Datasets:

andersonbcdefg

/

pubmed_pairs_part3

like 0

Characterization of human B lymphocyte specific antigens.

Anti-human B cell serum (ABS) was developed by sequentially absorbing a rabbit anti-human tonsil serum (AHTS) with human red cells, liver, serum, and thymocytes. AHTS was also absorbed quantitatively with tissues and cells from different sources. ABS was nontoxic for thymocytes but lysed the majority of chronic lymphocytic leukemia (CLL) cells. This also killed a number of tonsil and blood lymphocytes which bound erythrocyte-antibody-complement complexes but not sheep erythrocytes. In further studies, it was shown that phytohemagglutin-responsibility of peripheral blood lymphocytes was not affected by treating them with ABS and complement. Anti-B cell activity of AHTS was not changed by absorption with thymus, liver, kidney, and brain tissues but absorbed with tonsil and CLL cells. These data confirm the B cell specificity of ABS. Indirect immunofluorescence was carried out on tissue sections of human tonsils and lymph nodes, which indicated that ABS-reactive cells were concentrated in lymphoid follicles including germinal centers, while plasma cells and cells located in the thymus-dependent area were essentially devoid of immunofluorescence.

A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglycan.

A major portion of the humoral immune response to peptidoglycans is directed against the non-cross-linked pentapeptide side chains of these ubiquitous bacterial antigens. At present, no specific and sensitive assay for pentapeptide antibody determination is available. Therefore, a radioimmunoassay has been developed which employs the synthetic pentapeptide hapten L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala, labeled by the active ester method of Bolton and Hunter to high specific activities (6.74 to 18.18 muCi/mug) with 125I, and used as a reagent for measuring pentapeptide antibody. A-variant streptococcal antisera, known to contain pentapeptide antibodies as shown by quantitative precipitation, would bind more than 95% of the radiolabeled hapten in contrast to 2 to 3% by preimmune rabbit sera. Specificity of the binding reaction was demonstrated by inhibition experiments imploying various synthetic oligopeptides related or unrelated to the pentapeptide in the radioimmunoassay. Binding curves established with serial dilutions of peptidoglycan antiserum were linear from 15 to 500 mug/ml of antibody permitting pentapeptide antibody measurement within this range. Comparative data on pentapeptide antibody determinations by quantitative precipitation and radioimmunoassay are given and the time course of the production of this antibody in 14 rabbits hyperimmunized with A-variant streptococcal vaccine is reported.

Immunofluorescent detection of hepatitis B antigen in paraffin-embedded liver tissue.

Hepatitis B antigen (HBAg) has been demonstrated by the indirect immunofluorescent technique and by orcein staining in 20 liver biopsies fixed in Bouin's fixative and embedded in paraffin. The results were compared with those obtained previously by immunogluorescence on frozen sections of the same biopsies. Ten biopsies which were positive in frozen sections were also positive by immunofluorescence in parafin sections, whereas only six were positive by orcein staining. In orcein-stained sections, the cellular localization of HBAg was precisely in the same places as in the slides examined by immunogluorescence. The intessity of the fluorescence in paraffin sections was almost the same as in frozen sections. The localization of the antigen was histologically more precise in paraffin sections. Besides various advantages, indlucing aboidance of freezing aquipment and procedures, paraffin sections are more easy to handle and biopsies from distant hospitals can be processed. The advantages of the immunofluorescent test in comparison to orcein staining are its immunological specificity and higher sensitivity.

The oncornavirus glycoprotein gp69/71: a constituent of the surface of normal and malignant thymocytes.

The oncornavirus related proteins associated with the surface of normal and malignant thymocytes were studied. Three virion-associated proteins (gp69/71, p45, p30) were associated with lymphoma cells from about 70% of the tumors studied. Two virion-associated proteins (gp69/71 and p45 were associated with normal thymocytes form some but not all strains of mice. In gp69/71- mice, conversion to the gp69/71+ phenotype accompanied leukemogenesis. An interesting difference in the apparent molecular size of virus related antigens of the 70,000 dalton size class was detected in lymphoma cells present in involved spleens as compared to involved thymuses. Mice infected as neonates with Scripps leukemia virus make antibody to gp69/71 and some make antibodies to molecules associated with the surface of their own tumors. The significance of the restricted presence of antigens coded for by the viral genome to the surface of some differentiated cells is discussed in reference to (a) the relationship between virion, leukemia associated, and differentiation dependent markers, and (b) the possible consequence to the host of having similar antigenic determinants on three independent structures with replicative potential (virus, normal thymocytes, and tumor cells).

