Delayed response of ankle jerk time changes in decreasing thyroid function in comparison with the serum cholesterol changes. 1. Serum cholesterol, PBI and ankle jerk time (AJT) were measured simultaneously in a group of not yet treated patients, where a relatively steady state of thyroid function was expected, and in a treated group, where change to lower thyroid function was supposed (thyrostatic drugs, near total thyroidectomy, radioablation of thyroid). 2. Serum cholesterol correlated poorly with PBI (r = --0,4427), better correlation was achieved after logarithmic transformation of PBI values (r = --0,6839). 3. Reference line and covariance between log PBI and serum cholesterol in decreasing thyroid function did not differ significantly from reference line and covariance in steady state, showing that serum cholesterol accompanies closely and without greater delay the changing PBI. Great dispersion of individual values shows a relatively low diagnostic value of serum cholesterol for assessment of functional thyroid state in individual patients. Early reactivity of serum cholesterol level and highly significant correlation with log PBI makes this parameter useful for longitudinal evaluation of therapy in individual patients. 4. Reference line between AJT and serum cholesterol in decreasing thyroid function was shifted to lower AJT values, showing a delay of response of AJT. Tested by analysis of covariance this delay was marked in young patients (0,5% level of significance), present in medium age (5% level of significance), not detectable in person over 60 years. 5. Delay of AJT changes in decreasing thyroid function is important for clinical practice. Our findings are further in accordance with the assumption, that subcellular systems in skeletal muscle may be responsible for delayed response of AJT, being more important than concomitant changes in serum T3/T4 ratio.


[The metabolism of 4-14C-pregnenolone in tissue sections of human ovaries and their modification by chlormadinone acetate]. In vitro incubations with slices of two normal human ovaries and 4-14C-pregnenolone as precursor were carried out to study the possibility of a direct influence of chlormadinone acetate on the metabolism of pregnenolone. In agreement with our previous studies the incubations of the ovary from the follicle phase of the cycle yields a profile of steroids different from that of the ovary from the corpus luteum phase of  the cycle. Under the experimental condition chosen, the presence of enzymes of the steroidogenic pathway responsible for the synthesis of 17alpha-hydroxy-pregnenolone, DHA, androstenediol (basic metabolites) and androstenedione represents a characteristic profile of steroids of the ovaries from the follicle phase. After the addition of chlormadinone acetate to the incubation medium, the formation of androstenedione was inhibited, whereas the basic metabolites increased. The biosynthesis of progesterone, 17alpha-hydroxyprogesterone, estrone and estradiol represents a characteristic profile of steroids of the ovaries from the corpus luteum phase. After a addition of chlormadinone acetate to the incubation medium, the formation of this characteristic profile of steroids was inhibited. The influence of chlormadinone acetate on the two different profiles of steroids indicated, that chlormadinone acetate exerts an inhibitory effect on the 3beta-hydroxysteroid-dehydrogenase-delta5-4-isome


An apparently direct inhibitory effect of oestrogen on the human testis. In men suffering from prostatic cancer, i.v. administration of 12 g diethylstilboestrol diphosphate within 20 days resulted in a decrease of the LH serum level to about 50%  (P less than 0.05), whereas the total testosterone level decreased to less than 5% (P less than 0.001) and the apparently free testosterone level to less than 2% of the initial values (P less than 0.001). Hence, the "systemic antiandrogenic effect" of oestrogen can be explained (1) by indirect inhibition of testicular androgen secretion via diminution of hypophyseal gonadotrophin secretion, (2) by direct inhibition of testicular androgen secretion and (3) by elevation of the capacity of testosterone binding beta-globulin.


Pathogenesis of multiple sclerosis. Neurochemical and morphological studies on MS biopsies and autopsies. In the present study, six multiple sclerosis (MS) brain white matter biopsies were analyzed both morphologically and chemically. The purpose of this study was to test the reliability of the earlier postmortem observations. Special attention was given to proteins which were analyzed in polyacrylamide disc gel electrophoresis and to acid proteinase. The results show that myelin basic protein (BP) was present in all biopsies, although advanced demyelination was seen in one case as based on morphology. This finding is controversial to earlier observations. In addition to this, some acid proteins were decreased or lost in two cases. The activity of acid proteinases was increased from two- to fivefold in four of six cases. This increase of the activity was seen also in the normal-appearing MS autopsy white matter which served as control. The source of this activity is discussed. On the basis of the present results it is likely that the role of the BP in the breakdown of myelin in MS and the role of acid proteinases has to be studied in more detail.


Effect of Ca++ on Vmax measured in absence of external or internal load. Myofibrils 1 sarcomere wide (single myofibrils) were prepared from rat heart. Mean initial sarcomere length was 2.68 mu. Contraction was initiated with 20, 25 or 100 muM MgATP and terminated with EGTA and EDTA. Single fibrils shortened at the theoretical Vmax for the in vitro conditions until sarcomere length reached 2.05 mu. At this length they encountered a significant internal afterload. Reaction time, substrate, and temperature were adjusted so that final sarcomere length was greater than or equal to 2.05 mu in most experiments. Ca++ increased Vmax of single fibrils 2- 4-fold. Results were similar at 20 and 100 muM MgATP, and in reaction mixture containing 100 or 140 mM KCI. We conclude that Ca++ controls not only the number of cross-bridges, but also the rate at which the cross-bridges turn over.


Formaldehyde-Schiff's reagent as a nucleolar strain. The use of formaldehyde-Schiff's reagent as a nucleolar stain has been described. Using different digestion procedures, it was confirmed that the stain is specific for RNA. It can be suitably used as a nucleolar stain, particularly in plant materials after a short TCA extraction, which probably extracts the nonbound RNA.


[Mechanism of action of vasodilator agents on the collateral blood flow into an area of acute myocardial ischemia]. In acute experiments staged on dogs subject to investigation was collateral coronary circulation studied by registering the retrograde blood flow and the pressure in the ligated coronary artery. It is shown that atropinization (0.05 mg/kg) does not change the positive action of papaverine and diethyphen (2 mg/kg) on the inflow of blood into the area of myocardial ischemia and enhances the beneficial effect on euphylline (4 mg/kg). Preliminary introduction of octadine (5 mg/kg) had no material influence on the nature of the diethyphen action, but greatly weakened that of euphylline and reduced the effect of papaverine. The vasodilating agents attenuated for a short while a pituitrin-induced (0.5 U/kg) spasm of the collaterals and vessels in the ischemic area of the myocardium.


[Effect of uranium on iodine metabolic kinetics in the thyroid gland (short-term intervals)]. Administration of uranium nitrate to rats is seen to be followed by a rise of the radio-iodide organic transformation constant and by a reduction in the proportion of the radio-iodide fraction released into the blood from the throid. The ratio glandular radio-iodide/plasmic radio-iodide decreases.


[Experimental study of the catatoxic effects of steroid hormones]. Tests set up on mice and rats brought evidence that spironolactone, progesterone, testosterone-propionate, methylandostendiol, desoxycorticosterone-acetate, methyltosterone, diethylstilbestrol, preparation PCN, megesterol-acetate and mestranol disply a catatoxic action, whose degree depends upon the method of evaluation and the animal species. A pattern for selection of catatoxic agents is proposed and some aspects of their mode of action are discussed.


Antigen-specific T-cell factor in cell cooperation and genetic control of the immune response. Cell interactions between thymus-derived (T) and bone marrow-derived (B) lymphocytes in the antibody response appear to involve soluble T-cell mediators known as 'factors.' This paper describes the properties of a T-cell factor that has specificity for the inducing antigen, a synthetic polypeptide (T, G)-A--L, and is able to replace T cells in the thymus-dependent antibody response to (T, G)-A--L. Besides antigen specificity, the main features of the molecule are that it is nonimmunoglobulin; it has a molecular weight of about 50,000; and it is a product of the I-A subregion of the H-2 complex (the mouse major histocompatibility complex). These properties suggest that the factor is closely related to the T-cell receptor, which may, by inference, also be a product of the H-2 complex. The factor cooperates well with allogeneic B cells. It can also be absorbed by bone marrow cells and B cells. Studies on the genetic control of the immune response to (T, G)-A--L using the T-cell factor indicate that two immune response genes in the H-2 complex are involved in genetic control, one expressed in T cells and the other in B cells. This two gene hypothesis has been confirmed by showing that an F1 between two low responders to (T, G)-A--L can be a high responder.


Detection of surface and core antigens of hepatitis B virus in the liver of 164 human subjects. A study by immunoperoxidase and orcein staining. The surface (HBsAg) and core (HBcAg) antigens of hepatitis B virus (HBV) have been searched by optic microscopy in the liver specimens from patients hospitalized for various conditions and from 38 HGsAg chronic carriers. The study was done blindly using Shikata et al.'s orcein staining on fixed and frozen material and direct immunoperoxidase on frozen material with antisera specific for surface (anti-HBs) and core (anti-HBc) antigens of HBV. No liver staining could be found in the 98 HBsAg seronegative patients. Among the 28 HBsAg seropositive patients, only 3 showed positive staining: 1 patient with acute viral hepatitis showed nuclear staining with anti-HBc; 2 patients with postnecrotic cirrhosis showed cytoplasmic staining with anti-HBs and/or orcein, and one of them also showed nuclear staining with anti-HBc. In contrast, among the 38 chronic carriers, 25 showed positive cytoplasmic staining with anti-HBs and/or orcein, while one of them (with chronic aggressive hepatitis) also showed nuclear staining with anti-HBc. Anti-HBs and orcein staining are equally sensitive and specific for the detection of HBsAg in hepatocytes; discrepant results can be attributed to sampling error of distribution of HBsAg in small liver fragments.


