Characterization of a new serotype of Marek's disease herpesvirus. The HPRS-24 strain of Marek's disease herpesvirus was selected for closer study from a group of virus isolates that appeared apathogenic under standard test conditons. Protection studies revealed an immunological relationship between this virus strain and acute Marek's disease herpesvirus. In chicken kidney cell cultures, the HPRS-24 strain caused small and slowly developing plaques, and the proportion of infected cells was less than with other strains. In chicken embryo fibroblast cultures, this virus multiplied rapidly, yielding a comparatively high proportion of infected cells. Electron microscopic studies revealed that infected fibroblasts contained more enveloped virus particles than those infected with other strains of Marek's disease herpesvirus. Infectious cell-free virus was extracted from cultured fibroblasts with titres high enough for use in neutralization studies. Cross-neutralization, immunofluorescence and precipitin tests served for serological comparison of the HPRS-24 strain with turkey herpesvirus and representatives of acute and classical Marek's disease herpesvirus. Antibody titres were 4-10 times higher against the homologous than against the heterologous virus strains. Qualitative differences between precipitating "A" antigens were characterized by spur line patterns of precipitation bands. These results suggest that the group of Marek's disease and turkey herpesviruses consists of at least three serological types. One of them is represented by the HPRS-24 strain of apathogenic Marek's disease virus. The other two types comprise pathogenic strains of Marek's disease virus and their attenuated variants, and turkey herpesvirus.


T-lymphoblastoid cell lines from Marek's disease lymphomas. The establishment and continuous culture of two lymphoblastoid cell lines derived from Marek's disease lymphomas is described. Although the cells carried T-lymphocyte surface antigens, they had many features in common with cultured Burkitt's lymphoma lymphoblasts, which carry B-cell determinants. A small proportion acted as infectious units in tissue culture, and a similarly small proportion contained intranuclear immature herpesvirus particles. The cells did not respond to phytohaemagglutinin. One cell line possessed some graft-versus-host capacity, as measured by the induction of splenomegaly. It is concluded that the development of acute Marek's disease involves the malignant transformation of thymus-dependent lymphocytes by Marek's disease virus.


Immediate-type hypersensitivity to keyhole limpet haemocyanin in the dog. A model of immediate-type hypersensitivity (ITH) in the dog using keyhole limpet haemocyanin (KLH) as the antigen was studied. Long-lasting ITH was induced in fifteen of sixteen dogs by the i.v. (Kepron & Tse, 1975), intradermal (Rockey, Schwartzmann & Halliwell, 1971), inhalation (Patterson & Kelley, 1974) and i.d. plus inhalation (Rockey et al., 1971) routes. This ITH was demonstrated by direct skin reactivity, acute respiratory reactions and peripheral blood leucocyte and respiratory cell histamine release. The reaginic antibodies considered to mediate these reactions were larger than canine IgG, heat labile, 2-mercaptoethanol sensitive, and are probably canine IgE. In addition to cutaneous and respiratory reactions, these reagins were shown to mediate systemic reactions. In the course of these studies, an unexpected finding was the occurrence of ITH to KLH in three dogs which had had no prior experimental exposure to KLH. This ITH was of sufficient intensity to be associated with acute airway reactions to the inhaled antigen.


Radioimmunoassay of the WZ polymorphic antigens of normal human colon and their relationship with ABH antigenic determinants. Heat resistant, high mol. wt glycoproteins of the three WZ phenotypes (W+Z+, W-Z+ and W-Z-) were extracted from normal human colonic mucosa and labelled with 125I. More than 80 per cent of the radioactivity of the W+Z+ antigens labelled with I125, was precipitated by natural or immune anti-W antibodies. About 60 per cent of the W-Z+ 125I-labelled antigens were precipitated by immune anti-Z antibodies. No specific precipitation could be detected with W-Z- 125I-labelled antigens. All the precipitin reactions were quantitatively inhibited with the crude antigen extracts of the corresponding phenotypes. The W and Z antigens were only found in the intestinal mucosa and there was a 100-fold increase in the relative concentrations between the small bowel and the rectum. As opposed to this, the ABH antigens were found in all the digestive secretions studied, except those of the rectum, and their relative concentrations progressively decreased by about 100 between the small intestine and the recutm. The decrease in ABH concentration seems to be correlated to a WZ increase in the same regions.


Common antigenic structures of HLA antigens. VII. Selective combination binding of beta2-microglobulin with HLA large component in cultured human cell lines. The intracellular distribution of human beta2-microglobulin was examined in human cell lines (a Burkitt lymphoma cell line, a B-lymphoid cell line and an epighelial-like cell line). Freshly harvested cells were mechanically disrupted and separated into the nuclear, cell-membrane and cell-sap fractions. Nearly 90 per cent of the total beta2-microglobulin was recovered in the cell-membrane and cell-sap fractions. The cell-membrane fraction contained 75-88 per cent of the beta2-microglobulin recovered. The rest was in the cell-sap fraction. Most, 84-91 per cent, of the beta2-microglobulin in the cell-membrane fraction was present combined with membrane fraction was present combined with membrane components of about 38,000 daltons that carried the xenoantigenic activity characteristic of the HLA large component. These membrane components did carry HLA alloantigenic activity. No other membrane components were involved in binding beta2-microglobulin. The beta2-microglobulin in the cell-sap fraction was present in the unbound state. Thus, in the cell lines examined, the membrane component which was combined with beta2-microglobulin appeared to be exclusively the HLA large component and no larg excess of beta2-microglobulin over the HLA large component was found.


Relationship between E receptors and a T-specific surface antigen on human T cells. The aim of this study was the delineation of different antigenic surface determinants on the surface of adult peripheral T cells by means of a specific horse anti-human T-cell serum (ATS). It was shown that this serum reacts both with E receptors and (an) additional T antigen(s). While E receptors showed the already known susceptibility to trypsin, T antigens (as demonstrated in cytotoxicity tests) were resistant to trypsinization even at high concentration. Incubation of the trypsinized peripheral blood lymphocytes (PBL) in 5 per cent CO2 allowed the resynthesis of E-receptors. High concentrations of ATS (without complement) significantly inhibited rosette-formation. This suggests a close steric relationship between E receptors and T antigen(s). However, absorptions of ATS with trypsinized PBL left the E-rosette inhibitory capacity unaltered. Treatment of PBL with ATS in appropriate dilutions and indirect immunofluorescence tests under capping conditions followed by conventional rosette procedures showed that the E receptor and T antigens are separately mobile within the T-cell membrane.


Antigen site distribution among weak A' red cell populations. A study of A3, Ax and Aend variants. The distribution of the A receptors was studied among 'agglutinated' and 'free' populations of A variant RBC (A3, AX, Aend) known to be either partially or weakly agglutinated by human anti-A reagents. Following separation of the red cell populations and disaggregation of the clumps by mild treatment with soluble blood group substances, it was shown after appropriate controls, that among A3 ARBC,  the 'agglutinated' RBC have at least five times as 'free' RBC, these latter however being strongly A positive. The differences between the A antigenic content of the AX RBC were less pronounced. The most striking result was obtained with the Aend RBC, where two populations are clearly demonstrated; the first, including 5-10 per cent of the RBC, strongly agglutinates with anti-A and contains erythrocytes of high antigenic content (140,000 A receptors per cell). The second, including the majority of RBC could not be differentiated from the control O RBC. A wide heterogeneity of antibody binding capacity of the various populations of A3, AX and Aend red cells, was also demonstrated following ultrastructural examination by immunoelectron microscopy with peroxidase-conjugated antibodies. Such study reveals furthermore an heterogeneity of labelling from one cell to another in the same population of red blood cells. Comparison of 'week A' RBC and O RBC enzymatically converted into A RBC, demonstrates a similar pattern of reactivity between these cells, and supports the general relationship between antigen site density and red cell agglutination. It is concluded that the typical pattern of agglutinability of A3 and AX RBC arises both from their heterogeneous antigenic content and from the occurrence of an antigenic threshold below which red cells become non-agglutinable. The typical mixed-field agglutination pattern of Aend RBC merely reflects the occurrence of a probably true dual population of RBC. Finally, the mechanisms of inheritance of such well-known Mendelian characters, enabling the production of highly heterogeneous blood cell populations in the same individual, remains to be elucidated.


Cell-mediated and humoral immune responses in mice. III. Dynamic balance between delayed-type hypersensitivity and antibody response. Delayed-type hypersensitivity in mice to bovine serum albumin (BSA) which would be induced by a s.c. injection of BSA in Freund's complete adjuvant was interrupted by an i.v. injection of alum-precipitated BSA plus bacterial endotoxin (AP-BSA plus ET). In contrast, s.c. injection of BSA restrained the antibody response in the spleen of the mice receiving AP-BSA plus ET. Subcutaneous injection of either the adjuvant alone or an unrelated antigen in the adjuvant did not affect the anti-BSA antibody response in the spleen. The antibody response to other antigens in the spleen was not affected by the s.c. injection of BSA. The reduction of the antibody response in the spleen does not seem to be attributable to suppressor cells, since the suppressor cell activity against the antibody response of normal mice and that of irradiated recipients of primed spleen cells was not observed in the spleen cells of mice given s.c. immunization. The results indicated that the reduced antibody response in the spleen may be caused by the migration of some part of the antigen reactive cells or their precursors from the spleen to the s.c. region.


Functional maturation of B cells in vitro. Maturation of B-cell function was studied in a two-stage tissue culture system. In the first stage, cells were cultured in the absence of antigen and then transferred to microcultures where the frequency of hapten-specific plaque-forming cell (PFC) precursors was determined; Bone-marrow cells and spleen cells from 6--8-day-old mice mice were shown to act as sources of B-cell neogenesis in vitro. Both populations had very low initial frequencies of hapten-specific PFC precursors, but this increased ten- to seventeen-fold during a period of 72 h in the preliminary cultures. This increase could not be accounted for by selective cell death, nor by decay of a suppressor cell subpopulation nor by proliferation of pre-existing Fc-receptor-bearing B cells. The mechanism for the increase in frequency of functional B cells in cultures of bone marrow and neonatal spleen was thus the result of maturation of B-cell precursors to a state of immune competence during the culture interval.