Heterogeneity of the BALB/c antiphosphorylcholine antibody response at the precursor cell level.

Immune responsiveness to phosphorylcholine (PC) in BALB/c mice has been characterized by combining (a) usuage of highly sensitive radioimmunoassays for quantitation of antibody, heavy-chain class, and idiotype on a weight basis; (b) isolation of PC-specific B cells in fragment cultures; and (c) stimulation in a carrier-primed environment with the PC hapten coupled to carrier through a tripeptide spacer in order to maximize carrier recognition. The specificity and accuracy of the radioimmunoassays have veen verified by specific inhibition, lack of nonspecific binding, and excellent concordance of values for monoclonal antibody concentration obtained independently for Fab and idiotype content. The latter evidence also serves as strong confirmation of the monoclonality of in vitro monofocal responses as well as the preservation of the idiotype on antibodies of differing immunoglobulin classes. The results indicate that while B cells expressing the TEPC 15 idiotype predominate, other idiotypes may be represented by 2-50% of PC-specific precursors, and monoclonal antibodies even of the TEPC 15 idiotype are produced in both the IgM and IgG1 immunoglobulin classes. These findings are confirmed by the analysis of serum antibodies produced in carrier-primed mice immunized with hapten coupled through a tripeptide spacer, thus re-emphasizint the enhancement of primary responsiveness, particularly IgG1 production, by maximizing carrier recognition. The finding of idiotype diversity in the PC response, as well as diversity of expression in terms of quantity and immunoglobulin class of antibody synthesized by the clonal progeny of B cells within the TEPC 15 clonotype, emphasize the heterogeneity of the B-cell population both in terms of specificity repertoire and the physiological state of cells even within a single clonotype.

The effects of the H1 and H2 antihistamines on "allergic" histamine release and its inhibition by histamine.

Antigen-induced, IgE-mediated release of histamine from human basophiles is an in vitro model of allergic reacttions; it is blocked by extracellular histamine, presumably as a result of its ability to increase adenosine 3',5'-monophosphate (cyclic AMP) levels. The H1 antihistamines do not antagonize these effects of histamine but at approximately equal to 1 mM cause histamine release and at approximately equal to 0.1mM inhibit antigen-induced histamine release. The phenothiazine antihistamines are 10-30 fold more potent inhibitors than the rest; other tricyclic antidepressant drugs share this activity. The mechanism of this inhibition, which occurs in both the 1 degree and 2 degree stages of histamine release, is not known but it is not due to partial agonist activity since the anti-H1 drugs cause a significant fall in cyclic AMP levels. The anti-anaphylactic effects of the H1 antagonists probably play no therapeutic role but we suggest that drugs structurally similar to the phenothiazine antihistamines should be developed for clinical testing. The H2 antihistamines block histamine-induced inhibition of histamine release and the increase in cyclic AMP levels, but neither cause nor inhibit histamine release. The K-B values for the anti-H2 drugs (burimamide approximately equal to 5 muM); metiamide approximately equal to 0.5muM); are similar to those described for other H2 receptors.

[Certain practical problems of recognition and treatment of cardiac rhythm disorders].

Having briefly touched upon the problem of terminology and classification of arrhythmias the authors consider the diagnosis and clinical evaluation of some disorders, including the extrasystole (the significance of the extrasystolic interval), ectopic arrhythmias from the region of the atrio-ventricular junction (with simultaneous consistent or transent disruption of the intraventricular conduction), isolated atrial tachycardia, some variants of auricular fibrillation, flutter paroxysms, paroxysms of ventricular tachycardia, with continued auricular fibrillation in particular, and also the earlier described electrocardiographic phenomenon tentatively interpreted as sinistroatrial fibrillation with dextraatrial tachycardia, as well as major types of disrupted condution. The authors give a brief exposure of their views as to the principles of the treatment. Emphasis is placed on the importance of a comprehensive clinical approach to the diagnostic matters and to the evaluation of arrhythmias, as well as to the fundamental need to define more precisely the pathogenesis of the disturbed rhythm in a concrete patient so as to adopt an effective treatment.

A multidisciplined approach for the treatment of metastatic carcinoma of the breast.

