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The serum proteins in Sapphire mink from the experimental and control groups in 2 endotoxin experiments and in a group of normal mink of the Standard type, were separated electrophoretically on cellulose acetate membranes. In experiment No. 1, in which the experimental mink were given repeated injections of endotoxin, and the controls were untreated, significantly increased total protein and significantly decreased albumin concentrations in the experimental group compared to the normal group were demonstrated. The concentration of the alpha(1)-globulin was significantly elevated and the alpha(2)-globulin significantly reduced in the experimental and the control groups compared to the normal group. Significant differences between the gamma-globulin concentrations in the various groups were not found. In experiment No. 2, all the animals were inoculated intraperitoneally with a crude tissue suspension containing the plasmacytosis agent 10 days before the experimental animals received the first of a series of injections with endotoxin. Significantly increased concentrations of total protein and gamma-globulin and significantly decreased albumin concentrations were, compared with the normal group, demonstrated in sera collected from the experimental group on the 94th, 201st and 254th day after the first injection of endotoxin. In the control group, compared with the normal group, significantly elevated concentrations of total protein, alpha(2)-, beta- and gamma-globulins and significantly reduced albumin- and alpha(1)-globulin were found but only in the second set of samples, while significantly decreased albumin- and significantly increased gamma-globulin concentrations were found in the third set. The results showed no significant differences between the gammaglobulin concentrations or between the other serum fractions in the experimental and the control groups in the 2 experiments. A possible explanation may be that there is no direct interrelationship between hypergammaglobulinaemia and amyloidosis, and that a common basic mechanism may stimulate related stem cells which thereafter differentiate in different ways.

Aleutian disease virus (ADV) antigen was prepared by fluorocarbon extraction from ADV-infected mink tissue. Satisfactory ADV antigen was produced from spleen, liver and lymph nodes 10 days after infection, but not from kidney. The heat stability of ADV antigen preparations is believed to be affected by the state of the antigen in that non-reactive ADV antigen was activated by heating at 65° to 80°C while fluorocarbon-activated ADV antigen was labile at these temperatures. ADV antigen titers were not affected by storage for at least 180 days at 5°C or 22°C. ADV antigen was sedimented with 50% ammonium sulfate but not with 33%, and the antigen titer was not affected by dialysis for 48 hr. Antigen titers were not affected by treatment with 0.5% formalin, or ether, whereas 0.05 N NaOH or 0.05 N HCl completely destroyed the antigenicity. Antigen titers were somewhat reduced by treatment with 2 N urea, chloroform-ethyl alcohol mixture (2:1) or acetone. ADV had a buoyant density of about 1.36 ± 0.02 g/ml in cesium chloride. It is suggested ADV is a member of the parvovirus group.

A chronic disease spontaneously occurring in mink is described. This disease is characterized by a genetic predisposition for mink carrying the Aleutian gene. It is manifested by renal, hepatic, and possibly arterial changes. There is marked hyperglobulinemia. The pathologic features of the disease are reviewed. Current studies have extended the knowledge regarding the hematologic and biochemical abnormalities. Comparison of the abnormalities of this disease to human connective-tissue diseases is made. Es describite un morbo chronic que occurre spontaneemente in visones. Iste morbo es characterisate per su predilection a base genetic pro visones portator del gen aleutian. Illo se manifesta in alterationes renal, hepatic, e possibilemente arterial. Marcate grados de hyperglobulinemia es presente. Le characteristicas pathologic del morbo es revistate. Studios currentemente in progresso ha servite a extender nostre cognoscentias relative al anormalitates hematologic e biochimic. Es interprendite un comparation de iste morbo con le morbos de tissu conjunctive in humanos.

A comprehensive review of the present state of knowledge regarding the role of immune mechanisms in the pathogenesis of renal damage in human disease.

Spleen extracts from a mink with Aleutian disease were dialyzed against a 20-fold amount of distilled water. The material which diffused through the dialyzing membrane did not produce Aleutian disease in mink inoculated with this material. It is concluded that the Aleutian disease agent can not pass through an intact dialyzing membrane.