Tolerance induction in maturing B cells. A two-stage tissue culture system was used to test the concept of clonal abortion as a mechanism for tolerance induction in B cells. In the first stage, neonatal spleen cells or bone marrow cells were cultured for 72 h under conditions in which B-cell neogenesis occurred. Haptens coupled to various carriers, were introduced during this stage. Following this culture phase, the cells were washed and their competence to respond to hapten-POL was measured in microcultures where feedback effects were minimized. The results indicated that immature B cells were specially susceptible to tolerance but that the conditions under which hapten was presented were also important in determining the outcome of the cell-antigen encounter.


In vitro studies on H-2 linked unresponsiveness. 1. Normal helper cells to (T,G)-A-L and GAT in low and non-responder mice. Lymphoid cells from unprimed high responder (C57BL/10) and low responder mice (B10.Br, B10.A, CBA) to (T,G)-A-L and high responder (B10, B10.A) and non-responder (B10.G, DBA/I) mice to GAT can be induced to form antigen specific T-helper cells in vitro under identical culture conditions. The helper cells induced from high and low or non-responder mice appear to be identical in efficiency, antigen concentration requirement for induction and induction kinetics.


In vitro studies on H-2 linked unresponsiveness to synthetic polypeptide antigens. II. Induction of suppressor cells in both responsive and unresponsive mice to (T,G)-A-L and GAT;. Antigen-specific T-suppressor cells can be induced in vitro from unprimed lymphoid cells of high responder (C57BL/10) and low responder (B10.Br, B10.A, CBA) mice to (T,G)-A-L and high responder (B10, B10;A) and non-responder (B10.G, DBA/1) mice to GAT10. The suppressor cells induced from high and low or non-responder mice appear identical in efficiency, in the antigen concentration required for induction and in their induction kinetics.


Pathogenesis of respiratory Klebsiella pneumoniae infection in rats: bacteriological and histological findings and metabolic alterations. Gram-negative bacterial pneumonias have been increasingly important as nosocomial infections. The following model was developed to study the pathogenesis and evaluate therapy of such infections. Intranasal instillation of rats with a suspension of 5 x 10(6) Klebsiella pneumoniae caused bronchopneumonia with 24 h. Bacteria were isolated from the lungs in large numbers (greater than 10(5) colony-forming units [CFU] for at least 13 days after inoculation. Thereafter, the viable concentration decreased to about 10(3) CFU at 21 days but increased to 10(4) CFU at 25 days. Mortality rarely exceeded 25%. Plasma zinc concentration decreased, and plasma seromucoid, lysozyme, and alpha2-macrofetoprotein increased during respiratory K. pneumoniae infection in rats. There seemed to be a linear relationship between seromucoid concentration and the concentration of K. pneumoniae in the lung expressed in log10 units. Plasma zinc, alpha2-macrofetoprtoein, or lysozyme levels, however, did not change until the concentration of bacteria retrieved fron lungs exceeded 4 to 5 logs, Analysis of blood samples obtained serially from the orbital sinuses revealed that rats that succumbed to infection had significantly higher levels of seromucoid, alpha2-macrofetoprotein, and lysozyme and lower levels of plasma zinc than infected rats that survived. Progressive increases in seromucoid and particularly in lysozyme and alpha2-macrofetoprotein appeared to be predicative of death. It is postulated that the threshold effect observed for alpha2-macrofetoprotein and lysozyme reflect significant damage to lung tissue, and thus these two variables are good indexes of the severity of this infection. We propose that this model may be of value in elucidating the pathogenesis of respiratory K. pneumoniae as well as in assessing various models of therapy.


Extracellular antigens of Micropolyspora faeni grown in synthetic medium. Extracellular antigens were prepared by growing Micropolyspora faeni in a new synthetic medium (SMSL), and this antigenic preparation was compared with antigens obtained by the double-dialysis method. These two preparations were tested against pooled sera from farmer's lung patients and rabbits immunized with these antigens by the agar-gel double-diffusion, immunoelectrophoresis, and two-dimensional immunoelectrophoresis methods. The immunological cross-reactivity between M. faeni strains of diverse origin and between other species of thermophilic actinomycetes associated with hypersensitivity pneumonitis was also investigated. The usefulness of two-dimensional immunoelectrophoresis in the comparison and standardization of antigens is discussed.


Immune suppression induced by acetoacetylated antigen. Injection of acetoacetylated antigen into rabbits with an ongoing reagin response abrogated this response. The possibility that this phenomenon might be due to activation of suppressor cells was studied in mice. Thymus or spleen cells from animals which had been primed with acetoacetylated antigen were able to suppress the IgG and IgM antibody response upon transfer to syngeneic mice. Maximal suppressive effect was observed 12-14 days after priming with native as well as acetoacetylated antigen. The IgG and IgM responses were equally affected by the transferred suppressor cells.


Effect of disodium cromoglycate and Picrorhiza kurroa root powder on sensitivity of guinea pigs to histamine and sympathomimetic amines. Four weeks pretreatment with disodium cromoglycate (DSCG) and the powdered roots of the herb Picrorhiza kurroa, Benth, rendered guinea pigs less sensitive to histamine when compared with appropriate controls. The bronchodilator effects of isoprenaline and adrenaline were found to be markedly enhanced. The severity and duration of the allergic bronchospasm was significantly less in animals pretreated with the two drugs. Furthermore, the total histamine content of the lung tissue in animals pretreated with DSCG and P. kurroa was significantly less than that in the untreated controls. The pretreatment was also found to exhibit inhibitory effect on the immunological release of histamine and SRS-A from chopped lungs.


Inhibitory effect of alpha-globulins of different origin on the cell-mediated immune responses in mice. alpha-Globulins prepared from different biological sources (human and bovine sera, mouse ascitic fluid, human ovarian cyst fluid) were separated by ion exchange chromatography on DEAE-cellulose into fractions A, B and C. Whereas fractions A and B had no immunosuppressive activity, fraction C injected into mice shortly before antigen administration, but not after, significantly inhibited the contact sensitivity to oxazolone. Single high doses were preferential over divided doses given on several occasions. In transfer experiments when cells of animals sensitized to oxazolone were injected into recipients treated previously with fraction C, the response was also inhibited. Fraction C, when administered into F1 hybrid mice before injection of parental splenocytes, prevented the development of GVH reaction. The authors point out that the critical concentration of alpha-globulins at the time of antigen administration is necessary to make T-lymphocytes hyporesponsive, and when once triggered by antigen they become refractory to alpha-globulins action.


Modulation of the IgE antibody response in rats to Kentucky blue grass pollen allergens. The low molecular weight dialyzable fraction (D) prepared from the aqueous extract of Kentucky Blue Grass pollen was shown to suppress the formation of IgE antibodies in rats immunized with the nondialyzable fraction (R). In an attempt to establish the nature of the constituents responsible for this suppression, D was fractionated by gel filtration through Sephadex G-25. The first fraction eluted (DI) elicited skin reactions in rats sensitized with a rat reaginic serum to R and also gave a precipitate with a rabbit antiserum to R. A later fraction (DIII) was devoid of these two properties. To investigate the effects of these fractions on the antibody response, rats received either DI or DIII, administered in saline, prior to their immunization with R in presence of aluminum hydroxide. Pretreatment with DI resulted in a reduction of IgE antibody levels as compared with the IgE antibody response in control animals which had received pretreatment only with saline; however, pretreatment with DI did not affect the anti-R-hemagglutinating titers. In contrast, pretreatment with DIII enhanced both the IgE and the hemagglutinating antibodies. Hence, it is concluded that the composite fraction D contains one group of constituents capable of suppressing and another of enhancing the IgE antibody response.


Elevated serum alpha-fetoprotein and impaired immune response in malnutrition. Serum alpha-fetoprotein (AFP) estimated in 33 malnourished children, was found to be elevated in 11 of 17 fetally growth-retarded infants and in 5 to 16 with postnatal nutritional deficiency. The majority of those with increased serum AFP concentration had associated liver dysfunction. Nutritional deprivation of growing rats resulted in an increase in AFP levels, especially in the offspring of starved female animals. The administration of purified AFP was associated with reduced antibody formation to sheep red blood cells. It is suggested that AFP exerts a modulating influence on immune response.


Comparative studies on EBV antigens by immunofluorescence and immunoperoxidase techniques. Three groups of EBV antigens, VCA, MA and EA, were compared by the techniques of electron microscopic immunoperoxidase (IP) and immunofluorescence (IF). P3HR-1 and EBV superinfected Raji cells served as targets for labelled sera from patients with BL, NPC and IM or from healthy donors. 125I peroxidase-labelled antibodies were also prepared to determine, autoradiographically, the penetration of the complex into the cell system, and to monitor the incubation and washing procedures. The development of a gentle sedimentation technique proved critical in handling the fragile target cells. VCA and MA were readily identified and localized by both procedures without significant modification of the basic techniques. Indentification of early antigens by IP required modification of the fixation method to include a brief treatment in acetone. The diffuse early antigen (EAD) was found to be associated with cellular ribosomes.


Viruses and renal carcinoma of Rana pipiens. XV. The presence of virus-associated membrane antigen(s) on Lucké tumor cells. Virus-specific antigens were detected in Lucké tumor cells by indirect immunofluorescence using antiserum prepared against Lucké herpesvirus. Intracellular fluorescence, both cytoplasmic and nuclear, was observed only in acetone:methanol-fixed tumor cells that contained herpesvirus detected by electron microscopy. The number of positive cells correlated well with the number of cells containing virus. In contrast, both virus-containing and virus-free cells exhibited membrane fluorescence when viable unfixed tumor cells were tested. A striking reduction in the number of membrane fluorescent cells was observed with an increase in the length of time that tumor cells were in primary culture. No reaction was observed with a variety of normal R. pipiens cells. Absorption of the antiserum with normal frog kidney tissue had no effect on the number of positive cells whereas absorption with virus-free tumor reduced the reaction; absorption with virus-containing tumor eliminated it. These findings provide the first demonstration that the Lucké herpesvirus genome resident in virus-free tumor cells expresses a virus-associated membrane antigen(s).


