in mammary tissues and other organs.

The indirect immunofluorescence technique was used to investigate the expression of virus antigens in a Swiss albino mouse strain infected by mouse mammary tumour virus (MMTV). The antisera used were monospecific for: the glycopolypeptides gp160 and gp47, and the internal polypeptides p28 and p8. In mice infected with milk-borne MMTV, all four antigens were detected in the mammary gland during the first pregnancy and then throughout life, and in mammary tumours. Antigen gp160, located at the secretory border of all glandular cells, is shared by the plasma membrane of the mammary cell, whether virus-producing or not, and by the virus envelope, as shown by the use of absorbed antisera. The other three antigens are virus-specific. Anti-p47 serum stained the secretory border of cells whereas the fluorescence related to p28 and p8 consisted of a few large intracytoplasmic spots. The specific antibodies for p28 and p8 were only absorbed by disrupted virions, confirming that these polypeptides are internal antigens. With serial sections of glands from pregnant and old female mice, the alveoli stained with anti-p28 or anti-p8 serum were found to react also with anti-gp47 serum. However, during lactation, the presence of p28 and p8 could not be detected in the alveoli stained with anti-gp47 serum. Thus, infected alveoli express virus antigens differently. In the mammary glands of all mice studied, some alveoli did not display any staining when the latter three antisera were used. The specific antigenic pattern, maintained in differentiated mammary tumours, became a diffuse and unlocalized staining after transformation into the undifferentiated state. With the exception of the kidney, where gp47 was found in the glomeruli, all other organs of the mouse did not stain with any of the antisera. The presence of gp47 in the glomeruli, probably related to immune-complex deposits, increases with the age of the mouse and is most noticeable in tumour-bearing females. In mice free of milk-borne MMTV, examined during the first week of lactation, antigen gp47 could not be detected. Internal antigens p28 and p8 were detected in nearly all mammary cells as numerous small cytoplasmic dots. This suggests a limited expression of an endogenous virus genome; this expression would be modified when milk-borne virus is produced.

Distribution of mouse mammary tumour virus antigens in RIII mice as detected by immunofluorescence on tissue sections and by immunoassays in sera and organ extracts.

Organs of RIII mice at various physiological stages were tested for mouse mammary tumour virus (MMTV) antigen expression. Indirect immunofluorescence was used with three monospecific antisera to localize one envelope glycoprotein, gp47, and two core proteins, p28 and p8. These virus-specific antigens gave characteristic fluorescent patterns in the mammary tissues and were also detected in thymus and salivary gland sections of some mice. The amounts of antigens gp47 and p28 were measured by immunoassay in sera and organ extracts of corresponding samples of mice. Sera of mice of both sexes contained virus antigens from the suckling age onwards. Although ingested virus could be traced in suckling mice, weanlings were characterized by the absence of virus expression except in lymph nodes. This location points to the possible role of lymphoid tissue in producing the antigens of the blood and in disseminating the infection. In adult animals, virus antigens were present in salivary glands, digestive tract, lymph nodes, male genital organs and female mammary glands. Antigen expression, even found in the mammary glands of virgin mice, was strikingly increased by pregnancy, lactation and (or) ageing, the highest values being found in mammary tumours. The results for milk-borne MMTV infection in RIII mice are compared with those obtained previously in Swiss albino mice.

Natural infection of Swiss mice with mouse mammary tumor virus (MMTV): viral expression in milk and transmission of infection.

Quantitative determinations of gp52, the main envelope glycoprotein, and p28, the main core protein, of MMTV, have been performed in about 1000 individual samples of milk of breeding females from our colony of MMTV-infected Swiss mice, a line characterized by a moderate incidence of mammary tumors. A computer analysis of the results showed: 1-- an important individual variation, ranging from 0 to 120 micrograms per ml of milk for p28, and from 0 to 320 micrograms per ml of milk for gp52; 2-- a variation of the release of both antigens during a single lactation, with a maximum on the 7--8th day of nursing; 3-- an increase of the release of both antigens with parity up to the 6th lactation, followed by a marked decrease during later lactations; 4-- a higher degree of infection in the offspring of 2nd and 3rd litters. The possible dependence of viral expression and transmission of infection upon factors such as cyclic activity of the mammary gland and progressive immunization of mice against MMTV is analyzed. The status of our laboratory line of MMTV infected Swiss mice is discussed in comparison with high and low tumor incidence strains.