Hypergammaglobulinemia in mink was produced by the injection of crude tissue suspensions from mink with spontaneous Aleutian disease. The initiating factor was found to be resistant to 0.3 per cent formalin for 2 weeks but not 40 weeks at 5°C. Foreign antigens as well as formalinized normal mink tissue from homologous and heterologous genotypes did not cause a detectable change in the serum protein values. Mink homozygous recessive for the Aleutian gene were found to be significantly more susceptible to the experimental disease. Possible pathogenetic mechanisms as well as similarities between the mink disease and certain immunologic and connective tissue diseases of man are discussed.

IN 1955 a patient in the Brooklyn Veterans Administration Hospital died after a two-year illness that was undiagnosed despite intensive examination by many physicians. Autopsy revealed widespread, unusual anatomic alterations that were not recognized by any of the pathologists who examined the tissues as ever having been previously described. Helmboldt and Jungherr,1 in 1958, reported the anatomic pathology of a newly recognized disease in mink, which because of the particular susceptibility of Aleutian mink was named Aleutian-mink disease. The tissue changes in this disease were similar to those found in our patient. This is especially noteworthy because our patient raised . . .

Aleutian disease-affected mink, which are markedly hypergammaglobulinemic, show a decreased half-life of the serum gamma globulin indicating that the hyperglobulinemia is due to increased production. No evidence that the gamma globulin was antibody to the infectious agent, to autologous or isologous tissues, or to antigens the animal was responding to prior to development of the disease was obtained. The increased gamma globulin was found to be of the 6.4S variety, and gamma globulin containing protein-protein complexes of 9S to 17S and 22S to 25S classes were observed in serums of affected mink. The findings are most consistent with the Aleutian disease virus acting as a direct and somewhat selective stimulus to plasma cell proliferation. There is no evidence that the arterial and glomerular lesions of Aleutian disease have an immunologic pathogenesis. It seems possible that these vascular changes may be directly caused by the viral agent, or may be the result of the increased gamma globulin levels.

It is now possible to construct a reasonable hypothesis for an infectious etiology and pathogenesis of RA and related multisystem rheumatic diseases. This has been done here using evidence from the clinical course and natural history of RA, recently studied animal models, the many immunologic abnormalities in these diseases, the pathologic tissue characteristics, and the few data available on efforts at direct culture of organisms. It is proposed that an infectious organism, a viable external agent, is the initiating or persistent cause of RA, and that this agent directly and indirectly results in the phenomena of immune complex disease, hypersensitivity, and autoimmunity. The organism is probably nonspecific; i.e., the disease may be initiated by various agents, such as bacteria, virus, infective particle (“L” form), phages or slow viruses (some may enter cells and compromise the genetic apparatus, resulting in synthesis of “foreign” antigen by the host cells), mycoplasma, or even physical agents. Chronic viral infection may act as an adjuvant in the patient's immunologic apparatus, with consequent involvement of most tissues and organs, and so can be a multisystem disease. There are known instances of acute arthritis that closely simulates rheumatoid arthritis and that results from viral infection. Unfortunately, at present most of the supporting evidence for a clear infectious etiology of RA is circumstantial. However, the evidence is very extensive and persuasive and remains the most probable etiologic hypothesis and most promising investigative approach to these diseases that are so often disabling and tragic in their consequences.