The macrophage electrophoretic mobility test: Results on carcinoma of the colon and rectum. The macrophage electrophoretic mobility (MEM) test was performed on guinea-pig macrophages treated with the interaction products of encephalitogenic protein and peripheral lymphocytes from 44 patients with colorectal cancer and 33 "healthy" controls. In 54/60 tests involving patients, statistically significant reductions in electrophoretic mobilities were observed, compared with 12/33 in controls. Our overall results on the reductions in macrophage mobilities by lymphocyte products are in accord with the work of some other workers, but not all. In contrast to many other studies, the standard procedures used here to express the results should permit an exchange of data on an international basis and allow a more rapid, general appraisal of the MEM test.


Effect of BNU treatment on leukaemogenesis in lethally irradiated AKR mice restored with bone-marrow and spleen cells. The leukaemogenic effect of N-butyl-N-nitrosourea (BNU) was studied in normal and thymectomized AKR mice which were lethally irradiated and restored with either bone-marrow (BM) or spleen cells from (AKR X AKR/T1ALD)F1 donors. In some instances T1ALD thymic cells were added to the restorative inoculum. It was possible to determine the origin of the leukemic cells by the metacentric marker chromosomes of T1ALD. The T- or B-cell characteristics were further ascertained by the cytotoxicity test for theta antigen and the EAC rosette test. All leukaemias whether thymic (TLS) or extra-thymic (ETL), developed from donor bone-marrow or spleen cells and never from the injected thymic cells. In non-thymectomized animals BNU increased the percentage of TLS and shortened their latency. Most of TLS which occurred after BNU treatment of BM-restored mice were theta-negative whereas the majority of TLS which occurred in controls and in spleen-restored animals were theta-positive. This suggests that during their maturation process BM-derived T precursors transit through a theta-negative compartment. This compartment does not reach a similar size during the maturation process of the spleen-derived precursors. Adding thymic cells to the restorative inoculum enhanced leukaemogenesis and suppressed theta-negative TLS in BM-restored mice. Thymectomized mice, restored either by BM or spleen, had a low incidence of ETL which was not significantly increased by BNU treatment except in the case of mice restored with spleen cells. The leukaemic cells of one ETL were theta-positive whereas all the other leukaemias had no detectable T or B marker. The percentage of ETL was higher in thymectomized mice treated with BNU alone than in those previously subjected to irradiation and restoration. These results strongly suggest that a theta-negative T precursor could be involved in extra-thymic leukaemogenesis but the possible involvement of a B precursor cannot be rule out unless experiments are carried out with specific markers of T- and B-cell sub-classes.


Genetics of murine sarcoma virus (MSV)--induced tumors in AKR mice: Evidence that late progressing and early regressing tumors are controlled by different gents. The genetics of late appearing MSV tumors showing a progressive growth pattern in AKR mice was investigated. The late MSV tumor response in F1 hybrids depended on the genetic background of the non-AKR parent. Within the 4-month observation period following virus injection, (CBA X AKR) F1, (DBA/2 X AKR)F1, and (NIH X AKR)F1 developed progressing MSV tumors, which exhibited latency and growth behavior comparable to that seen in AKR mice, (BALB X AKR)F1, (B6 X AKR)F1, and (B10br x akr)f1 mice did not show any late MSV tumors. In contrast to early regressing M-MSV tumors, whose development is independent of Fv-1 genotype, late MSV tumor progression is largely a function of this gene, since all late tumors which appeared in (B10BR x AKR) x AKR were observed in Fv-1n homozygous mice, H-2k halotype is a further factor in the occurrence of late MSV tumors, at least in (B6 x AKR) x AKR mice. In crosses of AKR with Fv-1 compatible mice, tumor appearance was strongly associated with inheritance of AKR-Mulv, and MSV recovered from late tumors of first back-cross animals appeared to be a new pseudotype with the endogenous AKR-MuLV. It is suggested that the host genetic control in both early and late MSV tumors is exerted mainly on the helper component of the leukemia-sarcoma complex.


Effects of a new glucocorticoid, oxazacort, on some variables connected with bone metabolism in man: a comparison with prednisone. The urinary elimination of calcium, other electrolytes, and hydroxyproline and the oral absorption of 47Ca have been evaluated in three groups of 8 patients before and during a 15-day treatment with prednisone at daily doses of 25 and 50 mg and with oxazacort, a new glucocorticoid, at a daily dose of 50 mg. The results obtained demonstrate that oxazacort in short-term teatment with a high dose has no significant effect on the urinary elimination of calcium and hydroxypyroline in experimental conditions in which prednisone produces statistically significant and clinically relevant increase, both when given at the same dose and when given at half that dose. On the other hand, the oral absorption of 47Ca is decreased by oxazacort, but less than by prednisone at the same dose. As the antirheumatic activity of oxazacort appears to be only slightly lower than that of prednisone (activity ratio of about 0.84: 1), these findings may have interesting therapeutic implications.


Immunochemical analysis of soluble antigens of Mycobacterium lepraemurium. Soluble antigens obtained from purified M. lepraemurium were identified by SDS polyacrylamide gel electrophoresis as three different proteins, M-W 1 X 10(4), 1 X 10(5), and 4 X 10(6), and two polysaccharide-polypeptides. Three antigens were found to react with anti-M. leprae antiserum; a single antigen proved to be immunologically related to M. tuberculosis, M. similae, M. balnei and a few other mycobacteria, but not to Eubacteriales and fungi, as examined by immunoelectrophoresis. In the dermal hypersensitivity assay, the guinea pigs sensitized with soluble antigens of M. lepraemurium only responded to the antigens of M. lepraemurium, M. leprae and partly to M. scrofulaceum, but not to other antigens, including lepromin and leprous serum globulins.


Electrophoretic pattern of serum proteins in leprosy. Sera collected from 65 patients having various types of leprosy and 20 healthy human beings were studied for the determination of total serum proteins, paper electrophoresis for protein types and albumin globulin ratios. Significant changes in protein equilibrium with the presence of hypoalbuminemia and increases in various globulin fractions (except beta globulin) were observed in patients having lepromatous and dimorphous leprosy. An abnormal protein band was also detected in 3 of 20 cases of lepromatous leprosy. The patients suffering from tuberculoid and polyneuritic leprosy did not show much protein disequilibrium except for mild rises in serum alpha-2 and reductions in serum beta globulins.


Present status of MA-160 cell line. Prostatic epithelium or HeLa cells. This report reviews the background and the present status of the MA-160 cell line. This cell line was reported to have arisen from tissue cultures of a human, benign prostatic adenoma and consequently has been used for investigations on the prostat and on hormone-dependent cells. Recent studies using chromosome analysis by banding techniques, glucose-6-phosphate dehydrogenase (G6D) electrophoretic mobility, and a specific prostatic acid phosphatase test indicate that MA-160 is not of  prostatic origin but a HeLa cell contaminant, arising as a result of intraspecies cell contamination. It is suggested that whenever spontaneous transformation is observed in vitro and established cell lines are being used in the laboratory, the cells in question be thoroughly checked with the currently available chromosomal, biochemical, and cytochemical methods in order to establish their origin and to rule out interspecies and intraspecies cell contamination.


Reverse transcriptase inhibitors and chemically induced bladder tumors in mice. Recent immunologic and microbiologic evidence suggests that urothelial tumors may be caused by "C" type oncogenic viruses. Such viruses may exert their oncogenic potential in responce to stimulation by known chemical carcinogens. By means of a unique enzyme, reverse transcriptase, these viruses are able to incorporate genetic information into that of the host, and can thereby be transmitted vertically from generation to generation. An evaluation of the specific antiviral agents dimethylbenzyldemethl-rifampicin and streptovaricin-comples, which inhibit the enzyme reverse transcriptase, revealed no depay in the induction of bladder tumors by the chemical carcinogen, 2-formylamino-4-(5-nitro-2-furyl) thiazole (FANFT) in C3H mice. This observation suggests that the reproduction and release of virus may not be essential in the malignant transformation of bladder epithelial cells, but does not preclude the possiblity that inherited viral genetic information may be involved in the oncogenesis of bladder tumors.


Pig calcitonin in the treatment of localised osteoporosis. On the basis of positive results obtained in the treatment of Sudeck's atrophy with calcitonin, the authors extended their investigation to other forms of localised osteoporosis. Six patients were examined affected by osteoporosis secondary to immobilisation, three patients with osteoporosis of the lower limbs from paralysis of the sciatic nerve and six patients with migrant osteoporosis. Treatment was as follows: pig calcitonin (Calcitar) in doses of 160 u MRC/daily + calcium gluconate in doses of 3 gr/daily. The duration of treatment averaged forty five days. In osteoporosis from immobilisation and nerve lesions the calcitonin treatment did not influence the condition and there was no change in radiographic appearances nor was there any analgesic action. On the other hand, the results were clearly positive in migrant osteoporosis: in all the patients treated there was complete regression of pain, cutaneous trophic changes, and functional loss. At a later stage, normal radiographic appearances were restored.


Bandign isolated metaphase chromosomes by a sequential flurescent G/Q technique. G/Q-banding is a new, rapid, fluorescent technique for banding isolated chromosomes that incorporates characteristics of both G- and Q- banding. G-bands, while easily characterized, are often inconsistent when using isolated chromosomes, and Q-bands, while reliable, fade rapidly under UV exposure, making prolonged observation and photography difficult. G/Q-banding combines these techniques to sequentially utilize quinacrine staining over Giemsa banding to produce slow-fading fluorescent G/Q-bands. The background fluorescence in G/Q preparations fades quickly under continued UV exposure, while the chromosomes remain brightly banded and can be observed and photographed for at least five minutes. G/Q-banding was extended to whole cell chromosome spreads and produced results identical to those obtained with isolated chromosomes. Whole cell karyotypes indicate that G/Q-bands generally correspond to Q-bands. Advantages of G/Q-banding as a unique and universal technique over current double-staining procedures are discussed.