Immunological characterization of a mammary tumour virus from Swiss mice: multiple epitopes associated with the viral gene products.

The major antigens of a mouse mammary tumour virus (MMTV) isolated from the milk of exogenously infected MB+ Swiss mice were compared with the viral components of other MMTV strains by using the methodology developed by Teramoto et al. (1977 a). The anti-gp47 (Swiss) serum differentiated between type- and group-specific antigenic determinants on the major glycoprotein; two distinct type-reactivities were demonstrated, one of them being shared by the MMTVs (Swiss) and (RIII), and the other by the MMTVs (C3H) and (GR). The MMTVs (Swiss) and (RIII) also reacted identically in a 'type-specific' assay using an anti-p28 (Swiss) serum, but a distinct reactivity was observed with the MMTV (C3H). In the isologous test where an anti-(total RIII) serum was used to bind intact, externally labelled RIII virions, the Swiss virus was seen to possess a type-specific determinant on its surface which distinguished itself from both MMTVs (RIII) and (C3H). The Swiss/fC57BL mice (which are devoid of the milk virus and for this reason are referred to as MB-), expressed only the internal virus antigens in their mammary glands. Under the 'type-specific' assay conditions, the p28 antigen present in the mammary gland extracts of the MB+ mice was indistinguishable from the p28 antigen purified from the Swiss virion, but was clearly distinct from the p28 reactivity present in the mammary gland extracts of the MB- mice. The p28 (MB-) antigen may thus represent the expression of an endogenous virus sequence different from the milk virus genome.

Detection of virus antigens in Swiss albino mice infected by milk-borne mouse mammary tumour virus: the effect of age, sex and reproductive status. II. Radioimmunoassay of two virus components, gp47 and p28 in serum and organ extracts.

Extracts of various organs, mammary tumours and sera from milk-borne MMTV infected Swiss albino mice of different age, sex and physiological conditions were tested by radioimmunoassay for the presence of gp47, the main envelope polypeptide, and p28, the main core protein of the virus. Except in brain, ovaries and testes, both antigens were found in all organs of old animals and of females after the onset of their first pregnancy. Antigens were not present in organs of weanlings or in whole foetuses. Higher values were found in mammary glands, mammary tumours, epididymis and seminal vesicles. These organs also harboured a greater amount of gp47 than p28. The serum generally contained gp47 but rarely p28. This indicates that gp47 is not virion-bound in blood. Pregnancy, lactation and especially the presence of mammary tumours increased the concentration of gp47 in serum. The results do not allow localization of target organs of MMTV infection in the interval between ingestion of the virus by the suckling mouse and the first pregnancy. Moreover, results obtained with one group of mice devoid of exogenous virus show that, as endogenous MMTV genome expresses p28, it might account for part of the p28 detected exogenous MMTV-infected mice.

Among the human antibodies reacting with intracytoplasmic a particles of mouse mammary tumor virus, some react with MMTV p14, the nucleic-acid-binding protein, and others with MMTV p28, the main core protein.

Human IgG antibodies reacting in the indirect immunofluorescence test with clusters of intracytoplasmic A particles in mouse tissues were analyzed by means of both radioimmunoprecipitation and enzyme-linked immunoabsorbant assays. Generally, antibody-containing sera reacted with epitopes of p14, the nucleic-acid-binding core protein of mouse mammary tumor virus, corresponding to the protein Ap14 of intracytoplasmic A particles. Comparatively few sera reacted with epitopes of the main core protein p28 corresponding to the protein Ap28 of intracytoplasmic A particles. The two types of reactivity occurred independently of each other.

The ganglioside content of the milk fat-globule membrane and the mouse mammary-tumour virus isolated from the milk of infected mice. Partial characterization of a new disialoganglioside.

The milk fat-globule membrane and the mouse mammary-tumour virus isolated from the milk of infected Swiss mice have been investigated for their content in gangliosides. When compared on the lipid phosphorus basis, viral envelope is found to contain more than twice as much lipid-bound sialic acid as fat-globule membrane. The ganglioside patterns of these two structures appear rather similar, except for the occurrence in fat-globule membrane of a low ganglioside homolog, presumably GM2, not detected in viral envelope. A common and dominant trait is the presence in both structures, as the main ganglioside, of a component which has been so far characterized as a disialoganglioside, having the same neutral glycolipid moiety as GD1a, but with both sialic acid residues displayig to Clostridium perfringens and Vibrio cholerae neuraminidase, the susceptibility typical of terminal sialic acid residues.