A concise definition of slow virus diseases is difficult to achieve, as criteria for this group of diseases are unclear. It is likely that several types of agents are incorrectly considered as slow viruses at present. As more is learned about them there will probably be considerable reclassification of the diseases so designated. The term "slow infection" is not new. In the early 1950's Sigurdsson (1-3), an Icelandic pathologist, applied the name to maedi, a slowly pro­ gressive pneumonia of sheep, to rida, a chronic encephalitis of sheep (prob­ ably identical to scrapie), and to Johne's disease, a mycobacterial infection of the intestine of cattle. Sigurdsson's criteria were: (a) a long latent period lasting from several months to several years; (b) a protracted course, usually ending in death; and (c) strict host specificity and sometimes familial pre­ disposition for the specific disease. Other characteristics now known to be associated with slow viral in­ fections are persistence of the agent in the infected host for much or all of its life, and a long, continued host response which may in itself be part of the disease process (e.g., circulating antigen-antibody complexes). In general, however, the host responses are minimal. Although it was some time before Sigurdsson's ideas became widely accepted, the concept of slow in­ fections has exerted considerable influence on thinking about diseases of a progressive, degenerative nature with long course and obscure etiology. In­ terpretation of Sigurdsson's theory by comparative pathologists has opened an entirely new field of research in chronic diseases. This work has recently been reviewed by Abinanti (4), Field (5), and Gibbs & Gajdusek (6). I t has been said that there are no slow viruses, only slow virologists. This statement serves to illustrate the difficulty of understanding these important but mysterious diseases. The most perplexing aspect has been the time inter­ val of one, five, or even twenty years between initial exposure and entry of the infectious agent into the host, and eventual overt expression of the dis­ ease. Thus, even though it may be improper to refer to these as slow viruses, for some of them replicate rapidly, the delay in expression of the diseases has been the chief obstacle to understanding their pathogenesis. In retrospect, this possibility should have been anticipated, for extended

Affinity chromatography was used to isolate the Aleutian disease virus of mink. Dissociation of the immunoadsorbent-virus complex with 0.75 molar sodium chloride and then with a glycine-hydrochloride gradient released infective particles resembling picornaviruses. The elution profile suggests that two different types of virus-antibody complexes are formed, one dissociated by sodium chloride and another that requires glycine-hydrochloride in addition to sodium chloride for release of virus.

Aleutian disease (AD) of mink is an immunological disease and the immune response against the viral antigen initiates the pathological changes1–3. This “slow virus”, however, replicates as rapidly as do many viruses which cause acute disease4,5. The disease is characterized by the development of hypergammaglobulinaemia, systemic proliferation of plasma cells, glomerulonephritis and hepatitis and is invariably fatal6–10. Extremely high titres of antibody have been detected from serum of hypergammaglobulinaemic mink3,5,11–13. The role of the host immune response in causing the disease condition has been shown by the prevention of lesions by immunosuppression1 and by the enhancement of lesions following the administration of a killed virus vaccine or passive antibody2.

THE successful application of affinity chromatography to the isolation of Aleutian disease (AD) virus has provided an opportunity for characterization of the agent1. Although AD was described in 1958, the agent had remained incompletely defined and the various strains used by researchers uncharacterized2. Before the work reported here characterization of AD virus was difficult because the only available assay consisted of producing the disease in mink.

Aleutian disease virus usually produces a persistent infection and progressive immune comples disease in mink of the Aleutian genotype. Study of Aleutian disease virus infection in non-Aleutian mink showed that about one-quarter developed nonpersistent infections by the virus, and that the nonpersistence was not genetically determined by the host. The nonpersistently infected mink developed only a transient elevation of serum gamma globulin, and much lower specific Aleutian disease virus antibody titers than persistently infected mink. No lesions were found in the nonpersistently infected mink.

Twenty-four virgin female aleutian mink were infected with aleutian disease agent and after 24 hours, 12 of these were treated with a course of polyinosinic acid-polycytidilic acid (Poly IC) injections. After six weeks the gammaglobulin level was significantly lower in the treated group but at 12 weeks this difference was no longer present. Four of the treated mink had normal target organ histology when killed at 20 weeks. The untreated group all showed moderate to marked changes but this difference was not statistically significant. There was a marked increase in the reactive lymphocyte blastogenesis index during the first weeks of infection and the phytohaemagglutinin response was seen to fall progressively. The antiglobulin reaction usually became positive after infection but neither antinuclear nor antierythrocyte antibodies were found. Precipitating antibodies to several polynucleotides were frequently present and were unrelated to infection or to Poly IC treatment.