Reduction and azo coupling of quinones. A histochemical study of human cutaneous melanin and adrenochrome. Cutaneous melanin in formol fixed skin and adrenochrome in dichromate fixed monkey adrenal after adequate bisulfite or dithonite reduction were found to give definite azo coupling reactions. Weaker reactions were obtained on unreduced material, and these disappeared on ferric chloride oxidation. Both cutaneous melanin and adrenochrome appear to exist in a quinhydrone status. Prolongation of dichromate treatment weakens or abolishes azo coupling capacity of adrenochrome. The findings support the concept of quinonization and reduction to prevent and restore azo coupling of enterochromaffin cells and noradrenaline islets of the adrenal. The most effective diazos for melanin were p-nitrodiazobenzene, fast black K and the diazosulfanilic acid, pH 1 pyronin B procedure, for adrenochrome. Diazosafranin and 2-chloro-4-nitrodiazobenzene were also useful. Blue and violet coupling products from toluidine blue and methylene violet RR fail to yield sufficient contrast to be convincing.


Cis-dichloro-diammine platinum (II) as stain for electron microscopic preparations. Cis-dichloro-diammine platinum (II) provides a strong contrast to electron microscopic preparations by reacting with nucleic acids and proteins. This staining technique is easy and applicable to ultrathin sections embedded in Epon; various staining conditions have been tested.


Inhibition of bleomycin-induced [3H] thymidine 5'-triphosphate incorporation into liver and hepatoma nuclei by N-ethyl maleimide and daunomycin. The addition of bleomycin to a nuclear incorporating system results in an increased incorporation of 3H-thymidine 5'-triphosphate (3H-TTP) into the DNA of liver and hepatoma nuclei. Bleomycin added to the nuclear incorporating system also produces scissions of DNA as determined by sucrose density gradient centrifugation of the extracted DNA. The action of bleomycin is dependent on the presence of sulfhydryl agents in the incubation mixture. Two compounds, N-ethyl maleimide and daunomycin, inhibit the bleomycin-induced incorporation of 3H-TTP preferentially. N-Ethyl maleimide inhibits bleomycin-induced activity in liver and hepatoma 7777 nuclei equally. Lower levels of daunomycin inhibit the bleomycin-induced activity in the hepatoma 7777 nuclei than are required to inhibit the activity in liver nuclei. The two compounds inhibit the bleomycin effect by different mechanisms. The addition of N-ethyl maleimide to bleomycin in the incubation system prevents bleomycin from causing breaks in the DNA. The addition of daunomycin, despite inhibition of bleomycin-induced 3H-TTP incorporation, does not affect the bleomycin-produced breaks in the DNA. N-Ethyl maleimide acts by binding to the DNA and by competing with a sulfhydryl agent for bleomycin-sensitive sites on the DNA. Daunomycin apparently inhibits a repair enzyme that is responsible for the increased incorporation following bleomycin treatment.


beta-actinin, a regulatory protein of muscle. Purification, characterization and function. beta-Actinin, a minor regulatory protein of muscle, was purified from skeletal muscles of rabbit and chicken by DEAE-Sephadex chromatography. beta-Actinin consisted of two subunits, beta I and betaII, with chain weights of 37,000 and 34,000 daltons, respectively. The amino acid compositions were similar, though not identical. It appears that each of the two subunits is associated in solution. beta-Actinin had the following effects on actin: (1) inhibition of reassociation of F-actin fragments; (2) inhibition of network formation of F-actin; (3) inhibition of growth of F-actin fragments; (4) retardation of depolymerization of F-actin and (5) acceleration of polymerization of G-actin. All these actions of beta-actinin can be explained in terms of action as an "ending factor". Experimental evidence favored the view that beta-actinin is bound to one end of the F-actin filament, namely to the end opposite to the direction of polymerization. Fluorescence-labeled anti-beta-actinin stained the middle portion of the A band of myofibrils. Based on the finding that the stain was unchanged on removal of myosin, it is suggested that beta-actinin is located at the free ends of the I filaments of myofibrils. Thus is seems likely that beta-actinin functions as an ending factor for actin filaments.


Biosynthesis of enzyme-bound formylvaline and formylvalylglycine. A possible initiation complex for gramicidin A biosynthesis. A partially purified fraction (Component I) which catalyzed the ATP-PPi exchange reaction dependent only on valine and glycine among the five constituent amino acids of gramicidin A was obtained from crude extracts of Bacillus brevis ATCC 8185, which produces both gramicidin A and tyrocidines. This fraction was found to bind valine and glycine as thioesters. The addition of a formylTHFA synthesizing system to Component I brought about the formation of Component I-bound formylvaline and formylvalylglycine.


Instructional television graphics.

Instructional television graphics. Instructional television graphics are defined as artwork and photographs used to aid comprehension of a concept presented through the medium of television. In this paper television graphics are examined for educational effectiveness and technical reproducibility. Educational effectiveness of a graphic is facilitated by utilizing the same criteria or components of instructional design used in development of the program. Technical reproducibility is more likely assured if the artist understands the characteristics of television as medium.


Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake. Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.


Actions of insulin in fat cells. Effects of low temperature, uncouplers of oxidative phosphorylation, and respiratory inhibitors. When isolated rat epididymal fat cells were incubated with [125I]iodoinsulin for 5 min at 37 degrees, radioactivity accumulated in the plasma membrane fraction (Peak 1) and an unidentified particulate fraction (Peak 2) as reported previously (Kono, T., Robinson, F.W., and Sarver, J.A. (1975) J. Biol. Chem. 250, 7826-7835). This accumulation of radioactivity in Peak 2 (but not that in Peak 1) was greatly impaired when cells were incubated with iodoinsulin in the presence of a variety of metabolic inhibitors that reduce the cellular content of ATP. The reduction in the ATP level coincided with a disappearance of the stimulatory effects of insulin on sugar transport and the hormone-sensitive phosphodiesterase. In contrast, ATP depletion had no significant effects, at least during a 5-to 15-min incubation, on the intracellular water space and on the basal sugar transport and phosphodiesterase activities. When cells once depleted on ATP by treatment with 2,4-dinitrophenol (1 mM; 10 min) were washed and suspended in fresh buffer, the ATP level was recovered almost fully in 10 min. This recovery coincided with the restoration of responsiveness to insulin. When cells were incubated with [125I]iodoinsulin or insulin for 5 min at 15 degrees instead of 37 degrees, a negligible quantity of radioactivity accumulated in Peak 2 and insulin failed to activate sugar transport. In contrast, under the same conditions, radioactivity accumulated in Peak 1 and insulin stimulated phosphodiesterase considerably. These results suggest that ATP, or some other compound metabolically related to ATP, may be necessary for the actions of insulin on sugar transport and phosphodiesterase. ATP, or some other related compound, may also be necessary in the formation of the radioactive Peak 2, although the physiological function and cellular location of this peak are yet to be ascertained.


RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzyme. Three forms of the RNA-dependent DNA polymerase were isolated from highly purified avian sarcoma virus B77 grown in duck embryo fibroblasts, using sequential chromatography on DEAE-cellulose, phosphocellulose, and poly(U)-cellulose. One form, which sedimented with about 5.2 S, contained only one species of polypeptide, with a molecular weight of 63,000; a second sedimented with about 7.8 S and contained only one species of polypeptide with a molecular weight of 81,000; and a third form, which sedimented with about 7.3 S, contained two species of polypeptides with molecular weights of 63,000 and 81,000. The molecular constitution of the three enzyme forms were therefore alpha, beta2, and alphabeta. All three possessed almost the same specific activity with poly(rA)-oligo(dT) as the primer-template. Forms alpha and alphabeta of avian sarcoma virus DNA polymerase have already been described in the literature; form beta2 is a new form. All three forms possessed ribonuclease H activity, the relative specific activities of the alpha, beta2, and alphabeta forms being about 1:4:5. All three enzyme forms were inhibited by antiserum to the alphabeta form, but whereas the alpha and alphabeta forms could be inhibited about 95%, the maximum degree of inhibition of the beta2 form was about 80%. The three enzyme forms also differed with respect to heat stability at 46 degrees, the monomeric alpha form of the enzyme being only about one-half as stable as the two dimeric forms.


RNA-dependent DNA polymerase of avian sarcoma virus B77. II. Comparison of the catalytic properties of the alpha, beta2, and alphabeta enzyme forms. The alpha, beta2, and alphabeta forms of the RNA-dependent DNA polymerase of avian sarcoma virus B77 grown in duck embryo fibroblasts have been compared with respect to several kinetic properties. The following results were obtained. 1. The Km values for dTTP and dGTP for enzyme forms alpha, beta2, and alphabeta were 77, 39, and 74, and 6.8, 3.1, and 6.1 micronM, respectively. 2. The affinity of 70 S Rous sarcoma virus RNA for enzyme form alphabeta was about twice that for the other two forms. 3. The relative specific activities of the three enzyme forms on synthetic primer-templates such as poly(rA)-poly(dT) were almost the same. The viral 70 S RNA-dependent specific activities were 2 to 3 orders of magnitude lower and in the ratio of 1:3:5 for enzyme forms alpha:beta2:alphabeta. Addition of exogenous oligo(dT) stimulated the 70 S viral RNA-dependent activity of enzyme forms alphabeta and beta2 by a factor of 3, and that of enzyme form alpha by a factor of 30, so that it then became the most active transcriptase of viral 70 S RNA. 4. The largest transcripts formed by the three enzyme forms with 70 S viral RNA as primer-template were about 4,500 nucleotides long. About one-third of the total amount of polynucleotides polymerized by the alphabeta enzyme was in the form of such transcripts. This proportion was far higher than for the other two enzyme forms. 5. All three enzyme forms were capable of transcribing single-stranded into double-stranded DNA. 6. The 3-propylcyclohexyl piperidyl derivative of rifamycin SV, at a concentration of 100 microng/ml, inhibited enzyme forms beta2 and alphabeta by over 99.5 and 96%, respectively, but enzyme form alpha by only about 60%. 7. The beta2 and alphabeta forms of the enzyme were processive DNA polymerases, the alpha form a nonprocessive polymerase. 8. In general, these results indicate that in most respects the properties of the dimeric enzyme forms resemble each other much more closely than those of the alpha form. In some very important respects, such as affinity for viral RNA and the size of transcripts formed from it, the alphabeta enzyme form performs significantly better than either of the other two enzyme forms.