We have reviewed our experience in a multidisciplined breast cancer clinic where we have utilized hormonal, ablative, and chemotherapetuci modalities. Our experience seesm to be similar to that of other groups in that oophorectomy treatment produces approximately a 61 per cent response (regression and arrest) rate, androgen therapy produces a 47 per cent response (regression and arrest) rate estrogen therapy produces a 40 per cent response (regression and arrest) rate, and ablative treatment produces approximately a 50 per cent response (regression and arrest) rate. Adrenalectomy and hypophysectomy showed similar response rates. Until it can be shown that hypophysectomy clearly offers enhanced benefits, this will not be utilized by our group except in those patients who cannot tolerate abdominal surgery (that is, patients with poor pulmonary reserve). Of interest is the high response rate (65 per cent) to ablative treatment in patients in whom disease exacerbates on additive hormonal treatment, with an increased duration of response and survival. Survival is increased in patients who are rebound responders after estrogen withdrawal. We expect to report data with future follow-up of this group of patients. New protocols will be instituted after review of the data in the hope of increasing clinical benefit and survival in this group of patients. Carcinoma of the breast accounts for almost 90,000 new cases of cancer a year, with metastases eventually developing in at least half of these patients. All physicians must be aware of the many complex problems associated with this disease and, hopefully, arrive at a logical approach for its control. We believe this can be achieved with a multidisciplined group approach as established at the Lahey Clinic Foundation.

Faecal bile-acids and clostridia in patients with cancer of the large bowel.

Of 44 patients with cancer of the large bowel, 36 ( 82%) had high faecal bile-acid concentrations compared with only 15 (17%) out of 90 patients with other diseases. 31 (70%) of the 44 patients with large-bowel cancer had high faecal bile-acid concentrations in the presence of faecal clostridia able to dehydrogenate the bile-acid nucleus, compared with only 8 (9%) out of 90 patients with other diseases. Thes findings support the hypothesis that cancer of the large bowel is caused by high concentrations of bile-acid derivatives produced by certain anaerobic bacteria.

Refeeding-malaria and hyperferraemia.

During the Central African (Sahelian) drought, attacks of falciparum malaria were common in patients and their relatives shortly after their arrival in a hospital in Eastern Niger. A prospective study of 72 adult patients not admitted for malaria and 109 accompanying relatives was undertaken to investigate this observation. 23 attacks occurred in patients and 51 in relatives, with a peak frequency five days after arrival. On arrival, parasitaemia was low but reached a maximum by five days. Serum-iron and percentage saturation of transferrin were moderately increased initially, rose dramatically within forty-eight hours with near maximum saturation, and were falling by the fifth day. It is suggested that the early hyperferraemia, apparently related to refeeding, led to rapid multiplication of existing parasites and attacks of malaria. The results of experimental malarial infection of Wistar rats, half of which had been given intramuscular iron, supported this hypothesis.

Progress in fetal assessment.

The growth areas in fetal assessment in late pregnancy have been studied by making a quantitative review of the papers in four obstetric journals. Of the 130 relevant papers published, in 1973, the most common subject treated was the phospholipid test of fetal lung maturity. Other important tests reviewed were estrogen assays, ultrasonic studies, human placental lactogen and alpha-fetoprotein measurements. The clinical value of phospholipid tests was demonstrated; liquor creatinine assays are much inferior in assessing fetal maturity. Plasma estriol assays are likely to be of increasing importance in clinical practice. Some caution should be applied in the interpretation of ultrasonic measurements of biparietal diameter as a test of fetal development and well-being.

P-Chloramphetamine: Selective neurotoxic action in brain.

Injection of 2.5,5, 10, or 20 milligrams of p-chloroamphetamine per kilogram of body weight into rats produced evidence of cytopathological changes in sections of brain stained by a Nissl or silver method. As early as 1 day after drug injection cells demonstrated an intense Nissl staining, intense argyrophilia, cellular shrinkage, and perineuronal spaces. At 30 days after injection both stains revealed cellular debris and glial reactions characteristic of cellular dissolution. The neurotoxic effects of 2.5, 5, or 10 milligrams of p-chloroamphetamine per kilogram were primarily restricted to an area of the ventral midbrain tegmentum corresponding to the distribution of the B-9 serotonergic cell group. After 20 milligrams of p-chloroamphetamine per kilogram there was also evidence of neurotoxic effects on cells within the substantia nigra. These results confirm previous suggestions that the long-term reduction in serotonin content of brain, tryptophan-5-hydroxylase activity, and uptake of serotonin after injection of p-chloroamphetamine is due to a neurotoxic effect of the drug or some metabolite on serotonergic cell bodies.

