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Sera that contained autoantibodies to mitochondria (AMA) by immunofluorescence were examined by immunoblotting for reactivity with mitochondrial polypeptides from various mammalian species, yeast, and E. coli. Mitochondrial polypeptides were separated by polyacrylamide gel electrophoresis, were immobilized on nitrocellulose, and were exposed to sera. The sera tested included 18 AMA-positive sera from patients with primary biliary cirrhosis (PBC), two AMA-positive sera from patients without PBC, and 53 AMA-negative sera. All AMA-positive sera reacted with either one or the other, or usually both of two human mitochondrial polypeptides of 70 kilodalton (kD) and 45 kD. The 53 AMA-negative sera were not reactive with the 70 kD polypeptide, but six reacted with the 45 kD polypeptide. The reactivity of the 70 kD and the 45 kD polypeptide was destroyed by brief exposure to trypsin. The counterpart of the 70 kD reactive polypeptide in human mitochondria was a 65 to 70 kD polypeptide in rat and mouse mitochondria, and a 55 kD polypeptide in yeast and in E. coli. The apparent 45 kD polypeptide was similar in all mitochondrial preparations tested, but no counterpart could be identified in E. coli. Beef heart mitochondria were used to show that the reactive polypeptides were present in a semipurified preparation of the F1 portion of mitochondrial H+ ATPase; however, sera did not react with the beta subunit of ATPase, proposed as a candidate mitochondrial autoantigen. The present molecular characterization of two particular antigens should lead to the more precise identification of these antigens, and also to a clearer insight into the pathogenesis of PBC.

T-lymphocyte immune capacity in man was assessed semiquantitatively by two in vivo procedures: the primary type of response to dinitrochlorobenzene and the secondary type of response, representing memory, to a group of five uniquitous antigens. Controlling for degree of illness proved important in assessing immune capacity in specific diseases; thus, the number of responders and mean score of semiquantitated responses was significantly lower in groups of patients with cancer and multisystem autoimmune disease when comparisons were made with healthy persons, but less so when comparisons were made with a group of subjects with other incapacitating diseases. A notable finding was the lack of correlation in the results of tests of cell-mediated immunity between the two procedures described. Depressed cell-mediated immunity shown in multisystem autoimmune disease is relevant to both predisposition to infection and the postulated role of thymic dysfunction in the pathogenesis of autoimmunity.

An improved method is described for growing human T-lymphocyte colonies in agar medium containing phytohaemagglutinin (PHA) and sheep red blood cells (SRBC). Cluster and colony growth was obtained when blood mononuclear cells were plated directly in the agar-medium (one-step procedure) or after incubation of cells in liquid medium with PHA (two-step procedure). In the one-step procedure approximately 1 per 100 cells plated formed a cluster containing four to fifty cells. In the two-step procedure 1 per 20 cells plated formed a cluster or a colony (more than fifty cells). The proliferating cells were shown to be sheep-erythrocyte rosette-forming cells (E-RFC). Optimal proliferation was dependent on the presence of phagocytic cells in the cell suspensions cultured. No growth occurred in cultures depleted of E-RFC. Detailed studies of the cycle, velocity sedimentation, and density of the cells plated showed that the majority of cluster- and colony-forming cells were small non-cycline lymphocytes with a sedimentation velocity of 4 mm/hr, and a density between 1-069 and 1-077 g/cm3.

A study was made in inbred mice of genetic determinants of toxic liver cell injury, and the subsequent occurrence of autoantibodies to mouse liver-specific protein (M-LSP). Carbon tetrachloride was injected s.c. in sublethal doses to induce liver cell damage in 4 strains of mice, BALB/c, C3H, C57BL/6 and SJL/J. The degree of liver cell damage was assessed by blood cholylglycine levels and by semiquantitative histological analysis 1, 4, 7, 14, 21 and 45 days after dosing. Striking differences were observed among the 4 strains in degrees of liver necrosis, cellular infiltration and rate of removal of the necrotic tissue. BALB/c was the strain most susceptible to the necrotizing effects of CCl4. These mice showed confluent areas of hepatocellular necrosis from Day 1 and histological recovery was protracted up to 3 weeks, and was accompanied by pronounced macrophage activity and a cellular inflammatory response. Mice of the SJL/J strain were the least susceptible and showed minor hepatocellular necrosis, which resolved by Day 7, and a slight histiocytic response. C3H and C57BL/6 showed lesions which were intermediate between the other 2 strains. The autoantibody response to LSP was weak, transient and detected only in C57BL/6 mice. This study indicates the presence of genetic control, either H-2 or non-H-2 linked, over the degree of liver cell necrosis resulting from toxic liver injury, and the ensuing cellular infiltrate and rate of removal of the necrotic tissue.