F-antigen: nature, liver specificity, and release in experimental liver injury. F-antigen is a liver-specific antigen detected with antibody, raised in allogeneic (CBA) mice, to BALB/c mouse liver extract. The authors have confirmed the hepatic specificity of this antigen by showing it to be absent in extracts of extrahepatic organs of mouse and human origin. It is present in liver extracts of guinea pigs, rats, and rabbits, as well as in liver extracts from mouse and human sources. The antigen was present in the circulation of rabbits with acute carbon tetrachloride-induced hepatocellular injury. It was also demonstrated in the sera of 3 of 8 patients with acute hepatitis B antigen-positive hepatitis and in 1 of 4 patients with chronic active hepatitis. It was absent from the sera of 121 other patients with a variety of hepatic and nonhepatic diseases, and from the sera of 20 healthy subjects. The antigen is immunologically distinct from hepatitis B antigen, from the liver-specific lipoprotein antigen LP-2 of Meyer zum Buschenfeld, and from each of 15 individual human serum proteins tested.


[Investigations on the accumulation of radio-bleomycin in gynaecological tumors (author's transl)]. The indication for treatment with Bleomycin in gynaecological tumors is as yet not well defined. At present Bleomycin is primarily used for advanced gynaecological malignancies. Results of our investigations show that the individual affinity of gynaecological tumors to Bleomycin is very variable. In subsequent investigations it should therefore be determined whether the accumulation of radio-Bleomycin and the sensitivity of a gynaecological malignant tumor to Bleomycin therapy show a positive correlation. The probable chances of success of a course of chemotherapy with Bleomycin could then be predicted.


[Paraproteinemia in chronic lymphocytic leukemia]. In 10 from 305 patients affected with chronic lymphatic leukaemia a paraprotein was identified by paper electrophoresis in the blood, namely IgG 5 times, IgM 3 times, IgA once and 2 paraproteins IgG and IgM once. The paraprotein content laid between 0.24 and 2.0 g %. During a long-time observation the paraprotein concentrations varied only insignificantly and independently of the number of lymphocytes. In one patient M-gradient disappaered after 2 years. In 8 patients protein could be proved in the urine, in 5 of them 25-65% of the urine protein consisted of light chains. The course of the disease in these patients affected with paraproteinaemia scarcely differed from those patients without this protein anomaly. The conclusion may be drawn from this that the cell clon producing paraprotein is little aggressive.


Studies on blood components of an air-breathing siluroid fish, Heteropneustes fossilis (Bloch) in relation to body weight. Different haematological parameters have been studied in relation to the body weight of Heteropneustes fossil (Bloch). The erythrocytes and leucocytes number and haemoglobin concentration increases from lower to higher weight groups. The heart weight also increases along with other blood components. With the unit increase in the body weight of this fish, the heart weight, erythrocytes, leucocytes and haemoglobin increase by a fractional power of 0.85700, 0.13480, 0.13215 and 0.22876, respectively. This shows that haemoglobin increases at a higher rate than erythrocyte number. The coefficient of correlation between body weight and erythrocyte (r = 0.70015), leucocytes (r = 0.95861), haemoglobin (r = 0.96615) and heart weight (0.97577) indicate high degree of correlation. The erythrocytes and leucocytes count and haemoglobin concentration per gram body weight is higher in younger fishes and decrease as the animal grows in size. The haematocrit values and mean corpucsular volumes decrease from lower to higher weight groups, whereas mean corpuscular haemoglobin concentration increases with body weight. The erythrocyte cell surface has a decreasing trend from lower to higher weight group. Due to difference in the rate of decrease of greatest and least diameter of erythrocytes, the elliptical shape of R.B.C. which is common in younger animals, becomes circular in higher weight group. The non-granulocytes increase constantly while the percentage of granulocytes decreases from lower to higher weight groups. The lymphocytes constitute the main bulk of all the leucocytes. The total lymphocytes also increase with the body weight. Spindle cells and monocytes are relatively less in numbers. The percentage of eosinophils, basophils and neutrophils also decrease from lower to higher weight groups.


[The search for hematologic-immunological parameters with prognostic value in children with various tumors]. In a total of 35 children affected with malignant sufferings (except leucaemias) the haematological-immunological parameters of children surviving for a short time are compared with those surviving for a long time. Compared with children surviving for a short time those surviving for long time had a more "normal" finding initially and during the control examination made after more than 5 years of a survival time which was free of complaints and recidives. The immunologically intact condition of the organism is assumed to be decisive for this course of disease.


[Studies on the immunogenic activity of the Bua antigen]. The fact that 14 persons after contacting Bua-antigen did not develop anti-Bua, that 5 Bua(-) mothers with Bua(+) children did not carry anti-Bua and that 10 rabbits with a long immunization did not produce any heteroanti-Bua either, leads to the conclusion, contrary to previous researchers, that Bua-antigen has only a weak immunizing effect. Therefore, the use of the blood of such blood donors seems to have no enhanced risk of immunization for the patient.


[Immunological aspects in the L-asparaginase treatment of children with lymphoproliferative diseases]. A group of 20 children, including 14 with acute lymphoblastic leukemia and 6 with lymphosarcoma, was studied. 24 cures of l-asparaginase therapy were carried out. The increase of serum immunoglobulin (IgG, IgA, IgM) levels was found in children treated with smaller (from 300 to 500 I.U./kg b. w.) doses of asparaginase. In the group treated with higher doses (from 501 to 760 I.U./kg b. w.) the maximal increase of immunoglobulins was observed in the second half of the cure with l-asparaginase, followed by a decrease of the immunoglobulins levels at the end of treatment. The presence of anti-asparaginase antibodies in two children with anaphylactic shocks after l-asparaginase has been shown. In these two children and 6 others the lymphocyte count significantly dropped down on the day of shock before l-asparaginase injection.


[Cytogenetic studies following immune induction therapy]. This work deals with the question whether the immunisation with BCG-vaccine and leukemic cells leads to morphological changes of the chromosomes from children with acute leukemia. In respect of the small number of patients, and of the variant numbers in the single groups it is concluded, that immunological induction therapy is a possible gravitating factor in manifestation of isolate breaks and complex aberrations. It is not known whether one of them or both the components would be necessary, whether dosage and duration of vaccination have any influence, and how long after vaccination the gravitating influence may be seen.


[Cytogenetic studies after contact with juvenile blastic leukemias]. Persons with long professionell contact to acute childhood leukemia and first-degree relatives had a higher tendency of chromosome bursting off in the metaphases from cultivated lymphocytes. Since persons with contact and relatives for one thing and controls for another showed significant differences in the degree of hurtibility of metaphases, cultivation ane preparation could not be responsible for themselves. It is discussed whether the contact to childhood leukemia may lead to any reaction between peripheral lymphocytes and an infectious agent. This reaction itself leads to a higher susceptibility of metaphases from peripheral lymphocytes to the injuring laboratory conditions.


The lysosomal enzymes of lymphocytes in the premature and the term infant. Activity of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase was determined in peripheral blood lymphocytes of 29 premature and 20 term infants with the use of cytochemical methods. The results were expressed semiquantitatively and included the total count of enzyme-positive and the enzyme-negative lymphocytes as well as the intracellular content of enzyme-positive and enzyme-negative lysosomal granules. The premature infant exhibited significantly lower activity of all the studied enzymes than the term infants. It thus argues in favour of the opinion that the lysosomal apparatus in lymphocytes undergoes development in the course of fetal maturation of the immune system. Evaluation of the activity of lysosomal enzymes in lymphocytes can serve as an indicator of fetal maturity and immunological status.


[Cellular immunity in newborn infants with hyperbilirubinemia]. In order to identify the possibility of prenatal or perinatal bacterial contact with immunization of the cellular immunity system as underlying cause of the "idiopathic" newborn icterus (without blood group incompatibility) the lymphocyte transformation test with addition of streptolysin O or E. coli antigen was carried out in 68 newborns with a birth weight ranging between 1260 and 4200 g. The sensitization rate identified among the newborns with hyperbilirubinaemia did not differ significantly from those of the control group. Thus an ensured connection between a prenatal streptococcus or E. coli contact and the appearance of an idiopathic newborn hyperbilirubinaemia could not be established.


Nitroblue tetrazolium (NBT) test: criteria for false positivity and its use in practice. The criteria for false positivity of the NBT test were described including absence of classical clinical signs of bacterial infection, negative blood (and, if necessary, other) cultures, and lack of response of antibacterial treatment as the basis for appreciation of positive NBT test result as false-positive. A case of acute lymphoblastic leukaemia with all the criteria being fulfilled was described.


[On the production of anti-human-lymphocyte serum (ALS) by means of combined local and intravenous immunization in swine]. Lymphocytes of donors' peripheral blood were used for the study of pig antihuman ALS preparation by means of local and intravenous immunization. The results were compared with those obtained with exclusively local application of the same antigen. Antisera were tested from the aspect of activity (lymphoagglutination, lymphocytotoxicity, the rosette inhibition test) and for the presence of unwanted antibodies (haemagglutinins, thromboagglutinins, precipitins to serum proteins). Of all alternatives tested the following scheme proved to be optimal: subcutaneous application of 2 X 10(9) lymphocytes with adjuvans on day 0, followed by intravenous application of the same dosage on day 13, and serum harvesting on day 20. Antisera thus obtained displayed titres 1: 32 000 to 1 : 130 000 in the RIT - this being maximum level reached by exclusively local immunization only in some animals. Therefore, favourable immunosuppressive effect in vivo can be expected. Even more profound difference was observed in the level of unwanted antibodies that were found to be on markedly lower level after combined immunization. There is a good reason to suppose that with the help of further techniques, more likely absorption, these can be lowered to acceptable level. It is apparent from the results that with the help of a suitable immunization procedure not only highly active ALS can be obtained but also formation of nondesirable antibodies can be suppressed and, despite their thrombocyte contamination peripheral blood lymphocytes can successfully be used.


[Studies on cryoprecipitates. 5. Proteinchemical studies]. The total proteins are enriched 2.41 fold by cryoprecipitation. The protein fractions, however, have the same distribution as in the original plasma. With the help of radial immunodiffusion method haptoglobins were measured to have enriched to 351%, IgG to 252%, IgA to 307%, and IgM to 268%. The simultaneous increase of functional properties was systematically observed by taking the complete ABO-isoagglutinin titre as an example. The titre increase moves within the range of 1 to 2 degrees.