Role of protein and fatty acid adsorption on platelet adhesion and aggregation at the blood-polymer interface. Thrombus formation on a foreign surface is a complicated process, involving many factors. However, there is little doubt that a foreign surface adsorbs plasma proteins upon blood contact and that the nature of this adsorbed layer may determine the mechanism of platelet adhesion and aggregation. The adhesion and aggregation of platelets play an important role in the initial events of thrombus formation on a foreign surface. In this work, adsorption studies using human blood plasma were done on several polymer surfaces. Some drugs which prevent platelet adhesion were utilized to verify the proposed mechanism for platelet adhesion which includes glycosyl transferase reaction. Also, adsorption and release of fatty acid salts, including fatty acid-bonded albumin, were investigated at different polymer interfaces. It is postulated that adsorbed fatty acid salts are released from the surface upon contact with plasma to form a high local concentration of fatty acid, and that this fatty acid suspension would cause platelet aggregation at the interface.


Immunological procedure for the rapid and specific identification of Coccidioides immitis cultures. An immunological procedure for the rapid and specific identification of Coccidioides immitis isolates has been developed. The specificity of the procedure is based on the fact that C. immitis produces antigens that are not produced by morphologically similar fungi. The procedure involved the transfer of heavy mold-form inocula to flasks that contained small volumes of brain heart infusion broth. Shake cultures were grown at 25 degrees C on a gyratory shaker at 150 rpm. The concentrated supernatant antigens so obtained from broth cultures were tested in parallel with coccidioidin by a microimmunodiffusion technique against C. immitis antiserum. The ability of the immunological procedure to identify C. immitis cultures was evaluated by testing the supernatant antigens of 166 unknown isolates, many of which, by gross or microscopic examination or both, resembled C. immitis or belonged to the family Gymnoascaceae. Each culture was also identified by conventional laboratory procedures. Comparative evaluation showed that the immunological test for C. immitis was 100% sensitive and specific. The diagnostic precipitin bands that were produced by the interaction of the C. immitis supernatants and antisera were shown to consist of (i) a heat-stable antigen comparable to that active in the tube precipitin test and (ii) two heat-labile antigens, one of which was associated with complement fixation reactivity. With pure cultures, this immunological procedure permitted the identification of C. immitis isolates within 5 days.


Comparisons of the analgesic effects of oral and intramuscular oxymorphone and of intramuscular oxymorphone and morphine in patients with cancer. The relative analgesic potency of oral and intramuscular oxymorphone was evaluated in a double-blind crossover comparison of graded single doses in patients with chronic pain due to cancer. When both duration and intensity of analgesia are considered (total effect), oral oxymorphone was 1/6 as potent as the intramuscular form. In terms of peak effect, however, oral oxymorphone was only 1/14 as potent. These values are almost identical to those obtained in a previous study comparing oral with intramuscular morphine. The analgesic effect of oral oxymorphone rose to a peak later and had a longer duration than the effect of intramuscular oxymorphone. Intramuscular oxymorphone and morphine were also compared in a similar patient group. Intramuscular oxymorphone proved to be 8.7 times as potent as morphine in terms of total analgesic effect and 13 times as potent in terms of peak effect. In roughly equinalgesic doses, the occurrence of side effects was qualitatively and quantitatively similar for oral and intramuscular oxymorphone and for intramuscular oxymorphone and morphine.


Detection of immediate hypersensitivity in rabbits by direct basophil degranulation. The direct in vitro antigen-induced degranulation of rabbit basophils based on a simplified one-step method for the staining of these cells is reported. Degranulation was assessed by counting the number of basophils before and after exposure to antigen at 37 degrees C in the presence of Ca++. The degranulation was IgE-mediated, as shown by its passive transfer to nonimmunized rabbits by IgE-containing serum; also, the reaction was dependent on the presence of Ca++ ions and normal cell metabolism. The technique allowed the detection of serum factors capable of blocking the degranulation. It provided a means for following IgE sensitization at the cellular level, which is a better index of immediate hypersensitivity than the presence of circulating specific IgE.


Delayed cold-induced urticaria: a dominantly inherited disorder. A delayed cutaneous response to cold, characterized by areas of erythematous, edematous deep swelling at 9 to 18 hr after experimental ice challenge, was recognized in a 10-yr-old boy and several members of his family. Biopsy of the cold-induced lesion showed edema and an infiltrate of mononuclear cells; mast cells were normal, and immunoglobulins, complement factors, and fibrin were not detected by immunofluorescence techniques. Local cold challenge did not release histamine or induce alterations in the complement system or the enzymes, histaminase, and histamine methyl transferase. The delayed cutaneous response to cold could not be passively transferred with serum or tissue extracts to monkey skin. Family studies suggested an autosomal-dominant mode of inheritance.


Polymerization of mixtures of grass allergens. Mixed grass pollen allergens were precipitated from crude grass extract by 90% saturation with ammonium sulfate. The precipitate was dissolved and polymerized with glutaraldehyde. Polymerized allergens with a molecular weight range of 200,000 to 4,000,000 were isolated. The resultant grass allergen polymers had reduced allergenicity but were capable of absorbing out IgG antibody from sera of 2 patients treated with crude grass allergens. This alteration of allergenicity was intended to reduce the ability of allergens to react with IgE-sensitized cells rather than to modify or destroy antigenic determinants. Prior exposure of allergens to phenol used as a preservative inhibited the polymerization process probably by blocking combining sites with which glutaraldehyde reacts.


Acute urticaria and bronchospasm following radiographic contrast media in a dog. An immediate-type systemic reaction in a dog following intravenous radiographic contrast media (RCM) manifested by urticaria and wheezing on one occasion and urticaria alone on another occasion is described. This reactivity disappeared spontaneously and plans to study the mechanisms of such a reaction were not possible. If such reactivity is more persistent in certain other dogs, a model for study of immediate type reactivity to RCM would be available.


Pathological changes during aging in barrier-reared Fischer 344 male rats. Pathology, microbiology, and selected serum chemistries were evaluated in 144 male Fischer rats from 4 to 33 mo of age. The rats were reared and maintained under barrier conditions, which successfully excluded the introduction of major infectious disease agents throughout the entire study, including Mycoplasma pulmonis. A wide variety of pathology was found and tabulated, and many lesions were found to increase in severity and incidence with age. There was a high correlation of renal disease severity with increasing age, while alpha-1 globulin and cholesterol increased.


Structure and biologic functions of human IgD. XI. Identification and ontogeny of a rat lymphocyte immunoglobulin having antigenic cross-reactivity with human IgD. Chicken anti-human IgD antiserum (anti-delta) has demonstrated an antigenically cross-reactive homologue on rat lymphocytes. IgD and IgM are the only cell surface immunoglobulins detectable by the lactoperoxidase radiolabeling technique employed. The results indicate that, although rat surface IgD is antigenically distinct from rat IgM, the respective H chains co-electrophorese in 10% polyacrylamide-SDS gels. Rat delta-chain has an apparent m.w. of 73,000 daltons and exhibits a minor 65,000 dalton component which probably represents a partially degraded delta-chain. The ontogenic emergence of rat IgD occurs approximately 3.5 weeks after birth whereas IgM, in contrast, is apparent by 6 days of age. Thus, as in the human, IgM develops before IgD. IgD receptors are undetectable in the thymus but are present in increasing levels in spleen, blood, lymph nodes, and Peyer's patches.


Quantitative studies on the precursors of cytotoxic lymphocytes. II. Specificity of precursors responsive to alloantigens and to concanavalin A. A microculture system was used to examine the specificity of cytotoxic T lymphocytes (CL) derived from single precursors. Our data indicate that clones derived from single precursors of CL (CLP) are specific. Thus, RNC mice (H-2k) simultaneously stimulated by both H-2d and H-2b antigens produce clones which are specific for either H-2b or H-2d but not for both. In lymph node (LN), the frequency of CLP giving rise to clones specific for H-2d and H-2b is 1/480 and 1/860, respectively. Clonal specificity is also maintained when Con A is used to activate the precursors. With a nonspecific assay of CL, it was found that 2.5% of the cells in LN are stimulated by Con A to produce clones of CL. Among the CLP stimulated by Con A, 2.1% of the clones are specific for H-2d alloantigens. An unexpected finding was that 18% of the clones produced by Con A stimulation lysed EL4 tumor cells, which carry H-2b; however, only a small proportion of these clones were specific for the H-2b alloantigen. The antigen on EL4 which accounts for the high response has not been characterized.


Isolation of mouse C-reactive protein from liver and serum. Purified proteins have been isolated from the sera and livers of mice. These proteins are antigenically identical but share antigenic determinants in common with human C-reactive protein and do not share antigenic determinants in common with mouse or human immunoglobulins. The proteins interact with C-polysaccharide, are precipitated by calcium ions, migrate electrophoretically with gamma mobilities, and have isoelectric points of 4.8 and 5.62. Since these properties are characteristic of C-reactive protein of man, monkey, rabbit and dog, the pure proteins isolated from mice are designated mouse C-reactive protein.


Characterization of molecular heterogeneity and multispecificity in homologous idiotypic antisera. The molecular heterogeneity of homologous anti-idiotypic reagents was characterized by a novel isoelectric focusing procedure. Idiotypic antisera directed against the PC-binding plasmacytoma protein T15 were raised in CE and and A/J mice. These antisera were shown to be highly specific by hemagglutination with myeloma protein-derivatized sheep erythrocytes and by radioimmunoassay. Competition experiments performed with affinity-labeled T15 revealed that about 40% of the pooled CE antibody activity was directed against binding site-associated determinants. Further analysis of anti-idiotypic sera from individual animals with the use of isoelectric focusing disclosed heterogeneous populations of antibody molecules distinguishable by isoelectric point and by subspecificity. Each animal expressed a unique spectrotypic profile. In addition, clones reactive with binding site and non-binding-site determinants as well as some clones with specificity for other PC-binding mouse myeloma proteins were detected. These results emphasize the importance of careful selection and thorough absorption of idiotypic antisera.