In an attempt to document progression rate differences in the development of glomerular lesions in mink infected with Aleutian disease virus (ADV), the glomeruli of Aleutian and non-Aleutian mink experimentally infected with ADV were evaluated by light, fluorescent, and electron microscopy. The animals were also examined for the presence of interstitial infiltrate, neutrophils, and arterial lesions. One hundred percent of the Aleutian mink had glomerular cell proliferation and interstitial infiltrate, while 95% of the Aleutian and 41% of the non-Aleutian mink had neutrophilic infiltrates and arteritis, respectively. Of the non-Aleutian mink, 91, 83, 42, and 12.5% had glomerular cell proliferations, glomerular neutrophils, interstitial infiltrate, and arterial lesions in, that order. All the Aleutian mink had glomerular depositions of gamma-globulin (IgG) and complement (C3), whereas 75% of non-Aleutian mink had deposits of IgG and C3. One hundred percent of both genotypes had glomerular deposits of immunoglobulin M (IgM). Ultrastructural glomerular changes consisting primarily of depositions of granular electron-dense material on basement membranes were observed in Aleutian mink 6 weeks after infection and 12 weeks after infection in non-Aleutian mink. These findings document progression rate differences in the development of glomerular lesions in Aleutian disease-affected Aleutian and non-Aleutian mink. Further, they emphasize the need for exploration of pathogenetic mechanisms involved in progression rate differences in lesion development.

The specific antiviral antibody response of sapphire and pastel mink to Pullman strain of ADV has been examined. Sapphire mink inoculated with from 300,000-3 LD50 developed high levels of specific antibody and AD. Pastel mink inoculated with parallel doses of ADV also produced antibody but did not develop AD. The low incidence of AD in pastel mink inoculated with Pullman strain of ADV is probably related to factors other than antiviral antibody.

Aleutian disease is a chronic persistent viral infection of mink characterized by hypergammaglobulinema, generalized plasmacytosis, sclerosing glomerulonephritis, polyarteritis, and plasma cell hepatitis with bile duct proliferation. The development of hepatic lesions was studied both light- and electron-microscopically in mink experimentally infected with Aleutian disease virus. Fifteen normal and 99 mink experimentally infected with Aleutian disease virus were used. Experimental mink were killed in intervals from 3 weeks to 23 months after infection, and liver sections were processed for both light- and electron-microscopic studies. Experimentally infected mink developed portal and intralobular lymphocytic and plasmacytic infiltrates in the liver 3 weeks after infection. Four to five weeks after infection there was evidence of early bile duct proliferation that began as an outgrowth of the portal bile ducts. Three to five months after infection a marked bile duct proliferation was present in some of the portal triads and adjacent liver lobules; but there was no tendency of these lesions to progress into biliary cirrhosis. Ultrastructural characteristics of proliferating bile duct cells were marked deformation, formation of multiple cell layers, reduction in the number of microvilli and desmosomes, and infiltration of the epithelial cells by lymphoid cells and plasmacytes. The hepatic lesions either develop by direct virus stimulation or by the deposition of virus-antibody complexes.

ALEUTIAN disease of mink is an immune complex disease induced by viral infection. Major abnormalities include glomerulonephritis, arteritis, plasmacytosis and hypergam-maglobulinaemia1,2. Infectious virus-antibody complexes have been found in the serum of infected mink3, and deposits of viral antigen, antibody and complement have been demonstrated in glomerular and arterial lesions4–6. Detailed sudy of Aleutian disease virus (ADV) has been hampered by an inability to isolate and purify it by conventional techniques, mainly because ADV obtained from mink tissue is complexed with specific antibody7. The immune complexes are not sufficiently homogeneous for purification by physical or chemical properties. Applying techniques that dissociate antigen–antibody complexes to crude preparations of ADV, Cho and Ingram purified viral antigenic material8,9. With the electron microscope they observed virus-like particles in both immune precipitates and viral antigen from CsCl gradients. Although the immune precipitates were infectious for mink, lack of quantitative infectivity data on the CsCl gradient fractions prevented identification of these particles as ADV. We have now succeeded in purifying similar virus particles and have identified them as ADV by quantitative inactivity titration.