Factors controlling IgM and IgG antibody responses to denatured trypanosome DNA. Rabbits immunized with complexes of denatured calf thymus DNA and methylated bovine serum albumin (MBSA) produced only IgM antibodies to denatured DNA, confirming the results of previous studies. In contrast, both IgM and IgG antibodies were induced by MBSA complexes of Trypanosoma brucei DNA. A similar response was observed by Gruenewald and Stollar (1973) to T2 bacteriophage DNA, in which the normal base cytosine is replaced by hydroxymethylcytosine. T2 bacteriophage DNA is known to have a high adenine+thymine (A + T) content. It is suggested that the 7S antibody response to MBSA complexes of T. brucei DNA might be linked to the high A + T content in the DNA (especially kinetoplast DNA) of the subgenus Trypanozoon.


Comparison of in vitro immunogenicity, tolerogenicity and mitogenicity of dinitrophenyl-levan conjugates with varying epitope density. The mitogenicity, immunogenicity and tolerogenicity of various DNP-levan (DNP-LE) conjugates were compared using in vitro methods. Anti-DNP antibody synthesis induced by DNP-LE conjugates was related to the epitope density of DNP, BUT WAS NOT AFFECTED BY Macrophage dependent and was not influenced by the degree of hapten conjugation. These results imply that mitogenicity of an antigen is not necessarily related to the specific triggering of B cells.


Hapten-specific responses to contact sensitizers. Use of fluorodinitrobenzene to elicit migration inhibition and macrophage agglutination factors from lymph node cells of contact-sensitive guinea-pigs. Hapten-specific sensitivity of guinea-pigs sensitized to dinitrophenyl (DNP) contactants and to DNP-protein conjugates was investigated by skin test and by antigen-induced elaboration of migration inhibition (MIF) and macrophage agglutination factors (MAF) From lymph node cells. The delayed contact reaction was highly specific for low doses of contactant and markedly less so for conjugates; lymph node cells elaborted both lymphokines in response to brief exposures to dinitrofluorobenzene (DNFB) or prolonged exposures to DNP conjugates. Elicitation of MAF by DNFB or DNP conjugate was inhibited in the presence of DNP glycine; the activity of MAF induced by DNP conjugate (but not that induced by DNFB) was inhibited in the presence of DNP-glycine as well. These results suggest that contact sensitivity to DNP conjugates reflect two different types of hapten-specific cellular sensitivity mediated by populations of cells with different antigen receptors and possibly, functionally different lymphokines.


Cellular and humorial immune responses in mice. III. Acceleration of delayed hypersensitivity response by presensitization with suboptimal dose of antigen. Delayed hypersensitivity (DH) response in mice induced by subcutaneous (s.c.) injection of optimal dose of sheep red blood cells (SRBC) 10(8)) was accelerated by s.c. injection of the antigen of 10(3) or more doses, given 2 or more days earlier. The accelerated response appeared soon after the injection of optimal antigen dose, that is, 1 or 2 days earlier than the response of non-presensitized control. The acceleration was antigen specific. The accelerated response was generally accompanied by an acceleration and/or enhancement of humoral antibody response. Parallel to the acceleration of DH response, the proliferation of regional lymph node cells in the presensitized mice was induced immediately after the following injection of 10(8) SRBC, 1 day earlier than that of non-presensitized animals. These results suggest that presensitization of mice with the antigen induces DH-related memory cells which proliferate immediately after the following injection and function as effector cells for DH reactions, and that the development of DH-related memory cells occurs in close relation to that of helper thymus-derived (T) cells for antibody production.


Antigen-non-specific T-cell factor in B-cell activation. Origin, biological properties and failure to show a relationship to H-2. A population of T cells sensitive to ALS treatment release a non-specific factor (NSF) capable of replacing T cells in the response of nude spleen cells to erythrocyte antigens. This factor cannot be removed by immunoadsorbents with specificities directed towards products of the H2 complex, nor by lentil lectin, unlike certain specific T-cell factors. Furthermore, it functions across histocompatibility differences between mouse strains. It has been directly demonstrated that this factor can influence both proliferation and differentiation of B cells in response to a restricted group of T-dependent antigens, of which donkey and sheep erythrocytes are such special cases.


The natural heterohaemagglutinin in the serum of the toad Bufo regularis, and its relationship to lower vertebrate immunoglobulins. The serum of the toad Bufo regularis contains a natural heterohaemagglutinin for human erythrocytes, Which appears to have anti-(B + HP) specificity. Results of inhibition and absorption experiments indicate that only one agglutinin is present. The biochemical specificity of the agglutinin may be provisionally described as involving alpha-D-galactose residues linked (1-3) in the B determinant, of red cells possessing the H ANTIGEN. Unlike amphibian IgM, the agglutinin was insensitive to 2-mercaptoethanol treatment; moreover, it could be eluted from the alpha1 globulin region on cellulose acetate electrophoresis. These results suggest that this naturally occurring heterohaemagglutinin has a structure similar to that of plant and animal lectins. The relationship of this observation to the phylogenetic evolution of immunity is discussed.


Disaster procedures report. Report following the Moorgate train crash on 28 February, 1975. There is no substitute for the use of intelligence and common sense both in the drawing up and interpretation of a disaster plan; for compromise in dealing with other rescue services; for ingenuity in filling the gaps in the equipment with which you find yourself provided; and, finally, perhaps most important, for self-discipline. None of us is indispensible--there are always others equally able to make decisions.


Studies of mycobacterial antigens, with special reference to Mycobacterium leprae. Eight individual antigens were detected in soluble antigen preparations from Mycobacterium leprae bacilli by using pools of serum samples from lepromatous leprosy patients as antibody reagents in crossed immunoelectrophoresis. Two of these antigens were analyzed further. Antgent no. 1 gave an elution pattern on Sephadex G-200 corresponding to a molecular weight of 285,000. This antigen was also present in three slow-growing and eight fast-growing mycobacterial species. There was a reaction of complete identity in immunological tests using lepromatous serum pools as well as with rabbit antisera raised against M. leprae and M. smegmatis. Antigen no. 21 of M. leprae showed antigenic heterogeneity when compared with other species. Three types of antigenic determinants were detected; one, called 21A, was shared by all mycobacteria, another, called 21B, was limited to antigen no. 21 of M. leprae; a third, called 21C, was present in all mycobacteria except the leprosy bacillus. This submolecular heterogeneity may indicate a separate taxonomic position of M. leprae among the mycobacteria.


Increased vascular permeability during passive peritoneal anaphylaxis in the rat. The effects of disodium cromoglycate and a nitroindanedione. Following intraperitoneal sensitisation of rats with rat serum containing reaginic antibody, intravenous injection of blue dye and intraperitoneal challenge with antigen caused a release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and dye into their peritoneal fluids. The times taken to reach peak concentrations after challenge were less than 2 min for histamine and between 5 and 10 min for SRS-A, whilst concentrations of dye were still increasing after 2 1/2 h. The amounts of histamine released by antigen were sufficient to account for about 60% of this extravasation of dye. Disodium cromoglycate (DSCG) and a nitroindanedione (BRL 10833) inhibited extravasation by inhibition of mediator release. BRL 10833, unlike DSCG, was active after oral administration, and for a given inhibition of histamine release it produced a greater effect on extravasation when given orally than when injected intraperitoneally.


Reactive and nonreactive lymphocytes in experimental allergic encephalomyelitis. I. Possible role of macrophage-lymphocyte interactions. Previous studies have shown that peritoneal inflammatory exudate cells from guinea pigs with experimental allergic encephalomyelitis proliferate prominently when cultured with the sensitizing myelin basic protein. Peripheral blood lymphocytes (PBL) obtained at the same time responded little, if at all. In the present studies, recombination experiments using appropriate mixtures of peritoneal exudate macrophages and PBL show that the presence of such macrophages will not enhance in vitro reactivity of the PBL to basic protein. The oil-induced peritoneal inflammatory response appears to deplete the PBL somewhat of antigen-reactive lymphocytes, but does not totally explain the difference in in vitro responsiveness between the lymphocytes in the peritoneal exudate and in the peripheral blood.


The mechansim of tachyphylaxis to ICI 74,917 and disodium cromoglycate. Pre-incubation in vitro of sensitised peritoneal mast cells for 10 min with either ICI 74,917 (10-5 M) abolished the ability of either drug to inhibit histamine release when subsequently presented to the cells at the same time as antigen. In the case of disodium cromoglycate, tachyphylaxis was abolished by washing the cells after pre-incubation with the drug. The failure to abolish tachyphylaxis to ICI 74,917 was due to the high pre-incubation concentration employed, as at lower concentrations (10-8 M) tachyphylaxis to ICI 74917 was readily abolished by washing. Tachyphylaxis to these anti-allergic agents may be related to a physical blocking of drug receptor sites on or in mast cells.


Presence and anatomic location of species and organ-specific intestinal antigens. Several groups of antigens were detected in the ileum of rabbits, rats, and guinea pigs. An antigen present in the cytoplasm of the epithelial cells is shared by these species, but is not tissue-specific because it can be absorbed by the liver. Organ-specific ileum antigens are present in the goblet cell mucus. There are different ileum-specific antigens in the three species and they do not elicit autoantibody formation. Additional antigens are located in the basement membrane and some basement membrane antigens are derived from exogenous sources.


IgE antibodies in patients allergic to rifampicin. In sera of patients treated with rifampicin, who had episodes ascribable to rifampicin sensitization, IgE antibodies were found. These antibodies cross-react with rifamycin SV and with the chromophoric moiety of rifamycins, but not with the side chain of rifampicin.