Localization of growth hormone-release-inhibiting hormone (somatostatin) in the rat brain.

Utilizing an immunoperoxidase technique at the light microscope level, growth hormone-release-inhibiting hormone (somatostatin) was localized in the external zone of the median eminence, the subcommissural organ, the organum vasculosum of the lamina terminalis and the pineal gland. No positive reaction was detected in any other brain area.

Proliferation of non-neuronal cells in spinal cords of irradiated, immature rats following transection of the sciatic nerve.

Transection of a peripheral nerve not only elicits changes in the injured neurons but also results in an increase in non-neuronal cells, considered by most workers to be neuroglia, in the region of these neurons. Since studies in this laboratory have shown that the neuroglial population of spinal cords of immature rats can be reduced markedly by ionizing radiation, the present investigation was undertaken to determine if this reaction would occur in the irradiated spinal cord following transection of the sciatic nerve. In order to answer this question the sciatic nerve was sectioned unilaterally at 17 days of age (14 days post-irradiation). Sham-irradiated littermates served as controls. Light microscopic examination showed an increase in non-neuronal cells throughout the gray matter on the side of axotomy in spite of a decreased neuroglial population in the 2,000 R and 3,000 R groups. These cells were scattered in the neuropil or were adjacent to injured neuronal perikarya in the anterior horn. Qualitatively similar reactions occurred in the 500 R and 1,000 R groups and in shamirradiated controls. Whether the magnitude of response is the same in all groups is currently under investigation, as are questions dealing with the origins of the reactive cells.

Effects of erythropoietin on erythroid colony formation in uremic rabbit bone marrow cultures.

Erythropoietin-responsive stem cell (ERC) kinetics in anephric uremic rabbits were studied in vitro using the growth of erythroid colonies in a methyl cellulose system in cultures with and without the addition of erythropoietin (ESF). Approximately 68 hr after bilateral nephrectomy, an increase in BUN and decreases in hematocrit and marrow erythroid cellularity were seen. However, the numbers of erythroid colonies formed in response to ESF on plates inoculated with 2 times 10-5 cells were greater in anephric rabbit marrows than in normal controls. In addition, the numbers of erythroid colonies produced by the uremic and normal marrows in the presence of ESF were increased in proportion to the number of precursors plated. These findings suggest that, in uremia, the concentration of ERC is increased and that the ERC are capable of responding normally to ESF. The increase in the number of erythroid colonies of uremia may be due to the undisturbed flow of uncommitted hematopoietic stem cells into the ERC compartment in the presence of a delay of differentiation of ERC into heme-synthesizing nucleated erythroid cells due to a lack of ESF.

Fungal fimbriae. I. Structure, origin, and synthesis.

Fine hair-like appendages on the cell walls of the another smut Ustilago violacea are described. These hairs are termed fimbriae because of their close similarity to the fimbriae (pili) found on certain Gram-negative bacteria. Cells of U. violacea may carry more than 200 fimbriae varying in length from about 0.5 mum to over 10 mum, and having a diameter of about 60-70 A. Some fimbriae produce knobs similar to those found on bacterial sex fimbriae. Log-phase cells are the most densely fimbriated, while stationary phase cells are devoid of fimbriae. The cells can be defimbriated by sonication, high-speed agitation, or centrifugation through a 40% sucrose solution. The fimbriae can regenerate in these defimbriated cells in about 1 h. This regeneration is inhibited by both cycloheximide and rifampin, but not by chloramphenicol and therefore appears to depend on de novo protein synthesis on cytoplasmic ribosomes. Similar long fimbriae are found on U. maydis and Leucosporidium (Candida) scottii. Short fimbriae, about 0.5 mum long, were found on all the other species of yeast-like fungi examined (Rhodotorula, Saccharomyces, Schizosaccharomyces, Hansenula, Lipomyces, Nadsonia, and Torulopsis spp.).

Elevated concentrations of serum alpha-fetoprotein in rats with chemically induced liver tumors.