Aleutian disease is an immunological disease of mink caused by a persistent virus infection. Recently Aleutian disease viral antigen has been extracted from tissue of mink early in the course of infection and the Aleutian disease virus has been isolated. The virus particles were similar in morphology and size to certain parvovirus having a 23 nm diameter, spherical shape and icosahedral capsid. Recent developments in serological techniques, i.e. immunofluorescence, complement fixation, countercurrent electrophoresis and immunodiffusion, have enabled rapid progress toward understanding the nature of the disease. It is suggested that the hypergammaglobulinemia is due to overproduction of IgG antibody specific for the Aleutian disease virus. The glomerulonephritis, vasculitis, positive antiglobulin test, systemic proliferation of lymphocytes and generalized plasmacytosis in Aleutian disease are believed to be the results of a continuing host antiviral immune response, the persistence of the virus in the presence of high level of specific antibody and the formation and deposition of the immune complexes.

Persistence of virus and a marked hypergammaglobulinemia are consistent features observed in mink with Aleutian disease virus infection. These events may result from altered lymphocyte function in infected mink. To test this possibility we determined the ability of lymphocytes from infected mink to respond to phytolectins (PHA and Con A), and a specific antigen (KLH) in a lymphocyte transformation test and quantitated the number of lymphocytes bearing surface immunoglobulin. Infected mink had a significantly lower response than normal mink to PHA and Con A (P less than 0.025), and the difference in the responses increased progressively with time post-infection. Infected mink immunized with KLH also had a decreased ability to respond to this antigen when compared to normal immunized mink (P less than 0.001). The number of lymphocytes bearing surface immunoglobulin was significantly elevated in three of 10 mink infected for 10 weeks, and in all of seven mink infected 32 weeks (P less than 0.001).

Antibodies to the factor present in the homogenate of the organs of minks experimentally infected with aleutian disease (AD) were revealed in the sera of minks with AD and also in the sera of humans suffering from systemic lupus erythematosus, hepatitis and cirrhosis, scleroderma and dermatomyositis. Sera of sick minks and humans failed to interact with control homogenates of the organs of healthy minks. In turn, sera of practically healthy minks and humans did not react with the AD-homogenate.

Free DNA and various types of antibodies to DNA were determined in the blood serum and plasma of minks which contracted Aleutian disease (AD) spontaneously and of minks experimentally infected with this disease. Healthy minks and other animals served as controls. It was found that a higher incidence of antibodies to DNA of the 2nd and 3rd types in high titres (1: 80-1: 2560) was characteristic of the experimental group of animals. Besides, a free polymeric DNA was more frequently revealed in the experimental group of animals.

Mink that were homozygous recessive for the Aleutian gene (aa) were inoculated with Aleutian disease virus (ADV) and simultaneously treated with cyclophosphamide (Cy). Control mink were inoculated with ADV. All mink were injected with bovine serum albumin (BSA) and their anti-BSA antibody response was measured to monitor the influence of drug therapy on the humoral antibody response. Formation of anti-BSA antibody was markedly suppressed and the hypergammaglobulinemia and development of AD lesions was inhibited in the Cy-treated mink. The non-Cy-treated control mink developed characteristic signs and lesions including glomerulonephritis and arteritis. The nontreated ADV-infected mink, but not the Cy-treated ADV-infected mink, had glomerular deposition of C3 and gamma globulin. Both groups had high titers of virus in their blood. These results indicate that the development of ADV lesions can be prevented by immunosuppressive treatment and further implicate host immune mechanisms in the pathogenesis of Aleutian disease.