Prompt elevation of rat serum alpha-fetoprotein by acute liver injury following a single injection of ethionine. The mechanism of increased alpha-fetoprotein (AFP) production following a single injection of ethionine was investigated by using rats aged 5 weeks at the time of killing. Marked elevations of serum AFP concentrations occurred within 4 days in both male and female rats after administration of DL-ethionine or L-ethionine, although the increased levels of serum AFP and liver triglyceride in the adults were less marked in the male than in the female. No apparent necrosis of liver cells was observed in ethionine-treated rats. Frequent administrations of adenosine triphosphate after a single dose of ethionine prevented the increases in liver triglyceride and serum AFP levels. The increased concentrations of serum AFP, reaching a maximum level within 4 days, occurred before a slight  increase in incorporation of 3H-thymidine into liver DNA. The serum AFP from ethionine-treated rats was immunologically and electrophoretically indistinguishable from that of fetal, carbontetrachloride-treated or hepatoma-bearing rats. These observations suggest that the increased production of AFP in ethionine-treated rats is closely associated with hepatic injury and is not the consequence of liver cell regeneration.


Mycobacterial antigens in antibody responses of leprosy patients. A reference system for M. smegmatis antigens in crossed immunoelectrophoresis was used to study antibody activities in serum samples of 91 leprosy patients. All polar and borderline lepromatous patients were positive. Mean numbers out of 14 M. smegmatis antigens involved were 4.3 and 3.5, respectively. Precipitins against antigen no. 1 were seen in all lepromatous cases. Antibodies against this antigen were detected in 50% of tuberculoid (polar, subpolar and borderline) cases. Antibody activity against M. avium and M. duvalii antigens was also detected using a staphylococcal radio-immuno-assay. Borderline and polar lepromatous cases showed elevated levels. Antigenic comparisons were made between four slow growing mycobacteria, fourteen fast growing mycobacteria and the leprosy bacillus using lepromatous serum pools as antibody reagents. Four of the antigens detected in M. leprae were also found in slow growing as well as fast growing species indicating a common occurrence among mycobacteria. Antigen no. 1 of M. duvalii, with an apparent molecular weight of 290,000, showed nonprotein characteristics. Further analysis of antigen no. 21, using lepromatous serum pools as antibody reagents, indicated the existence of at least two groups of antigenic determinants. In addition to determinants shared by all mycobacteria, there were antigenic structures apparently unique to M. leprae.


The effect of interruption of the short posterior ciliary arteries on slow axoplasmic transport and histology within the optic nerve of the rhesus monkey. Tritiated leucine was injected into the vitreous of rhesus monkey eyes to make it available for protein synthesis by the ganglion cells. The short posterior ciliary arteries were cut three hours later or several weeks prior to the leucine injection. A reduction of labeled protein within the retrolaminar optic nerve was seen in all eyes so treated. Autoradiography revealed a diffuse reduction of axoplasmic transport into these optic nerve heads. There was consistent evidence of focal obstruction of labeled protein at the interface between the lamina scleralis and retrolaminar optic nerve. Vacuoles appeared in the most severely affected areas. These histologic changes were followed by gliosis in the areas of ischemic damage. Glaucomatous cupping of the optic nerve head was not seen within six weeks following the induced ischemia.


Acid-fast properties and pyridine extraction of M. leprae. The reportedly unique pyridine extractability of acid-fastness as an identifying characteristic for M. leprae was examined in the leprosy bacilli and in eight other strains of mycobacteria. The initial findings were, in general, in accord with previous reports except that M. smegmatis and M. phlei likewise demonstrated two hour pyridine extractability of acid-fastness. Perhaps, more significantly, it was found that this characteristic in M. leprae is related to aged, probably nonviable bacilli. Some other strains of mycobacteria when tested in aged cultures showed the same phenomenon while M. leprae cultivated in vitro in a recently developed medium resisted pyridine extraction up to three weeks of growth, but thereafter as the culture aged pyridine extractability became characteristic. It is concluded that this pyridine extractability of acid-fastness is a characteristic of aging or nonviable bacilli. As such it is not definitive in the determination of whether or not in vitro cultivation of M. leprae has been achieved.


Evaluation of topical application of ronnel solution for generalized demodicosis in dogs. Of 20 dogs with generalized demodicosis, 18 responded favorably to treatment with 8.5% ronnel solution applied to one-third of the body every 3rd day, in rotation. Duration of treatment was from 5 to 20 weeks. In 2 dogs, the demodicosis recurred and required additional treatment with ronnel. Two dogs died during the study. One death was attributed to ronnel toxicosis; the other was unexplained. Eight of 10 control dogs treated orally and topically for pyoderma associated with demodicosis still had demodicosis 1 year after initiation of the study.


Gross, microscopic and ultrastructural study of the intestinal tube of Xenodon merremii Wagler, 1824 (Ophidia). The small and large intestines of Xenodon merremii have a similar structure. They are separated by a sphincter of thickened circular muscle. The mucosa of the proximal part of the small intestine is raised into a honeycomb pattern, but distally there are only longitudinal folds. The lining epithelium throughout is of a simple columnar type, with absorptive, goblet, argentaffin and argyrophil cells, but no Paneth cells, villi or crypts of Lieberkühn are present.


Glycoproteins of Sendai virus: purification and antigenic analysis. Solubilized envelope antigens (glycoproteins) of Sendai virus were purified in a DEAE-Bio-Gel A column. Monospecific antisera were prepared against the antigens, designated HN and F glycoproteins. These glycoproteins are antigenically distinct. Immunodiffusion analyses of the envelope antigens of three representative paramyxoviruses (Sendai, Yucaipa, and Newcastle disease virus)did not reveal any cross-reactions among them with antisera specific for Sendai virus glycoproteins.


Cardiac transplantation: eight years' experience. Eight years' experience has allowed for standards to be set for the nursing care of the heart transplant patient. Early diagnosis of rejection by cardiac biopsy has improved the treatment of the patient. Cardiac transplantation will be continued at Stanford as a life-saving operation for those cardiac patients who need and want it.


The use of 5-(125I)iodo-2'-deoxyuridine for monitoring DNA synthesis in organ culture. 5-[125I]iodo-2'-deoxyuridine (125IUdR) has been shown to be effective in monitoring DNA synthesis in murine tumour and human benign prostatic hyperplasia explants maintained in organ culture for 2 days, thus making possible the rapid screening of antitumour drugs. Uptake was linear for at least 24 hours in the case of the murine tumour and for 6 hours in the case of the prostate. Hydroxyurea almost completely blocked uptake by the murine tumour at doses above 10(-4) mole. Fluorodeoxyuridine (10(-6) mole) markedly increased the uptake of 125IUdR into the human prostate explants. Fixation of the murine tumour explants in alcoholic Bouin after culture did not alter the radioactive uptake and so routine histological examination of the tissue can be carried out after counting, which is a major advantage over [3H]thymidine.


Mast cells of the human stomach.

Mast cells of the human stomach. Mast cells were examined from various sites in the normal human stomach and in the stomachs of patients with gastric ulceration. The distribution of the different types of mast cell granules was determined in the subepithelial region of the normal human stomach. There was a significant difference in this respect between the mast cells at the incisura angularis as compared with those high on the lesser curve or high on the greater curve. Mast cell degranulation (i.e. the shedding of intact granules) and vacuolation were examined with the electron microscope. Degranulation and vacuolation were observed in subepithelial mast cells, whereas only vacuolation was seen in intraepithelial mast cells. The significance of degranulation and vacuolation is discussed.


An interaction between bleomycin and human serum. The reaction of Ehrlich cells and Triticum plant roots to some antineoplastic drugs alone and in combination with human serum. In two growth tests, one employing EHRLICH ascites tumor cells in suspension culture, the other employing intact roots of wheat (Triticum sativum Lam), the growth inhibiting action of bleomycin (BLM) was shown to be intensified by human serum. Practical consequences of the finding are suggested. No such serum-drug interaction has been demonstrated with other anti-tumor compounds, including methotrexate (MTX) and threosulphan (TRSF). Isolation of the active factor(s) in serum has not yet been attempted.


Isolation and characterization of T-even ghost-tolerant mutants of Escherichia coli. Mutants of Escherichia coli tolerant to the ghosts of T-even phages (T2, T4, and T6) have been isolated from a strain supersensitive to T6 phage. First, T6 supersensitive mutants were isolated from mutagenized E. coli W2252 by replica plating to T6 phage-overlaid agar. One of them, strain NM101, was mutagenized again, grown, and then plated with a high multiplicity of T4 and T6 ghosts. Surviving cells were checked for tolerance to ghosts and adsorption of phages. One such ghost-tolerant mutant, strain GT29, was tolerant to ghosts of both T4 and T6 phages and sensitive to T2 ghosts. This mutant was also sensitive to ethylenediaminetetraacetic acid and penicillin G and intermediately sensitive to acriflavine, sodium dodecyl sulfate, sodium deoxycholate, actinomycin D, and lysozyme. Another mutant, strain GT62, was tolerant not only to T4 and T6 ghosts but also to T2 ghosts. It was sensitive to sodium dodecyl sulfate, sodium deoxycholate, penicillin G, acridine orange, actinomycin D, phenethyl alcohol, and novobiocin and intermediately sensitive to acriflavine and lysozyme. Spontaneous revertants of strain GT62 were isolated with a frequency of 2.7 X 10(-9). It is suggested that ghosts attack host bacteria indirectly through the cell surface by a mechanism similar to the transmission hypothesis that was originally adopted by Nomura (1967) to explain the mechanism of the action of colicins, and that our ghost-tolerant mutants presumably have defects in the cell surface.


Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties. Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia.


Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form. In the envelope of Escherichia coli, is a lipoprotein of molecular weight 7,200 as a major envelope protein. This lipoprotein was previously shown to exist in two different forms in the outer membrane of E. coli: the free form and the boundform, which is covalently linked to the peptidoglycau. The free form of the lipoprotein has been purified and paracrystallized by adding acetone to a sodium dodecyl sulfate solution in the presence of magnesium ion. The paracrystals were needle shaped. An electron micrograph of the negatively stained paracrystals showed a highly ordered ultrastructure. The chemical structure of the free form was compared with that of the bound form by (i) the amino acid composition, (ii) the fatty acid composition, and (iii) the peptide analysis after cyanogen bromide cleavage. The alpha-helical content of the free form of the lipoprotein was measured from the circular dichroism spectrum of the lipoprotein in 0.01% sodium dodecyl sulfate and found to be 87%. Using the purified lipoprotein as antigen, antiserum against the free form of the lipoprotein was obtained. Immunoprecipitation of the lipoprotein with the antiserum was found to be very specific, since only the free form of the lipoprotein was found as a major peak when the antiserum was reacted with the whole envelope proteins solubilized in 0.2% sodium dodecyl sulfate, and the immunoprecipitate thus formed was analyzed by polyacrylamide gel electrophoresis.


A biocommunication centre in embryo.

A biocommunication centre in embryo. Biocommunication and educational technology centres are established features of North American and British campuses. As yet New Zealand Universities have felt little of the impact of educational technology and such centres have been set up here only comparatively recently. This paper describes the Audio Visual Learning Centre of the University of Otago-its aims, its resources, and the strategies which have been selected for translating these aims into realities. Hopefully, this Centre will succeed in building on the achievements of others while avoiding some of the more obvious pitfalls.


Biocommunication courses for medical students. Properly designed elective courses in audiovisual techniques and biophotography can help the medical student become a more effective communicator. After graduation, whether a practicing physician, teacher, author, researcher, or simply a postprandial speaker at a civic club meeting, he or she will be able to influence the thinking and behavior of others in a much more efficient and interesting manner.


Importance of full size complementary DNA in nucleic acid hybridization. The size of the DNA product synthesized by RNA-directed DNA polymerase (isolated from avian myeloblastosis virus) was found to be important for complementary DNA (cDNA)-mRNA hybridization reactions. Incomplete cDNA to rabbit reticulocyte globin mRNA formed poor hybrids and presumably lacked sequences needed for hybridization. The size of the cDNA synthesized was influenced by the reaction conditions used. The complementary DNA product contained 10 S material when synthesis was done at high deoxynucleoside triphosphate concentrations (greater than 50 muM) while the product was smaller than the template when synthesis was at lower concentrations. The concentration and size (oligo(dT)6 to (dT)10) of primer had little or no effect on the product size. Increasing the concentration of 10 S globin mRNA caused the cDNA product to contain more small material. The cDNA synthesized at high deoxynucleoside triphosphate concentrations was fractionated into heavy, medium, and light fractions by alkaline sucrose density centrifugation. All hybridized to globin mRNA. The larger cDNAs had a higher TM when hybridized to globin mRNA, a lower dTMP/dCMP ratio (indicating that the poly(dT) region constituted a smaller fraction of the molecule), and gave increased protection of 125I-labeled mRNA from nuclease digestion. The full size cDNA was especially useful for studying the RNA transcribed from chromatin by RNA polymerase. The complement of the 5' end of the mRNA is contained only in full size cDNA; the 5' end is the part of the mRNA first transcribed by the RNA polymerase assuming correct transcription. Thus, full size cDNA can hybridize more effectively to the short RNA transcripts that are obtained than partial cDNA. RNA transcribed from rabbit bone marrow chromatin by Escherichia coli RNA polymerase hybridized twice as efficiently to complete cDNA as it did to partial cDNA demonstrating the usefulness of full size cDNA.


Effects of sex hormones on the level of the messenger RNA for the rat hepatic protein alpha 2u globulin. Alpha 2u Globulin is a protein synthesized in the liver, secreted into the serum, and excreted in the urine of mature male rats. The effects of androgens and estrogens on the level of the messenger RNA coding for the male rat hepatic protein have been investigated. Castrated male rats have reduced levels of alpha 2u globulin in serum and liver cytosol, as measured by a radial immunodiffusion assay. The livers from these castrated males were found to contain similarly reduced levels of the mRNA coding for alpha 2u globulin, as measured in an mRNA-dependent wheat germ cell-free translational system. Administration of dihydrotestosterone to castrated males resulted in increased levels of alpha 2u globulin in liver and serum and this increase in the level of the protein following androgen administration was accompanied by a parallel increase in the functional level of alpha 2u globulin mRNA. Administration of estradiol-17 beta to intact male rats gradually diminishes the levels of alpha 2u globulin in liver and serum. The livers from these estrogen-treated males were found to contain alpha 2u globulin mRNA at similarly reduced levels. The time course of the disappearance of the alpha 2u globulin mRNA following estrogen treatment parallels the disappearance of the protein in liver cytosol and serum. These results indicate that sex steroids affect the synthesis of the hepatic protein alpha 2u globulin by acting pretranslationally, possibly at the level of transcription. Although the liver had not been considered a primary target tissue for sex hormones, these results indicate that sex steroids can affect certain hepatic functions in a manner consistent with the accepted model for the action of steroid hormones on their target tissues.


Regular structures in unit membranes. III. Further observations on the particulate component of the suckling rat ileum endocytic membrane complex. Further morphological observations on the particulate components decorating the lumenal surfaces of membranes of the endocytic complex of the epithelial cells of the suckling rat ileum are presented. The particles each measure approximately 7.5 nm across and give the appearance of the capital letter H in frontal view. They consist of the enzyme n-acetyl-beta-glucosaminidase (NAG). They are arranged in rows called "decorated strips" with the symmetrical lateral bars in register and spaced approximately 14.5 nm apart. Decorated strips lie side-by-side in the external (lumenal) surface of the membrane. They are parallel and sometimes spaced approximately 14.5 nm apart making an orthogonal lattice. The lateral spacing between the decorated strips under certain conditions is reduced and sometimes there is shear between the adjacent ones. Occasionally, shear is present within the decorated strips themselves, with slight displacement of the two sides of each H-shaped particle. A purified preparation of these membranes has been studied by electron microscopy using thin sectioning, negative stain, Markham translation and optical diffraction computer image reconstruction methods. The individual particles comprising the array can be seen in the membrane surface in profile view when dried in a pool of negative stain. They appear either triangular or diamond-shaped in such views. If triangular, they appear to consist of three domains at the corners of an equilateral triangle. One side of each triangular figure is parallel to the membrane surface but separated from it by a dense band of negative stain approximately 2 nm thick that runs along the surface of the membrane. Sometimes a fourth symmetrical domain is visible within this dense band, giving a diamond-shaped figure. This fourth domain connects the particle to the membrane. Thus, each H-shaped particle is a double structure, with each half in profile view appearing as a diamond figure of four symmetrical domains. Each H-shaped particle is believed to consist of either two or four molecules of NAG.


The crystalline glycoprotein cell wall of the green alga Chlorogonium elongatum: a structural analysis. Members of the Chlamydomonaceae, mostly single-celled green algae, have been shown to contain a crystalline glycoprotein cell wall component. Most of the species examined fall into a class of algae whose walls have an identical crystalline unit cell. Chlorogonium elongatum has been chosen as a representative of this class in order to investigate in more detail its cell wall structure. The alga has a spindleshaped cell wall which retains its asymmetric shape on isolation. Sections from walls fized in the presence of tannic acid clearly reveal a regular subunit monolayer, about 20 nm thick, within the wall. Sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis shows the presence of at least 2 major glycoprotein species in the wall. Negatively stained purified cell walls demonstrate the crystalline nature of the cell wall. Optical diffraction of bright-field images and direct electron diffraction both give clear diffraction patterns whose spacings extend out to 3 nm and fall on a reciprocal lattice whose vectors describe a 2-dimensional unit cell within the wall 21.5 nm X 7.0 nm and an included angle of 80 degrees. Lattice defects within the cell wall are revealed by both negative staining and surface replication. Through-focal series were used to choose images with the optimal degree of underfocus for image processing. Linear integration and optical filtering of such images gave essentially the same result. A similar image was also obtained by computing the autocorrelation function of the amplitudes in the electron-diffraction pattern and the optical-diffraction pattern of the in-focus image. On the basis of these data a 2-dimensional model of the crystalline cell wall layer is presented.


Radioimmunoassay for measuring antibodies specific for group B streptococcal types Ia, Ib, Ic, II, and III. The object of this study was to develop a test that would measure antibodies directed against group B streptococcal types Ia, Ib, Ic, II, and III. The type-specific carbohydrate antigens were purified, labeled with 125I, and used to develop a radioimmunoassay. This procedure should be particularly useful in testing human sera for group B type-specific antibodies, since it requires very small quantities of antigens and measures primary type antigen-antibody interactions.


The immunopathology of herpes gestationis. Immunofluorescence studies and characterization of "HG factor". Nine skin biopsies from seven herpes gestationis patients were studied by immunofluorescence (IF) techniques. Basement membrane zone (BMZ) deposition of C3 and properdin was present in all nine skin specimens, while IgG deposition was apparent in only one. With in vitro C3 IF staining, positive BMZ staining (HG factor activity) was noted with all seven of our patients' serum samples tested. By standard indirect IF staining, however, only one of these serum samples contained BMZ antibodies of the IgG type. Two cord serum samples, tested by these same methods, yielded positive in vitro C3 staining (HG factor activity) but negative indirect IF staining (IgG). HG factor activity was found to be stable at 56 degrees C for 30 min and in two of three specimens at 56 degrees C for 1 h. Treatment of the complement source (normal human serum) used in the in vitro C3 staining assay with Mg2-EGTA or use of C2-deficient serum as the complement source inhibited HG factor activity. HG factor blocked the specific staining of the BMZ of normal human skin by labeled bullous pemphigoid antibodies. By sucrose density gradient ultracentrifugation and gel chromatography (Sephadex G-200), HG factor activity eluted with IgG-containing fractions. The highly purified IgG fraction of two herpes gestationis sera was also positive for HG factor activity. Our studies suggest that HG factor is an IgG antibody that may not be demonstrable by conventional IF methods, but which activates the classical complement pathway.