The study was undertaken to determine whether aflatoxin B1 (AFB1)-induced liver tumors in rats produced alpha1-fetoprotein (AFP) and whether the age of the animals would influence such as appearance, a finding suggested by data seen in man. Other liver carcinogens (N-hydroxy-N-2-fluorenylacetamide, N-2-fluorenylacetamide, and diethylnitrosamine) were tested for their ability to induce liver tumors producing AFP. The presence of AFP. The presence of AFP in the serum was determined by double diffusion in agarose and by comparison also by quantitative radioimmunoassay. Using double diffusion, AFP was detected in the majority of tumor-bearing rats that had received either N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide. Sera of diethylinitrosamine-treated rats with liver tumors were all positive, whereas sera of rats bearing AFB1-induced tumors were positive in only a few cases. However, all sera of tumor-bearing rats examined had elevated AFP levels by radioimmunoassay. Nonetheless, the average level of AFP in the sera of rats bearing AFB1-induced tumors was considerably lower, compared to the sera of rats with tumors caused by diethylnitrosamine, N-2-fluorenylacetamide, or N-hydroxy-N2-fluorenylacetamide. Rats started on AFB1 when 6 weeks old had more mixed liver tumors with neoplastic hepatocytes and bile ducts and higher AFP levels than did rats started at 26 weeks of age. However, the histological grade of differentiation of inducted tumors did not seem to influence the AFP level.

Retardation of development including immunogenic expression of histocompatibility antigens in mice--by postnatal administration of antiandrogenic steroid.

A specific antiandrogenic steroid cyproterone acetate was administered daily to mice of three different inbred strains starting from the day of birth until the age of 30 days. The total dose per mouse was 17.2 mg. This treatment resulted in developmental retardation which was manifested in a number of ways: at the age of 30 days, the weight of the body was well as spleen, testes and particularly thymus was significantly reduced; histologically, the normal proportion of the red and white pulp in the spleen was changes; spermatogenesis (but not oogenesis) was markedly retarded corresponding to the age of 12-15 days in normal males; also skin displayed a persisting immaturity as reflected by an abundance of mast cells. Minor signs of toxic changes were seen in the liver. Skin grafts from CA-pretreated donors had a subnormal immunogenicity; when transplanted across the MSA-barrier, they survived significantly longer than control grafts and about 23% took. Significantly prolonged survival was also observed with H-3 incompatible skin grafts from CA-pretreated donors, particularly from male donors. Across the barrier dicated, but did not reach a level of significance. The present study extends our previous observations concerning the androgen dependence of a normal immunogenic expression of H-antigens. The antiandrogenic effect of CA is comparable to the more complex effect of neonatal orchiectomy in terms of the subnormal immunogeneity of MSA-incompatible skin grafts from 30-day-old males which seems to be arrated at a stage typical for 1-2-day-old normal males.

Biological properties of a canine distemper virus isolate associated with demyelinating encephalomyelitis.

A canine distemper virus (CDV), DESIGNATED R252, originally recovered from a dog with demyelinating encephalomyelitis was shown to reproduce this disease in gnotobiotic dogs with a high incidence as compared to other CDV strains, which produced an acute fatal infection. In this investigation, R252 was propagated for the first time in Vero cells and compared to two known strains of CDV, Snyder-Hill (SH) and Onderstepoort (Ond). The results of this study revealed that intracellular R252 accumulated more slowly than either SH or Ond. There was extensive destruction of Vero monolayers infected with either R252 or SH. Each virus induced the formation of intracytoplasmic and intranuclear inclusions. Ond infection resulted in minimal cytopathic changes and intracytoplasmic inclusions. Immunofluorescence studies indicated that the spread of R252 infection within the monolayers was intermediate between the rapidly spreading SH and slowly spreading Ond. R252-infected cells developed characteristic immunofluorescent cytoplasmic inclusions. Initially, each stained homogeneously and later appeared as a non fluorescent body surrounded by a fluorescent ring. This characteristic pattern of fluorescence was observed only infrequently in thelate stage of SH infection and was absent in Ond-infected cultures. Reciprocal neutralization studies indicated that the three strains are of one serotype. These findings suggest that R252-CDV has biological properties which differ from two other strains of CDV and which may have bearing upon the in vivo capability of this virus to produce demyelinating encephalomyelitis.

Role of the chondrocytes in the breakdown of pig articular cartilage induced by complement-sufficient antiserum to pig erythrocytes.