Selective proteolysis of the J chain component in human polymeric immunoglobulin. To clarify the losses that have been observed in the J chain portion of human IgM and IgA, were carried out studies on the enzymatic susceptibility of the J polypeptide. When Waldenström macroglobulins and myeloma IgA polymers were subjected to limited proteolysis with various endopeptidases, only subtilisin was found to attack the J chain component. The pattern of cleavage was a function of the polymer species. The J chain in IgM was highly susceptible to digestion, quantitative cleavage being achieved at very low enzyme to IgM ratios and without significant changes in the remaining pentamer structure. Analyses of the digestion products showed that the initial cleavage occurred at an exposed region midway in the J sequence and was followed by extensive degradation of the carboxy-terminal segment. These findings indicated that the observed loss of the IgM J component can be explained by the inadvertent introduction of subtilisin in vitro or by the attack of in vivo enzymes with a specificity similar to subtilisin. In contrast, the IgA  J chain was found to be much more resistant to subtilisin proteolysis; its cleavage required higher enzyme concentrations and was accompanied by significant degradation of the alpha-chains. Thus, it appears unlikely that the IgA J polypeptide is degraded by either in vitro or in vivo enzymes unless its accessibility is first enhanced by changes in the IgA Fc structure.


Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized. Spleen cells were treated with TNBS in order to determine if cell surface H-2 antigens are derivatized with TNP. By labeling the cell membrane of the TNP-modified cells with 125I, followed by detergent lysis and immune precipitation with anti-TNP, it was determined that no H-2 antigenic activity remained in the supernatant. Further, by the use of an antibody-induced antigen redistribution assay it was found that previous exposure to TNP-modified cells to anti-TNP in the absence of complement rendered these cells resistant to lysis by anti-H-2 in the presence of complement. Together these data indicate that at the concentration of TNBS used for modification, H-2 antigens are derivatized with TNP. However, in addition to H-2, other proteins including immunoglobulin were also derivatized with TNP. Anti-TNP cytotoxic effector cells were blocked from their cytotoxic activity by anti-TNP antiserum. These data indicate that TNP directly couples to H-2 antigens on the cell surface of TNP-modified cells and that TNP is associated with the antigenic determinant that the cytotoxic T cell recognizes.


Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. II. Correlation between derivatization of H-2 antigens with trinitrophenyl and the ability of trinitrophenyl-modified cells to react functionally to the CML assay. Spleen cells were modified with varying concentrations of trinitrobenzene sulfonic acid and then assayed for both their ability to stimulate syngeneic spleen cells into displaying a cytotoxic effect against TNP-modified target cells and for the extent of TNP derivatization of H-2 antigens. It was found that there was a direct correlation between the extent of derivatization of H-2 antigens and the ability of such derivatized cells to act as stimulator cells in the TNP-CML assay. Thus, these data lend support to the altered self or interaction antigen hypothesis as the explanation for the H-2 gene restriction of syngeneic CML. Target cells were also modified with TNBS at varying concentrations to determine the optimal concentration required to permit lysis in the CML assay. The results of these experiments indicate that similar concentration ranges of TNBS are required to create antigenic determinants on the target cells as well as immunogenic determinants on the stimulator cells that can be recognized by cytotoxic T cells.


Quantitative measurement of mouse IgG subclasses with the use of heteroantisera: the importance of allotype considerations. Double antibody radioimmunoassays have been used to determine the quantities of IgG1, IgG2a and IgG2b in samples of normal serum IgG from BALB/cJ, AKR/J and C57BL/6J inbred mice. The assays employed subclass-specific goat antisera which had been prepared with BALB/c myeloma proteins as immunogens and as immunoabsorbents. 125I-labeled BALB/c myeloma proteins were used as probes. Results indicate that partial resolution of mouse IgG subclasses was achieved by ion exchange chromatography on DEAE-Sephadex. Nearly all of the protein in BALB/cJ and AKR/J IgG fractions could be accounted for as IgG1, IgG2a and IgG2b, and IgG2a was the predominant species observed. However, considerably less protein in C57BL/6J IgG fractions of purity similar to the BALB/cJ fractions could be accounted for as these three subclasses, and virtually no IgG2a was detected. Furthermore, an IgG2a myeloma protein bearing the C57BL/6 allotype failed to inhibit the IgG2a-specific assay significantly. Thus the IgG2a-specific antibody in the goat heteroantiserum employed appeared to consist nearly exclusively of antibody to BALB/c Ig-1a allotypic determinants. These findings point to the importance of allotype considerations in the use of heteroantisera to quantitate IgG subclasses.


Induction of cell-mediated immunity to chemically modified antigens in guinea pigs. I. Characterization of the immune response to lipid-conjugated protein antigens. Bovine serum albumin (BSA) conjugated with a lipid, dodecanoic acid, is capable of inducing strong delayed-type hypersensitivity (DTH) in guinea pigs. This paper reports experiments on the nature and specificity of this hypersensitivity. The response to lipid-conjugated BSA (L-BSA) was found to be classical DTH, as evidenced by its ability to be transferred passively by immune cells, but not by serum. In addition, special histologic examination of skin test sites demonstrated the characteristics of DTH rather than cutaneous basophil hypersensitivity. Similar results were obtained when lipid-conjugated purified protein derivative of tubercle bacilli (L-PPD) was used. The increased immunogenicity of L-BSA was not caused by the presence of protein aggregates, but seemed to be related to the hydrophobic nature of the conjugated side chains. A series of cross-reacting serum albumins was used for a study of the specificity of the antibody and DTH responses to BSA. It was found that the degree of enhancement of immunogenicity for DTH caused by lipid conjugation varied for different antigenic determinants on BSA.


Ia antigens on murine thymocytes: detection by cellular radioimmunoassay. An indirect radioimmunoassay was used to detect Ia antigenic determinants at the cell surface of thymic and splenic cells. An A.TH anti-A.TL serum was used as a source of anti-Ia antibodies which could bind to H-2a or H-2k target cells. Bound alloantibody was then detected with 125I anti-mouse IgG1 antibody. This assay system revealed Ia antigens on thymic T cells which are not usually detectable by cytotoxicity assays, and has the potential for quantitative measurement of such determinants on cells. Critical adsorption studies were performed to confirm that Ia antigens were in fact the targets of the alloantiserum tested.


The xenogeneic effect. III. Induction of cell-mediated cytotoxicity by alloantigen-stimulated thymocytes in the presence of xenogeneic reconstitution factor. A supernatant from human mixed leukocyte cultures (MLC), Xenogeneic Reconstitution Factor (XRF), was added to one-way murine MLC. This supernatant greatly enhances the generation of cytotoxic cells from C57BL/6 or DBA/2 responder thymocytes, as assayed by a standard 51Cr-release assay. This enhancement is shown 1) to be dependent upon the presence of BDF1 semi-allogeneic stimulator cells and 2) to result in the generation of cytotoxic cells that are specific for the H-2 type of the stimulating alloantigen. In some experiments, alloantigen-stimulated murine spleen cells, when cultured with XRF showed increased induction of cell-mediated cytotoxicity. These experiments show that XRF contains a cytotoxic-cell activating factor(s) that is functionally similar to those found in murine-derived supernatants, and that this factor can participate in the generation of cytotoxic cells from precursors present in the murine thymus.


Inhibition of the mixed lymphocyte culture response with anti-Lyb-4.1 serum 1,2,. C57BL/Ks anti-L1210 serum, which recognized a non-H-2-linked B cell alloantigen, designated Lyb-4.1, specifically blocked the mixed lymphocyte culture (MLC) response to allogeneic cells that expressed the Lyb-4.1 determinant. Anti-Lyb-4,1 serum blocked the MLC response across H-2 and MLC disparities. To test that this effect was not the result of a toxic or nonspecific cell-coating action, the response of parental cells to F1 lymphocytes was studied in combinations in which only one parent expressed the recognized allele. MLC stimulation was blocked only when the responding parental cell recognized on the F1 cell H-2 or MLs disparities which were derived from the parent which possessed the Lyb-4.1 antigen. Several DBA/2 tumors were characterized by cytotoxic and quantitation absorption assays for the presence of the B cell antigen. The presence of the antigen correlated with the ability of a limited number of tumors to stimulate the MLC response of H-2d identical BALB/c lymphocytes. An increased representation of the B cell alloantigen was found on the transformed B lymphoblast cell line in comparison to splenic B lymphocytes.


Topographic determinants on cytochrome c. I. The complete antigenic structures of rabbit, mouse, and guanaco cytochromes c in rabbits and mice1. Rabbit, mouse, and guanaco cytochromes c differ from each other by only two amino acid residues. The identification is described of all of the antigenic determinants of mouse and guanaco cytochrome c that elicit an antibody response in rabbits, and those of the rabbit and guanaco proteins that elicity antibodies in the mouse. All except one of these sites center around single amino acid residue differences between the antigen and the host cytochrome c. The corresponding antibody popylations bind only to the areas of the protein in which the substitutions occur. Such antigenic determinants manifested in rabbits by quanaco and mouse cytochromes c are centered around residues 62 and 89, and residues 44 and 89, respectively. Similarly, the mouse recognizes sites containing residues 44 and 62 in guanaco cytochrome c, and residues 44 and 89 in rabbit cytochrome c. In none of these instances has a change in sequence failed to produce an antibody response. Each of these determinants appears to elicit and bind to its antibody, independently of other determinants present on the protein. In addition, two different autoantigenic responses have been detected. The antibodies produced against the determinant formed by glutamyl residue 62 of the guanaco protein in both rabbits and mice, the cytochromes c of which carry an aspartyl residue in that position, also bind to the aspartyl-containing region but with lower affinity. However, mouse and rabbit cytochrome c also elicit antibodies to the area of residue 62 in rabbits and mice, respectively, and these antibodies still bind more strongly to the glutamyl-than to the aspartyl-containing determinant. This last response occurs only when there are residue substitutions elsewhere in the molecule, because mice and rabbits fail to respond to their own cytochrome c. Antibodies produced in mice against the change from alanyl to valyl residue 44 by rabbit and guanaco cytochromes c also bind to the alanyl-containing determinant, except less tightly than to the valyl region. Conversely, antibodies raised in rabbits against the change from valyl to alanyl residue 44 only bind to this region when it carries an alanine. It is suggested that antigenic determinants that arise as a result of amino acid residue substitutions between the immunizing and the corresponding host protein, without a change in the spatial arrangement of the polypeptide backbone, be termed topographic determinants.