Comparison of four techniques for the routine diagnosis of Trichomonas vaginalis infection. Specimens from 495 patients attending Johannesburg hospitals and family planning clinics were examined for Trichomonas vaginalis by microscopy of Giemsa (GS), Papanicolaou (PAP), and acridine-orange (AO) stained smears, and by culture in Feinberg-Whittington medium. Culture, Pap and GS stained smears from vaginal swabs yielded fewer positives than AO stained smears. Although Pap-stained cytological smears gave the highest number of positives, in 30% of these cases the presence of T.vaginalis could not be confirmed by examination of vaginal swabs. Of the positive AO-stained smears, 93% were also positive by at least one other technique.


Evaluation of a new technique for the demonstration of gonococci and other micro-organisms in host cells. A method (Gram-MGPLG) for demonstrating micro-organisms was compared with Gram and four other known methods. Each method was tested on tissue infected with Staphylococcus aureus, Pseudomonas aeruginosa or Neisseria gonorrhoeae, which were then fixed in Bouin's formol saline, formol sublimate, or Van de Grift solutions. Gram-positive organisms in tissues were easily seen even at low magnification when stained by several of the methods tested. Gram-negative organisms, however, are very difficult to locate when stained by Gram's method because tissue components and the organisms are all shades of red, whereas the Gram-MGPLG provided easier location of organisms because these are stained red while the nuclei are blue and connective tissue is green. All methods are markedly affected by fixation; better preservation of cytological detail and improved staining reactions were produced by fixative containing mercuric chloride.


The development of the isthmo-optic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons. The development of the centrifugal projection to the chick retina in the isthmo-optic tract (IOT) has been studied by the retrograde transport of the enzyme marker horseradish peroxidase (HRP) injected into the eye at various times during the incubation period. Some neurons in the isthmo-optic nucleus (ION--the cells of origin of the IOT) can first be labeled from the eye following injections on the tenth day of incubation; after injections on the twelfth day or later, about 95% of the neurons can be so labeled. It follows from this that the axons of virtually all the neurons in the ION (including the 60% which normally degenerate between the thirteenth and seventeenth days of incubation) reach the contralateral eye. Since in 12-day old embryos the IOT is between six and seven millimeters in length and HRP can be identified in the perikarya of ION neurons within three and one-half hours, the rate of retrograde transport in the system must be of the order of 48 mm/day. A similar time is required for HRP to appear in the perikarya of ION neurons in post-hatched chicks in which the length of the IOT is estimated to be about 14 mm. This suggests that at some time during the latter half of the incubation period there is a significant acceleration in the rate of retrograde transport, similar to that found for anterograde axonal transport in the chick and rabbit visual systems.


Automated histamine analysis for in vitro allergy testing. I. A method utilizing allergen-induced histamine release from whole blood. A sensitive, automated, histamine assay system has been developed and applied for in vitro allergy testing. Nine common pollen and environmental allergens were used at three log dilutions for in vivo studies utilizing small volumes of blood (15-20 ml). The clinical evaluation was correlated with the results of the histamine release. two different procedures were utilized. The first is the commonly used histamine release from washed leukocytes. There was excellent correlation between the clinical evaluation and the results of histamine release from washed leukocytes in 17 different individuals. The second and simpler method utilized whole heparinized blood which might better reflect the immunologic reaction which occurs in vivo. Aliquots of blood and allergen were incubated for 1 hr at 37 degrees C and each supernatant was then analyzed for histamine release. There was excellent correlation between the two tests in 29 patients tested by both the whole blood and washed leukocyte methods. There was also good correlation between the clinical evaluation of the patients amd the intro tests. The precision, accuracy, and sensitivity of the automated histamine assay make feasible its routine application in the clinical study of allergic patients.


Allergy to insect sting. III. Allergenic cross-reactivity among the vespid venoms. Recent reports have indicated that venoms may be more beneficial than whole body extracts for the diagnosis and treatment of Hymenoptera sensitive patients. These studies were undertaken to determine the cross-reactivity among the vespid venoms. Eighteen patients who were anaphylactically sensitive to vespid venoms were studied using in vitro leukocyte histamine release. The results (venom concentration for 50% histamine release) were analyzed by linear regression analysis; there was no allergenic cross-reactivity between any of the venoms, except for a modest association between yellow hornet and white hornet venom. In spite of this result 13 of the 18 patients studied were sensitive to three or four of the venoms tested. There is no clear explanation for this observation, but it suggests the existence of multiple major allergens in the vespid venoms, some of which are cross-reactive. Since immunotherapy with inappropriate proteins may lead to the development of IgE and the possibility of clinical sensitivity and since the majority of patients were not sensitive to all venom preparations, we suggest that appropriate diagnostic studies be carried out before the institution of therapy.


Hereditary vibratory angioedema: confirmation of histamine release in a type of physical hypersensitivity. A new syndrome called hereditary vibratory angioedema (HVA), characterized by pyruritis and angioedema at the site of a vibratory stimulus, was recently reported as a model of nonimmunologic immediate hypersensitivity. The biochemical mediator of this syndrome was not defined initially. This study now demonstrates that in vivo histamine release occurs in conjunction with the local and systemic clinical manifestations following a controlled vibratory stimulus. Histamine is suggested as the major mediator in this inherited physical hypersensitivity state.


Removal of dentin by fuchsin staining.

Removal of dentin by fuchsin staining. A 0.5% basic fuchsin-propylene glycol solution was applied to the carious dentin of extracted and vital human teeth, and the depths of the fuchsin-stained dentin and the bacterial invasion were compared on histological sections. Excavation guided by this staining method was always deeper than the bacterial invasion, with differences between the depths being greater in acute decay than in chronic decay. This staining is considered to be a reliable clinical guide for the complete removal of deteriorated dentin without causing failure to remove the infection completely. The depths of the images of dentin caries on the radiographs before treatment and after excavation guided by the fuchsin staining method were also compared. The image on the radiograph was always shallower than the excavated cavity. Dentists must expect to excavate caries cavities somewhat deeper than radiographic images.


Histochemical identification of myosinlike substance in cells of the human dental pulp. In this study of the human dental pulp, substances were found in smooth muscle cells, myoendothelial cells, fibroblasts, and odontoblastic processes that demonstrate staining and extraction characteristics identical to myosin in muscle. It is concluded that the myosinlike material similar to that found in cells elsewhere in the human also occurs in cells of the dental pulp.


Contributions to ecological chemistry CVII1. Fate of lindane-14C in lettuce, endives and soil under outdoor conditions. In seven successive outdoor experiments, lindane-14C was applied to lettuce or endive leaves as an aqueous formulation (about 12 mg on 20 plants for each experiment). The growing periods varied between 21 and 37 days. After this time, between 4.5% and 13.9% of the applied radiocarbon was recovered from the plants. Conversion rates to soluble metabolites as well as to unextractable residues appeared to be dependent on weather conditions. During the summer months, the radiocarbon in plants consisted of 36% soluble metabolites and of 30% unextractable residues (average of 4 experiments); in autumn, the conversion rates were much lower. The following metabolites were identified in both plant species by gas chromatography/mass spectrometry: a polar group (a free trichlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol, conjugates of the latter two compounds, and unidentified water-soluble products) amounting to 35% of the radioactivity in plants cultivated in summer, and a nonpolar group (a dichlorobenzene, 1,2,3-trichlorobenzene, 1,2,4-trichlorobenzene, 1,2,3,5, and/or 1,2,4,5-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, and gamma-pentachlorocyclohexene) amounting to 1% of the radioactivity in plants cultivated in summer. The 20 cm top-soil layer had about 14% of the total radioactivity applied to all plants. Six % of the radioactivity recovered from the soil was soluble metabolites and about 50% was not extractable. The soluble metabolites comprised a polar group (free and conjugated 2,3,4,6-tetrachlorophenol, pentachlorophenol, and unidentified water soluble products) amounting to 5% of the radioactivity in the soil as well as a nonpolar group (1,2,3-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, 1,2,3,5 and/or 1,2,4,5-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, and gamma-pentachlorocyclohexene) amounting to 1% of the radioactivity in the soil.


Residues of lindane and its metabolites in eggs, chicks, and body tissues of hen pheasants after ingestion of lindane-14C via treated wheat seed or gelatin capsules. Studies were carried out to investigate possible contamination of pheasants with residues of lindane used as seed dressings for the control of wireworms in cereal crops. One group of laying hen pheasants was fed 20 mg of lindane-14C in gelatin capsules while another group was fed wheat seed treated with 100 ppm of lindane-14C for 15 days. Residues in eggs were monitored for about 70 days. Residues in chicks hatched from the eggs were also measured. Concentrations of residues in muscle, liver, brain, and fatty tissues were determined at various times. 14C-labeled residues in eggs increased sharply in the capsule treated group and reached an average maximum of about 19 ppm in 8 days. This level decreased to less than 0.5 ppm in about 50 days. Residues in eggs from the pheasants fed treated seed increased gradually to an average maximum of 17 ppm 22 days after commencement of the feeding program. This level decreased to less than 0.5 ppm 50 days later. Residues in hatched chicks were significantly lower than those in the eggs. Highest concentrations of residues were found in fatty tissues which decreased to non-detectable level in 6 months. Several chlorobenzene metabolites were identified in egg yolk and chick tissues. Chlorophenolic metabolites were indicated only in chicks.


Metabolism of O,O-dimethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate in sheep and rats and of 3,5,6-trichloro-2-pyridinol in sheep. Sheep and rats metabolized single oral doses of O-O-dimethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate (I) to three major metabolites that were excreted in the urine (approximately 70% of the 14C). These were the glucuronide of 3,5,6-trichloro-2-pyridinol, O-methyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate, and 3,5,6-trichloro-2-pyridinol. The latter two metabolites and the parent compound were isolated from sheep feces. Sheep plasma contained the same metabolites that were found in sheep urine, and no parent compound was detected in the plasma. Tissue residues from I were determined. Visceral fat contained the highest concentration of I-equivalents (11.8 ppm). Sheep excreted a single oral dose of 3,5,6-trichoropyridinol (II) unchanged in the feces and as II-glucuronide in the urine.