Previous work showed that in organ culture pig articular cartilage is not affected by complement-sufficient antiserum to pig erythrocytes (AS plus C') unless it is associated with soft connective tissue. In the presence of synovial tissue the matrix becomes depleted of proteoglycan and may finally disintegrate. Immunoglobulin fails to enter normal cartilage matrix, but penetrates depleted matrix and reacts with the surfaces of the chondrocytes. The present experiments were made to see whether the breakdown of partially depleted cartilage matrix would progress in AS plus C' after removal of the synovial explant. Affronted explants of articular cartilage and synovial tissue were cultivated in AS plus C' for 10 days (primary cultures). The synovial tissue was then removed and the isolated cartilage maintained for a further period either in AS plus or C' or in one of a variety of control media (SECONDARY CULTURES). The behavior of the isolated cartilage during the secondary culture period depended on the degree of breakdown attained at the end of the primary period. If only proteoglycan but not collagen had been seriously depleted, degradation of the matrix did not progress in AS plus C' and slight regeneration of metachromatic materal sometimes took place; regneration of matrix was greater after transfer to control medium. If collagen as well as proteoglycan had been destrobyed, the chondrocytes assumed a fibroblastic form, their surfaces no longer reacted with IgG antibody, and in secondary culture they failed to regenerate new matrix.

A cleavage-associated neoantigenic marker for a gamma chain site in the NH2-terminal aspect of the fibrinogen molecule.

The E fragment, derived from the NH2-terminal aspect of fibrinogen by plasmin cleavage (fg-E), possesses two generically distinct sets of antigenic expressions. The major set of antigens is expressed by the parent molecule as indicated by the capacity of a major subpopulation of antibodies present in antiserum to fg-E and reactive with fg-E to: (a) react with fibrinogen, and (b) be specifically absorbed by fibrinogen but appears following proteolysis with plasmin. These cleavage associated neoantigens (fg-E-neo) specifically react with a minor subpopulation of antibodies present in antiserum to fg-E.E fragments isolated after varying exposures to plasmin all expressed fg-E-neo, but early E fragments exhibited quantitatively less neoantigenic expression than more extensively degraded E fragments. The entire fg-E-neo expression is recovered on a single isolated constituent chain of the E fragment, and immunochemical analysis with antiserum to the isolated constituent chain-bearing fg-E-neo identifies it as a derivative of the gamma chain constituent, exhibits marked stability to physicochemical denaturation and enzymatic degradation. These properties suggest that the neoantigen may be associated with a specific amino acid sequence which is exposed by the cleavage process. The identification and localization of fg-E-neo provides a specific molecular marker site for the characterization of structural and conformational changes associated with catabolism and function of fibrinogen.

Variation with age and disease of an amyloid A protein-related serum component.

Using the radioactively-labeled alkaline-degraded acid-soluble fraction of amyloid ([ 125I ]DAA), we developed a radioimmunoassay for the previously described amyloid-related component of the human serum (SAA). Screening the sera of 228 normal individuals and of 297 patients with a variety of illnesses, we found that SAA is a component of all human sera, including cord blood (mean 94 plus or minus 57 ng/ml). The concentration of this component increases significantly with the aging process, reaching very high levels in the eighth and nine decades. It is also elevated in all cases of amyloidosis (except for those associated with nephrotic syndrome) as well as in many patients with myeloma, macroglobulinemia, lymphoma, carcinoma, rheumatoid arthritis, and tuberculosis. A marked increase was noted in the early stages of a variety of acute inflammatory and infectious states with a return to normal levels paralleling clinical improvement and faster than the erythrocyte sedimentation rate. The possible implications of this component in the genesis of amyloid and in the immune process are discussed.

Lack of B lymphocyte depletion from murine spleen cell populations by a human gamma-globulin, anti-human gamma-globulin column system.

It has been reported previously that enriched populations of mouse thymus-dependent (T) lymphocytes could be prepared by the use of a column procedure that is believed to remove selectively cells with receptors for antigen-antibody complexes. In our hands, this procedure does not selectively remove B lymphocytes but rather masks the surface immunoglobulin. Analysis of the column-passed cells revealed an apparent decrease in the percentage of immunoglobulin-bearing cells but little decrease in either the percentage of complement receptor lymphocytes or the mitogenic response to lipopolysaccharide. Furthermore, rabbit immunoglobulin could be detected on the surface of 35 to 45% of the cells emerging from the columns prepared with rabbit anti-human gamma-globulin (HGG). After overnight incubation, 30 to 40% of the emerging cells reexpressed mouse immunoglobulin on their surfaces. Adsorption of the antisera used to make the columns with mouse gamma-globulin (MGG) diminished the depleting capacity of the columns. We conclude from these observations that antibodies in the anti-HGG sera, cross-reactive with mouse immunoglobulin, account for the spacious depletion of B lymphocytes observed when cells are passed over these antigen-antibody complex columns.