Colony-forming T-lymphocytes were studied in the blood of twenty-two patients with systemic lupus erythematosus (SLE) by the technique of plating phytohaemagglutinin (PHA) stimulated lymphocytes in soft agar. The mean count of T-lymphocyte colonies in SLE of 70+/-64 (1 s.d.)/mm3 of blood was considerably less than the count of 218+/-102/mm3 for healthy young adults and of 139+/-99/mm3 for aged persons. In SLE, counts of T-lymphocyte colonies correlated with low counts of T-lymphocytes in blood and with other indices of T-lymphocyte impairment. The data point to derangement of factors which influence the capacity of T-lymphocytes to proliferate, but it cannot be stated to what degree this is a cause and/or result of the autoimmune disease process associated with SLE. Cloning of peripheral blood T-cells could help determine whether the essential defect in SLE is in the T-lymphocyte or one of its sub-sets, or in factors produced by adherent cells which are essential for growth in vitro of T-lymphocyte colonies.

Thymocytes from twenty-two human foetal and post-natal thymuses were separated according to their buoyant density. Thymocytes from eight were separated into multiple fractions by means of continuous gradients of bovine serum albumin (BSA), pH 5.1 and iso-osmolar with human cells, and thymocytes from fourteen were separated into two fractions of density less than and greater than 1.068 g/cm3. Fractions were tested for antigen-binding lymphocytes (ABL) to 125I-labelled human thyroglobulin, for response to phytohaemagglutinin (PHA) and for rosette-forming cells (RFC) using sheep red blood cells. For subjects of all ages there was a pronounced enrichment of both ABL and thymocytes responsive to PHA amoung low or 1.064-1.065 g/cm3 density thymocytes. In older subjects there was a second enrichment of ABL among high or 1.072-1.073 g/cm3 density thymocytes. RFC were distributed over a wider range of densities, and although they did not form a discrete subpopulation they predominated among high density thymocytes.

Guinea-pigs injected with thymus or muscle in complete Freund's adjuvant developed `experimental autoimmune thymitis'. Thymitis was defined by accumulations of lymphocytes around Hassall's corpuscles in the thymic medulla and by distortion and compression of reticulin fibres towards the cortico-medullary junction. Thymitis occurred in five of eight animals injected with thymus and four of four animals injected with muscle.

An immunogenetic study of autoimmune chronic active hepatitis (CAH) showed the relative risk (RR) for this disease was 11.6 for patients who were HLA-B8, 11.7 for patients who were DR3 and 2.3 for patients who were Gma+x+. Moreover, the Gm haplotype Gma+x+ was present in 18 of 40 (45%) patients with HLA-B8, but in none of 10 patients negative for HLA-B8, whereas in 180 healthy controls Gma+x+ was evenly distributed among those positive (24%) and negative (18%) for HLA-B8. The RR was lowest in patients lacking HLA-B8 but positive for Gma+x+. Relative to this low-risk group, the risk was increased 39 times in subjects with both HLA-B8 and Gma+x+, 15 times in subjects with HLA-B8 who were not Gma+x+ and twice in subjects who were neither HLA-B8 nor Gma+x+. Statistical analysis indicated that the three-factor effect (disease risk affected by non-additive effects of HLA-B8 and Gma+x+) was significant (P less than 0.01), as were the main effects of HLA-B8 (P less than 0.001) and Gma+x+ (P less than 0.02). Thus in the presence of HLA-B8, genes linked to Gma+x+, an immunoglobulin CH allotype, may contribute to the development of autoimmune chronic active hepatitis; in the absence of HLA-B8 these same genes appear to be inactive. This may indicate interactions between MHC gene products and VH gene products in the presentation and recognition of autoantigen(s) in autoimmune hepatitis.