Light microscopic localization of labile calcium in hypertrophied chondrocytes of long bone with alizarin red S. A method which localizes labile 5% ethylene glycol-bis-(beta-amino-ethyl ether)N-N'-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pericellular matrix using alizarin red S (ARS) is described. Fresh blocks of epiphyseal cartilage approximately 1 mm thick were immersed into 0.5-2% ARS solution containing 7% mounted on glass slides in 7% sucrose or in glycerol-gelatin. The stained tissue blocks were also dehydrated in acetone, cleared in xylene and mounted in Preservaslide. The ARS precipitated ionic calcium as red Ca-ARS salt which was birefringent in polarizing microscope, stable in water at pH 4-9 and in nonpolar organic solvent but soluble in polar solvents, especially in dimethyl sulfoxide. In contrast, ARS-stained insoluble calcium phosphate was stable even in dimethyl sulfoxide. Calcium in the hypertrophied chondrocytes, therefore, was thought to be present in a readily ionizable state instead of as insoluble calcium phosphate. Since addition of 7% sucrose retained as well as improved ARS localization of cellular calcium, the calcium was believed to be present in an osmotically sensitive, membrane-bound cytoplasmic compartment. The ARS-positive labile calcium in spherules which develop in the hypertrophied chondrocytes as well as in the pericellular matrix at the zone of provisional calcification suggested a preparatory stage in the process of cartilage calcification.


The effects of glutaraldehyde treatment and horesradish peroxidase conjugation on the immunoreactivity of bovine myelin encephalitogenic protein. The effects of the immunoreactivity of bovine myelin encephalitogenic protein (EP) of treatment with glutaraldehyde and conjugation with horeradish peroxidase (HRP) were investigated by complement fixation and immunohistochemistry. Both glutaraldehyde treatment and HRP conjugation of EP decreased but did not abolish the reactivity of EP with rabbit anti-EP. Conjugation of EP to HRP by the two-step method of Avrameas had less detrimental effects on the immunochemical reactivity of EP than did the one-step procecure. The use of EP-HRP conjugates to probe for antibodies to EP is an immunochemically sound system with the major limitation that the number of antibody-producing cells is likely to be underestimated due to the failure to detect cells producing low levels of antibody or antibodies directed toward determinants altered by the modification procedures.


Plasma membrane localization of alkaline phosphatase in HeLa cells. The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.


Electron histochemistry and ultrastructural localization of carbohydrate-containing substances in the sheath of Volvox. The location and characteristics of carbohydrate-containing structures within the intact sheath of Volvox were studied by 3,3'-diaminobenzidine tetrahydrochloride-osmium, colloidal iron, colloidal thorium, ruthenium red and periodic acid-silver methenamine staining. The sheath consists of external and internal fibrillar layers separated by a tripartite structure. The external layer reacts positively with 3,3'-diaminobenzidine tetrahydrochloride, colloidal iron, colloidal thorium and ruthenium red, indicating that it contains acid mucosaccharides. Staining in the external layer is abolished by Ba(OH)2 treatment. The tripartite structure and internal fibrillar layer contain periodic acid reactive groups which do not occur in the external layer. Under certain conditions, reactions between the cationic dyes and the internal material were also observed. It is postulated that the internal matrix of the sheath contains glycoproteins or a mixture of acid mucosaccharides and glycoproteins. Possible functions of the sheath material are discussed.


A phenomenon of photoenhancement of acridine orange-induced fluorescence. It is a common experience that, with exposure to exciting radiation of the fluorescence microscope, the acridine orange-induced red fluorescence of the nucleus, produced by Feulgen hydrolysis, fades with a concomitant shift to green. The present investigation reports a phenomenon of photoenhancement observed in the hydrolyzed cytoplasm where pale green fluorescence increases in intensity with exposure to exciting radiation. The phenomenon has been noticed in Rhizobium, Oscillatoria, tomato root tip cells and human buccal epithelial cells. It is tentatively concluded that the gain in fluorescence yield is due to certain conformational changes of the acridine orange-protein complex induced by ultraviolet light flux.


Protection against experimental allergic encephalomyelitis with peptides derived from myelin basic protein: presence of intact encephalitogenic site is essential. Experimental allergic encephalomyelitis (EAE) is a cell-mediated autoimmune response directed toward a component of central nervous system (CNS) tissue, myelin basic protein (BP). Injection of animals with either whole CNS tissue or purified BP in complete Freund's adjuvant (CFA) induces severe and usually fatal disease. Preimmunization of animals with BP in incomplete Freund's adjuvant (IFA) prevents EAE. We have examined the relative abilities of whole guinea pig BP and its fragments to protect guinea pigs from subsequent EAE induction. The data suggest that the presence of the intact encephalitogenic site (residues 113-121) in the molecules used for preimmunization is necessary but may not be sufficient for complete protection against EAE induction.


An improved method for isolation of H-2 and Ia alloantigens with immunoprecipitation induced by protein A-bearing staphylococci. Mouse alloantigens, including the histocompatibility (H-2) and immune response linked (Ia) antigen molecules, can be readily isolated by a new precipitation technique utilizing protein A-bearing, fixed Staphylococci. The antigens are prepared by radiolabeling mouse spleen cells with 3H-leucine, solubilizing with the non-ionic detergent Non-Idet P-40, and purifying by affinity chromatography with lentil lectin coupled to Sapharose. The antigen preparations are mixed with appropriate alloantisera, and immune complexes formed in the mixture are then bound by the protein A sites on the Staphylococci. The organisms, Staphylococcus aureus, Cowan I strain (ATCC 12598), are heat inactivated and fixed, and are a stable, uniform IgG-binding reagent, especially when stored frozen. The antigen-antibody complexes are easily eluted from the organisms for electrophoretic analysis. The precipitation mediated by the organisms is more efficient, rapid, and artifact-free than the traditional "sandwich" precipitation technique involving anti-globulin reagents.


Blocking of MLC stimulation by anti-Ia sera: studies with the virus plaque assay. The development of congenic mouse strains identical at the H-2K and H-2D loci but differing by I-region associated (Ia) determinants has permitted an association to be established between Ia determinants and stimulation in mixed lymphocyte culture reactions (MLR). The present experiments were undertaken to establish whether the Ir-coded control of MLR operated at the level of recognition or of stimulation. Reciprocal MLR were established between A.TH and A.TL mouse spleen cells in the presence or absence of anti-Ia sera directed either at determinants of the stimulating or responding cells. The number of T cells responding was assessed by the virus plaque assay. Anti-Ia sera directed against the responding cells were no more inhibitory of the MLR than normal mouse serum. In contrast, anti-Ia sera directed against determinants of the mitomycin-treated stimulating cells markedly inhibited activation of T cells in the MLR.


Characterization of an antiserum specific for cell surface antigens of rat mast cells. Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.


Induction of specific suppressor T cells in vitro. We describe conditions for generating sheep red blood cell-specific suppressor T cells in Mishell-Dutton cultures. The production of specific suppressor cells is favored by increasing antigen dose in the initial culture but can be produced by transferring more cells when lower doses of antigen are used. Transfer of small numbers of cells cultured with low doses of antigen leads to a specific helper effect. Transfer of large numbers of educated cells leads to nonspecific suppression. Suppression can be effected by the effluent cells from nylon wool columns which do not make detectable PFC. A fraction of these cells become resistant to treatment with anti-T cell sera and complement after culture. The suppressor cells are radiation sensitive and must be able to synthesize protein to suppress. They take 2 to 3 days of education to reach maximum suppressive efficiency and will not suppress cultures if added 2 to 3 days after culture initiation. Their production is favored by the absence of mercaptoethanol, suggesting that the observed suppression is not "too much help". The ability to generate specific suppressor cells in vitro should be of great benefit in determining the factors that regulate their appearance in vivo.


Basophil-sensitizing antibody response to herpes simplex viruses in rabbits. Antibodies to herpes simplex virus type 1 and type 2 were detected in the sera of rabbits by release of histamine from basophils sensitized in vitro with the sera. The time course of the appearance of the antibodies, the dose-response curve of the release of histamine in relation to antigen concentration, the sedimentation characteristics of the antibodies in sucrose gradients, and the ability to destroy the sensitizing capacity of the sera with heat suggest that the antibodies being assessed were of the IgE class. These antibodies were induced in animals injected intradermally, intramuscularly, and i.p. with live virus. The antibodies were detected 1 week after primary injection and a similar time course of antibody appearance was observed after a second or third injection. The same cross-reactivity between type 1 and type 2 virus observed with IgG antibody was also observed with IgE antibody.


Measurement of IgG-blocking antibodies: development and application of a radioimmunoassay. A radioimmunoassay for measuring blocking antibodies has been developed. We used the ragweed antigen E system to show that the same blocking antibodies (IgG) measured by inhibition of antigen-induced leukocyte histamine release were precipitated in the binding assay (r8 = 0.96, p  less than 0.001), thus validating a widely applicable technique for measuring blocking antibodies. Binding of phospholipase-A (Phos-A), the major allergen in honey bee venom, was also shown to correlate significantly with inhibition of histamine release. Hymenoptera (insect) hypersensitivity was used as a model to demonstrate application of the binding assay. Sera obtained from patients undergoing whole body extract therapy contained negligible amounts of specific blocking antibodies. Significantly higher blocking antibody titers to both whole honey bee venom and Phos-A were measured in sera drawn from patients immunized with whole venom. The use of the binding radioimmunoassay should facilitate management of allergic disease processes in which blocking antibodies are thought to be protective.