Effects of carrageenan on immune responses. Studies on the macrophage dependency of various antigens after treatment with carrageenan. Carrageenan, a sulfated polygalactose having macrophage toxic properties, elicited a marked suppression of IgM response to T cell-dependent antigens such as sheep red blood cells (SRBC), dinitrophenylated bovine serum gamma-globulin (DNP-BGG), and trinitrophenylated concanavalin A (TNP-Con A). In contrast, carrageenan did not inhibit antibody responses to such T cell-independent antigens as trinitrophenylated DEAE-dextran (TNP-DEAE-dextran), trinitrophenylated polyvinyl pyrrolidone(TNP-PVP), and trinitrophenylated Ficoll (TNP-Ficoll). Compared to total spleen cells, spleen cells from which macrophages had been removed by adhesion to plastic Petri dishes had less effect on the production of antibody against T cell-dependent antigens, but no change or a rather stimulated effect was observed in in vitro antibiody synthesis against T cell-independent antigens. These results strongly suggest that macrophages are involved in antibody responses to T cell-dependent antigens but not in those to T cell-independent antigens. However, the antibody response to trinitrophenylated lipopolysaccharide (TNP-LPS), a T cell-independent antigen, was inhibited by carrageenan treatment, suggesting that the response is macrophage dependent. Moreover, antibody response to higher doses of dinitrophenylated phytohemagglutinin (DNP-PHA), a T cell-dependent antigen, was shown to be macrophage independent by carrageenan treatment, although the antibody response to low doses of the antigen was macrophage dependent. Considering all these results, carrageenan treatment seems to be a very useful method to determine whether immune response to various antigens are macrophage dependent or not.


Immunosuppressive activity of IRA on the plaque-forming response to SRBC in vitro: reversal with educated cells. The alpha-globulin-rich fraction of Cohn Fraction IV, designated IRA, suppresses the in vitro antibody response to sheep red blood cells (SRBC) without cytotoxicity. IRA was effective if added before or up to 24 hr after antigen exposure. The suppression could be reversed after 24-hr treatment by washing the cells two to three times; after 48 hr of IRA treatment, however, suppression could only be partially reversed. The addition of a population of thymus-derived cells educated to the antigen SRBC could effectively reverse the IRA-induced inhibition of antibody production, whereas BSA-educated T cells could not.


In vitro secondary MLR. I. Kinetics of proliferation and specificity of in vitro primed responder cells. We have examined the kinetics and specificity of secondary in vitro mixed lymphocyte reactions (MLR). With limited numbers of primed responder cells (PRC) in the presence of "excess antigen" it was possible to obtain proliferative responses that were proportional to the number of PRC initially placed in culture. The responding cells, after an initial lag period, seem to grow exponentially until day 3 of culture. The responses of PRC (with the strain combinations and culture conditions described in this report) seemed to be directed toward stimulator cell determinants whose expression was determined by genes in the I region of the MHC. In one case, the relevant incompatibilities could be further restricted to the I-A region. Although PRC responded best to stimulator cells sharing the I region with the priming stimulator cell, apparent cross-reactivity could be observed by restimulating PRC with stimulator cells that did not carry the MHC haplotype of the priming stimulator cell. The rate of proliferation (measured as 3H-thymidine incorporation) in these apparent cross-reactions was reproducible and comparable to the rate observed in response to the priming stimulator cell. It was possible, therefore, to estimate the proportion of PRC that reacted in the presence of third party stimulator cells compared to the response of these PRC to the priming stimulator cells. We have estimated that the response of A (B6) PRC against H-2d and H-2s haplotype stimulator cells is about half of the response of these PRC to H-2b, the priming stimulator cell.


A human alloantiserum precipitates Ia-like molecules from chronic lymphocytic leukemia cells. Alloantigens specific for human B lymphocytes can be identified with selected antisera. These antigens have similarities to murine Ia antigens in that they are found on human B lymphocytes and are controlled by genes linked to genes controlling HLA. Chronic lymphocytic leukemia cells bearing B cell antigens were labeled with 3H  leucine and the membrane components reacting with the B cell antisera isolated by immunoprecipitation. These membrane components had m.w. of 33,000 and 24,000 daltons similar to the murine Ia antigens. The results complete the homology of murine Ia and human B cell alloantigens.


Experimental allergic encephalomyelitis in the Lewis rat: farther delineation of active sites in guinea pig and bovine myelin basic proteins. Highly encephalitogenic peptide (37-88), derived from the guinea pig myelin basic protein by peptic digestion, was treated chemically to destroy its tyrosyl and histidyl residues and enzymatically to remove its C-terminal sequence Val-His-Phe. Neither of the modifications resulted in loss of activity in Lewis rats. The enccephalitogenic region within peptide (37-88) was located by examination of derivative peptides obtained by selective proteolytic cleavage. The results showed that peptide (61-88), like peptide (43-88), was fully active at the level of 0.02 nmole whereas peptides (72-88) and (72-84) were fully active at levels of 0.5 and 2.5 nmole, respectively. In contrast, peptides (43-71) and (75-88) were completely inactive. These results demonstrated that the undecapeptide Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro (residues 72-84), although not as encephalitogenic as peptides (43-88) or (61-88), does contain the elements essential for the induction of disease. At the levels tested (10.8 and 2.2 nmole) only peptides (43-88) and (61-88) were capable of inhibiting the induciton of disease by passively transferred lymph node cells; this inhibition, however, was less than that achieved by the intact guinea pig basic protein. Further studies on the encephalitogenicity of the bovine basic protein in Lewis rats demonstrated that the active site in the C-terminal half of this protein is present in its entirety within residues 89 to 115.


Genetics of cell-mediated lympholysis in man. Cell-mediated lympholysis (CML) was studied in a family containing two siblings in who genetic recombinaiton had occurred in the HLA comples. In one sibling, recombination occurred between the HLA-A locus and the HLA-B locus. In the second sibling recombination occurred between the HLA-B locus and the HLA-D locus. Strong CML activity was generated in mixed lymphocyte cultures (MLC) when stimulator and responder cells differed in HLA-A, B, and D antigens. MLC involving HLA-D differences alone did not generate CML. Weak, but definite CML activity was generated during MLC with cells differing at HLA-A and HLA-B but sharing HLA-D. HLA-B antigens were good targets for lysis in all combinations studied. HLA-A antigens were poor targets in some but not in all combinations. However, combinations where HLA-A antigens seemed to be good targets could have involved HLA-B differences due to polymorphism of HLA-B7 antigens each inherited from a different parent. HLA-D antigens did not serve as targets for lysis. In three cell experiments, excellent CML activity was generated when responder cells were stimulated by HLA-D antigens and by HLA-A and B antigens present on separate stimulator cells.


Immunochemistry and polypeptide composition of hepatitis B core antigen (HBc Ag). Core particles were isolated from a nuclear extract of a hepatitis B-infected liver labeled with 125I by using chloramine-T and further purified by rate zonal sedimentation on sucrose gradients. Iodinated HBcAg was used as a ligand in a sensitive double-antibody radioimmunoprecipitation (RIP) assay for antibody to HBcAg. The specificity of the RIP reaction was evaluated using defined anti-HBc sera and paired sera from six well-documented cases of hepatitis B infection. The polypeptide composition of the iodinated antigen was examined by SDS-polyacrylamide gel electrophoresis of solubilized complexes of 125I-HBcAg and anti-HBc. Two major polypeptides with apparent m.w. of 17,000 and 35,000 daltons were observed and designated as cP-1 and cP-2, respectively.


Antigen-binding lymphocytes in guinea pigs. I.B cell expansion to the monovalent antigen L-tyrosine-p-azophenyl trimethylammonium (tyr(TMA)) in the absence of antibody production. The monofunctional antigen L-tyrosine-p-azophenyltrimethylammonium chloride, tyr(TMA), and the polyfunctional antigen, TMA-human gamma-globulin (TMA-HGG), were used to investigate the antigen structural requirements necessary for clonal proliferation of B cells. This clonal expansion was characterized with respect to receptor immunoglobulin class and affinity maturation. Antigen-binding analysis revealed that inoculation of tyr(TMA), although only of m.w. 344, triggers clonal expansion of B lymphocytes 9-fold in the absence of any apparent antibody production. There does not appear to be any maturation with respect to antibody class since greater than 90% of the tyr(TMA)-specific B cells bear the micron receptor in the nonimmune and immune state. However, the average avidity of the B cells for this antigen increases with time after immunization. In contrast, immunization with TMA-HGG results in an 18-fold increase in B lymphocytes with significant amounts of anti-TMA antibody production. With time after immunization, both maturation of average avidity and class of Ig receptor (micron leads to gamma shift) occur. These findings indicate that the functionally T cell-specific antigen tyr(TMA) can trigger clonal B cell expansion and affinity maturation at the receptor level in the absence of detectable antibody production.


Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release. The number of IgE molecules bound to human basophils was calculated from direct measurements of the IgE dissociated after exposing leukocytes to pH 3.7 acetate buffer in the cold. In 18 donors studied, cell-bound IgE ranged from 4000 to 500,000 molecules/basophil and correlated with the serum IgE concentration (r = 0.89, p less than 0.001) which ranged from 5 to 3,000 ng/ml. Sensitivity of these cells to anti-IgE was tested to explore the relationship between cell-bound IgE and the concentration of anti-IgE required for histamine release. Cells from some nonatopic donors (4000 to 100,000 IgE molecules/basophil) were as sensitive as cells from allergic donors (100,00 to 500,000 IgE molecules/basophil). Moreover, cells from donors having approximately the same cell-bound IgE concentration varied widely in their sensitivity to anti-IgE. We conclude that an intrinsic property of human basophils ("releasability") is an important parameter in determing mediator release.