Identification of a butanol-extractable human placenta-specific antigen with alkaline phosphatase activity. N-Butanol extracts of whole-term placenta from different individuals were prepared, and used as immunogens to raise heterologous hyperimmune sera in rabbits. Upon immunoelectrophoresis the anti-placenta antisera could recognize at least six antigenic components in the placental extract even after they had been completely absorbed with pooled male serum proteins. However, the antisera so absorbed, designated (-PMS) antisera, could still react strongly with several normal adult tissue extracts including kidney. Systematic and quantitative absorptions of the (-PMS) antisera were thus further carried out with individual butanol extracts of normal adult liver, lung, intestine, stomach, kidney, bone, pancreas, spleen, heart, cerebrum, cerebellum, breast, and packed red cells, as well as a composite extract containing equal amounts of each of the 13 adult tissue extracts. Of the six antigenic components in the placental extracts reacting with the (-PMS) antisera the only one which retained its reactivity with the antisera throughout exhaustive absorptions was associated with alkaline phosphatase activity. This immunologic and enzymologic identity was confirmed with homogeneous placental alkaline phosphatase. Extracts from each of three placentae injected into three pairs of rabbits all produced an identical antibody reaction with the unique determinant(s) of placental alkaline phosphatase. The same identity of precipitin reaction was also found with extracts of 14 other placentae against each of these antisera. It thus firmly establishes that placental alkaline phosphatase is a characteristic placenta-specific fetal protein.


Histochemical staining in lymphocyte suspensions complexed with beta-galactosidase as an antigen. Unspecific staining of spleen cells by indigo. Spleen cells from mice immunized with E. coli beta-galactosidase were incubated with the enzyme and with 5-bromo-4-chloroindolyl-beta-galactosidase as a histochemical substrate. Some cells are stainable in this reaction, however, not all of them carry specific receptors for beta-galactosidases. Some spleen cells can take up dye and thereby become unspecifically stained. This system is therefore not suitable for the detection of antigen binding to cells.


Comparison of radio-immunoassay and lymphocytotoxicity inhibition techniques for the determination of beta2 microglobulin. Rabbit anti-human beta2 microglobulin antisera can lyse human lymphocytes in the presence of rabbit complement. Inhibition of the lymphocytotoxic reaction by highly purified beta2m was applied to the measurement of beta2m concentration in biological fluids. Parallel determinations were also performed using a radioimmunoassay. Lymphocytotoxicity inhibition is simple and more sensitive than radial immunodiffusion but less sensitive than the radioimmunoassay. beta2m was measured by these two techniques in serum and urine from normal individuals, uremic or transplanted patients.


The effect of antigen suicide on numbers of cells binding defined antigenic determinants. Lymph node cells from guinea pigs immunized to oxidized ferredoxin (O-Fd) were treated in an antigen suicide procedure designed to inactivate lymphocytes binding the haptenic peptide determinants of the ferredoxin molecule. O-Fd-induced DNA synthesis and cells binding either peptide determinant were examined both before and after allowing suicide. The proliferative response to O-Fd and the number of determinant-binding cells were specifically and markedly decreased after antigen suicide.


In vitro induction and measurement of hemolytic plaque forming cells in man. Following co-cultivation with sheep red cells or ovalbumin, Hypaque-Ficoll-separated human tonsillar lymphocytes were demonstrated to generate specific hemolytic PFC with maximum numbers at day 5-7. PFC were enumerated on poly-L-lysine coupled red cell monolayers in Microtest-II-plates. Plaque formation appeared to be puromycin-sensitive, complement-dependent and showed clear specificity for the antigen present during the inductive culture. Treatment of PFC with mu-chain specific antisera and complement resulted in complete inactivation of PFC; gamma-chain antisera had no effect. The development of such a simple and sensitive assay system permits the analysis of cellular interactions required for the induction of PFC responses in man.


An improved quantitative plaque assay for lymphocytes bearing antigen-specific cell receptors. An improved method for the determination of the number of lymphoid cells bearing antigen-specific receptors is described. The method is based on the use of hapten-coupled bacteriophage (dinitrophenyl-T4) and detection of lytic plaques formed by the action of DNP-T4 on a target E. coli strain. The method is highly specific (up to 90% specific binding) and can be adapted for use with other antigenic determinants chemically attached to an active bacteriophage.


Quantitative studies of Gm allotypes. I. Reappraisal of the method using an autoanalyzer. In this study, various parameters for the autoanalyzer hemagglutination-inhibition circuit applied to Gm studies, were examined. Details are given of modifications of this circiut and related methodology, that improve its precision and the reproducibility of results: use of a cells counter, effects of temperature, PVP, Tween 20, anti-Gm concentration, etc. The effects of age, storage and freezing on quantitative Gm allotypes antigenicity are also stressed.


What can be measured with RAST?

What can be measured with RAST? A theoretical study of the basic principles involved in Radioallergosorbent test (RAST) showed that: 1) When a given serum is tested, the significance of the numerical value obtained with RAST depends upon the serum assayed and the allergosorbent preparation, in a rather unpredictable way. Three factors can be measured: a) The percentage of specific IgE antibodies among all allergen-specific antibodies; b) The specific IgE antibody level; c) The product of the specific IgE antibody level and its affinity constant. 2) Simple graphical techniques allow a straightforward determination of all these factors if four dilutions of each serum are assayed at the same time. The results are expressed in two constant parameters (arbitrary IgE unit and allergosorbent capacity). It is concluded that these theoretical calculations may give a fair account of a lack of correlation between specific IgE antibody levels (as assayed with RAST) and several clinical and biological parameters. Furthermore, they provide a simple procedure which makes such tedious manipulations as specific IgE antibody purification quite necessary.


Immunoenzymatic methods for the study of surface bound immunoglobulins in mice. Methods are described for the study of lymphocyte surface bound immunoglobulins (Ig) for light microscopy experimentation. a) Live peripheral lymph node cells (LNC) or spleen cells were reacted in suspension under capping conditions with rabbit anti-mouse Ig antibodies. After washing the cells were cytocentrifuged, fixed and relabeled with either peroxidase-(PO) of fluorescein-(Fl) conjugated anti-rabbit IgG antibodies (Method SC). The staining was heterogeneous and three main categories of lymphocytes were distinguished on the basis of a characteristic surface label distribution (1) all label within distinct caps (caps), (2) all label evenly distributed over the cell surface (rings), and (3) an intermediate pattern where only part of the label was concentrated over one pole (polarized). b) LNC or spleen cells were treated for the simultaneous detection of lymphocyte surface Ig and cytoplasmic Ig of plasma cells. For this, live cells were cytocentrifuged, and after fixation were labeled with PO-conjugated antibodies. Lymphocytes stained in a continuous ring and were easily distinguished on morphological grounds from the intense cytoplasmic stain of plasma cells (Method D). Positive lymphocyte counts done by this method compared favorably with those obtained by Method SC. Comparison of Fl- and PO-conjugated reagents were done by either Method SC or D, by scoring total positive cells. Although either reagent gave closely similar percentage values, the lymphocytes stained by the immunoenzymatic technique contrasted sharply from negative cells and were as a result more easily counted. Other practical advantages of immunoenzymatic staining over immunofluorescence are (1) the simultaneous visualization of cell morphology and type of label distribution over the cell surface, (2) cell scoring can be done rapidly on a large population sample, and (3) stained slides can be reexamined on subsequent days. A common advantages of Method SC for both immunofluorescence and immoenzymology is that antibody labeled slides can be stored before the final staining step.


Antigen and antibody purification by immunoadsorption: elimination of non-biospecifically bound proteins. Immunoadsorption is a simple and effective method for purifying antigens and antibodies. The major interfering effect is non-biospecific binding, especially when crude preparations of antibodies or antigens are coupled to the matrix. In this paper various possibilities for eliminating these effects are compared and analysed. It was found that for antigen (AFP) purification pre-elution with a pH 10.0 buffer of high ionic strength (1.0 M) is effective. A major problem in purifying antibodies in the non-biospecific binding of IgG. This can be eliminated by using a non-biospecific immunoadsorbent column, where specific antibodies are not, or only weakly bound and IgG with irrelevant specificity but exhibiting high non-biospecific interaction is retained. This has been applied to preparation of sheep anti-pepsinogen II group protease antibodies.


Chromosomal assignment of the HL-A common antigenic determinants in man-mouse somatic cell hybrids. In the study presented here, man-mouse somatic cell hybrid clones were examined by means of radioimmunoassays for the presence of both beta2-microglobulin (beta2m) and the HL-A xenoantigenic determinant. In addition, the clones were examined for their karyotype and the expression of enzymes with known chromosomal assignments. The results obtained indicate that the gene coding for the HL-A xenoantigenic determinant is carred on chromosome 6. The data obtained provides a direct demonstration that the gene coding for beta2m segregates independently of that coding for the alloantigenic polypeptide chain of the HL-A molecule, and that the gene coding for beta2m is carried on chromosome 15.


A mouse plasma substance carrying beta2-microglobulin activity and lacking in H-2 alloantigenic activity. A mouse plasma substance carrying beta-2-microglobulin activity and lacking in H-2 alloantigenic activity was separated from the blood plasma of A/J strain female mice. The plasma substance had a molecular size of about 300,000-400,000 daltons and an electrophoretic mobility of alpha-globulin. The plasma substance was split by papain digestion to a fragment of about 50,000-60,000 daltons that still carried beta2-microglobulin activity. The papain-split plasma substance was devoid of H-2 alloantigenic activities and Thy-1 and TL alloantigenic activities as well, but yet had a two-component structure that was similar to papain-solubilized H-2 molecules. It contained a 37,000-dalton component linked non-covalently to an 11,00-dalton component, i.e. mouse beta2-microglobulin. This plasma substance appears to be different from Ss protein or Slp protein, both found in mouse serum.


High density lipoprotein (HDL) polymorphisms in rabbit. II. A study of the inherited Hl 1 and R 67 antigens in relation to HDL polypeptides. Rabbit high density lipoprotein (HDL) has been isolated by ultracentrifugation delipidated by ether and ethanol, and treated with urea prior to separation of the different polypeptides by gel filtration. The two different genetic polymorphisms known in rabbit HDL were found to reside in two different polypeptides. This conclusion was reached in experiments where a quantitative radial immunodiffusion technique was employed. The results of polyacrylamide gel electrophoresis in SDS indicate that the Hl 1 antigen resides in a polypeptide chain with a molecular weight of 40,000. whereas the R 67 antigen resides in a chain with a molecular weight of 17,000.