Binding of affinity cross-linked oligomers of IgG to cells bearing Fc receptors. The binding of covalently cross-linked oligomers of rabbit IgG antibodies to tumor cells resembling macrophages and lymphocytes and to normal spleen cells has been measured. With all cells trimeric IgG binds with greater affinity than does the dimer, which in turn binds more tightly than does the monomer. However, as the oligomers increase in size, the individual monomeric subunits bind with decreasing energy. The macrophage tumor line. P388D1, binds oligomers with greater affinity than does the lymphocyte line, AKTB-1, but the differences in affinities are not great, differing by at most a factor of 5 in equilibrium constant. Normal spleen cells bind oligomers in the same concentration range as the tumor cells. The kinetics of binding do not occur as single first or second order reactions and suggest a multistage mechanism for oligomer binding. The presence of large concentrations of monomeric IgG tends to weaken oligomer binding and increases the exchange rate of bound oligomer from the cells. Since plasma and extracellular fluid also contain large concentrations of monomeric IgG, it is suggested that many immune complexes will bind weakly to the types of cells examined here and will rapidly exchange with IgG from the external medium.


Priming for a cytotoxic response to minor histocompatibility antigens: antigen specificity and failure to demonstrate a carrier effect. Spleen cells from normal mice do not give a detectable in vitro cytotoxic T cell (CTL) response to minor H antigens. Spleen cells from animals primed in vivo with minor H antigens give a strong CTL response when boosted in culture with the appropriate stimulating cells. Here I have studied the requirements for priming a CTL response to minor H antigens. It is shown that priming is just as antigen specific as is cytolytic effector function. That is, priming cells have to carry the same minor antigens as the challenge cells. Inducing a graft-vs-host reaction in vivo does not nonspecifically allow spleen cells to respond to minor H antigens in vitro. Using minor H congenic mice (congenic for H-Y and/or H-7) I have also tried, and failed, to demonstrate a carrier effect in priming. Female mice primed to H-Y were challenged in culture with cells bearing H-Y and H-7 antigens in the hope that a helper response to H-Y would augment a CTL response to H-7. This did not happen, however. Such primed and boosted cells gave a strong secondary CTL response to H-Y but none to H-7. It is concluded that in order to prime for a detectable in vitro response to minor antigens it is necessary to expose the CTL precursors to antigen in vivo. This either expands the size of the pool of precursors by cell division or changes them in some qualitative way.


Long-term effects of in vitro sensitization on immune reactivity of lymphocytes: generation of suppressor and helper T cells. Mouse spleen cells were cultured for 5 days with or without HRBC. Cultured cells were 'parked' in irradiated syngeneic recipients for 3 weeks and then tested for their immunologic reactivity in vitro. We found that spleen cells from recipients of HRBC-sensitized cells (S) as well as spleen cells from recipients of control unsensitized cells (U) possessed radiosensitive suppressor and radioresistant helper activities. Suppressor activity was observed by the capacity of unirradiated S and U spleen cells to inhibit the in vitro generation of IgM and IgG PFC by spleen cells primed in vivo to HRBC or to LacKLH. Helper activity was shown by the capacity of the irradiated S and U cells to restore IgM and IgG PFC responses of in vivo primed, T-depleted spleen cells to HRBC, LacHRBC, and LacCRBC. Both suppressor and helper activities were mediated by T cells. The possibilities that immunologically specific or nonspecific mechanisms account for these phenomena are discussed.


Additional rabbit IgA allotypes, f69, f70, g76,g77: control by the C alphaf and C alphag loci. Genetic and immunochemical studies have led to the identification of four additional rabbit IgA allotypes controlled by the Calphaf and Calphag loci. The folowing linkage combinations of the VHa and the 'new' alleles were observed among the populations of rabbits studied: a1f70g76, a1f69g77, and a2f69g77. Cross-reactions among g74, g76, and g77 molecules with various anti-g anti-allotype antisera indicate that the IgA-g allotypic specificities are comprised of multiple antigenic determinants. These studies provide a basis for further understanding of the evolution and gnetic control of the immunoglobulin heavy chain chromosomal region.


Genetic control of the humoral response to an H-2 public specificity. The humoral response of mice to an H-2 public specificity, termed H-2. '28' was found to be under genetic control. The genes determining this specificity were mapped to both the H-2K and H-2D regions, suggesting possible structural homologies between the products determined by these two regions. Genetic analyses indicated that a single non-H-2-linked gene regulates the anti-H-2. '28' response. In a backcross study, no linkage was detected between this putative H-2. '28' Ir gene and either the V H region or the Ly-2 locus to which the K-light chain locus is linked. Thus, a regulatory rather than a structural genetic locus seems a more likely basis for differences in response to this antigen. Our data further indicate that control of the humoral response to H-2. '28' is determinant specific since responses of the same backcross mice to other K and D alloantigens were not found to be subject to the same control.


H-2 homology requirements for secondary cell-mediated lympholysis and miced lymphocyte reactions to TNP-modified syngeneic lymphocytes. Secondary cell-mediated lympholysis (CML) and mixed lymphocyte reactions (MLR) were generated in a tissue culture system against trinitrophenyl (TNP)-modified murine syngeneic spleen cells. H-2 homology between primary and secondary TNP-modified stimulating cells was required in order to restimulate in the secondary CML. Strong proliferative responses (MLR) were detected only in the secondary cultures, for which H-2 homology was also required between TNP-modified primary and secondary immunogens. Intra-H-2 mapping for the secondary MLR indicated that the relevant regions of homology were I, D, and K and/or I-A. Homology throughout the entire major histocompatibility complex or at K plus I-A gave stronger MLR than did cultures in which there was homology between the primary and secondary phases at I or D only.


Mitogenic activity of Actinomyces viscosus. I. Effects on murine B and T lymphocytes, and partial characterization. Actinomyces viscosus homogenate (AVIS) contins substance(s) which cause spleen cells from conventional and germfree mice to undergo increased DNA synthesis. This mitogenic effect is primarily on B cells since spleen cells from nude mice or T-depleted spleen cells from conventional mice respond as strongly as conventional (T + B) spleen cells. Mouse thymocytes do not respond mitogenically to AVIS. It is unlikely that the mitogenic acitivity is due to the presence of LPS, since A. viscosus is Gram-positive and is not known to have an LPS cell wall component. Also, AVIS is not inactivated by polymyxin B, as are some preparations of LPS, and C3H/HeJ mouse splenocytes respond strongly to AVIS but not to LPS. The activity is heat stable, is not lost upon dialysis, and is not affected by lysozyme. Mitogenic activity is partially lost when AVIS is digested with nonspecific bacterial protease or treated with metaperiodate. Sodium hydroxide treatment completely abolishes mitogenic activity. Actinomycotic lesions are characterized by a long-tern inflammatory response involving a dense plasma cell infiltrate. We suggest that B cell mitogens form Actinomyces may play a role in the elicitation of the plasma cell component of these lesions.


In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors. Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.


Ala-1: murine alloantigen of activated lymphocytes. II. T and B effector cells express ala-1. Ala-1 (activated lymphocyte antigen-1) is a murine alloantigen expressed only on activated peripheral T and B lymphocytes. The presence or absence of Ala-1 on specific functional lymphocyte subsets was determined by treating the relevant cell population with anti-Ala-1 and complement, and assaying for residual functional activity. By this method, Ala-1 was shown to be on in vivo primed killer T cells cytotoxic for allogeneic tumor cells. It was also found on helper T cells generated in vivo to sheep red blood cells, and on IgM and IgG plaque-forming cells (PFC) to sheep red blood cells. In contrast, splenic precursors of helper cells and of IgM PFC to sheep red blood cells were completely resistant to treatment with anti-Ala-1 and complement. These findings indicate that effector cells can be distinguished from their nonactivated precursors by their expression of Ala-1.


Substrate specificity of antinuclear antibodies in scleroderma. Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) and 4 of mixed connective tissue disease (MCTD) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guineapig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE and MCTD, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate, Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.


Rapid detection of antibodies to cytomegalovirus by counterimmunoelectrophoresis. A new method for the detection of precipitating antibody to cytomegalovirus by counterimmunoelectrophoresis (CIEP) is described. Fourteen of 15 adult sera (95%) with IgM-specific antibodies (as detected by immunofluorescence) and complement-fixing antibodies to cytomegalovirus in high titers gave positive reactions by this method. Control sera from 156 patients and 40 normal subjects were negative by CIEP. One of 32 individuals acutely infected with other members of the herpesvirus group gave a positive reaction (3%). False-positive reactions were restricted to a group of sera containing rheumatoid factor (10 of 31), but this activity could be eliminated by preliminary absorption of the sera with aggregated gamma-globulin. The present findings demonstrate that CIEP is useful for the detection of precipitating antibodies to cytomegalovirus in sera from acutely infected patients and promises to be a rapid, inexpensive screening procedure of diagnostic value.


Osteosarcoma of the upper jaw.

Osteosarcoma of the upper jaw. 8 cases of osteosarcoma of the upper jaw from a personal series of tumours are described, and their treatment discussed in the light of past experience. Of the cases, four are still alive and well 6 months to 11 years after treatment. A case is put forward for the initial use of radical surgery, especially maxillectomy with orbital exenteration. The addition of routine postoperative Adriamycin therapy is recommended in the light of its known activity against sarcomas. The palliation of advanced cases is also discussed.


Bleomycin-methotrexate-chemotherapy in combination with telecobalt-radiation for patients suffering from advanced oral carcinoma. 20 patients suffering from advanced oral carcinoma (T3NXMO) were treated simultaneously with the cytostatics Bleomycin and Methotrexate and Telecobalt-radiation. 14 of them (70%) showed complete remission. Four relapses were treated by surgery. These patients are still alive and tumour-free. One patient with a tumour is also still alive. Five died of their tumours or other causes. As a preliminary result, overall survival of about 80% after the first year seems to be much higher than with other therapeutic procedures.


Evolution of human cytogenetics: an encyclopedic essay. III. The second decade after 1956: banding techiques. Unequivocal establishment of the correct diploid chromosome number in 1956 started the modern era of human cytogenetics. The next impetus came when the peripheral blood leukocyte culture technique for the chromosome preparation was described in 1960. Discovery of special staining procedures - banding techniques - in early seventies not only saved it from early senescence but played decisive roles in broadening the horizons of modern human cytogenetics.


Reticulum stain for frozen-section diagnosis of pituitary adenomas. Technical note. The authors describe a simple, quick, and reliable stain for use on frozen-section diagnosis of anterior pituitary microadenomas.