Broad antigenic relationships among rhinovirus serotypes revealed by cross-immunization of rabbits with different serotypes.

The rhinoviruses are a large group of at least 89 serotypes and extensive cross-relationships have been demonstrated among these serotypes using both rabbit and human antisera. The extent of these cross-relationships was further investigated by sequential intravenous immunization of rabbits with different virus serotypes, known to be both related and unrelated. In all cases in which a relationship had previously been demonstrated with monovalent antiserum, after heterotypic stimulation with the related virus, the antibody titer to the virus used as primary immunogen showed an anamnestic response, whereas the antibody response to the secondary immunogen was good, but resembled a primary response. In some instances when immunized rabbits were injected with an unrelated antigen, previously unsuspected relationships were revealed. A number of clusters of related rhinoviruses were demonstrated: 5, 17, 42; 3, 4, 6, 14; 9, 32, 67; and 13, 14, 41. Heterotypic immunization proved to be a powerful biologic probe for detecting relationships among the rhinoviruses, and together with newly available epidemiologic data it offers a possible approach for design of a vaccine for the common cold.

Structural studies on induced antibodies with defined idiotypic specificities. I. The heavy chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

Amino acid sequence analysis has been performed on three groups of heavy (H) chains of A/J mice. H chains derived from unimmunized animals were compared to anti-p-azophenylarsonate (anti-Ar) antibodies which were further subdivided into those possessing and those depleted of a cross-reacting idiotype (CRI). It was found that anti-Ar antibodies bearing the CRI are homogeneous through the first hypervariable region of the H chain. The same sequence was obtained for pooled antibody isolated from the ascites fluid of 18 A/J mice or from a single mouse. The H chains appear to belong to a minor V-H subgroup. In the first 30 positions Anti-Ar antibodies depleted of the CRI had the same sequence as those containing the CRI (with small amounts of heterogeneity at some positions), but contained a mixture of sequences in the first hypervariable region of the H chain. These studies indicate that antibodies with similar specificity and with identical framework sequences, but which differ in their hypervariable regions, contain different idiotypic determinants, and support the concept that the idiotypic determinants reside primarily within hypervariable regions.

Cardiac histamine-ouabain interaction: potentiation by ouabain of the arrhythmogenic effects of histamine.

Cardiac effects of histamine include stimulation of sinus rate and ventricular contractile force, impairment of atrioventricular conduction and increase in ventricular automaticity. Atrioventricular block and increase in ventricular automaticity are common features of digitalis toxicity. The purpose of the present investigation was to study the influence of low concentrations of ouabain on the cardiac effects of immunologically released and administered histamine. Hearts excised from guinea pigs passively sensitized to penicillin antigens responded to antigen with sinus tachycardia, atrioventricular conduction block, increase in ventricular automaticity, decrease in coronary flow rate and histamine release. During anaphylaxis in the presence of ouabain, 10-9 and 3 times 10-9 M, the duration of conduction arrhythmia and the incidence of ventricular automaticity were greatly increased. Dose-response studies for the cardiac effects of exogenous histamine were conducted in vitro in the presence of ouabain 10-9 and 3 times 10-9 M. Ouabain, in a concentration-dependent fashion, potentiated histamine-induced prolongation of the P-R interval, but not the increases in sinus rate and in ventricular contractile force. Oution block and idioventricular also greatly increased the incidence of histamine-induced atrioventricular conduction block and idioventricular rhythms. Our results clearly identify a histamine-Ouabain interaction leading to severe disruption of atrioventricular conduction and to increased ventricular automaticity.

Light-microscopical demonstration of different cell membrane structures of human peripheral blood lymphocytes (PBI).

Labelling of membrane structures in human peripheral blood lymphocytes is achieved by a Fe-(III)-hydroxide-glycane-complex. According to the distribution of labelled structures, coarse-granular clustered (about 80%) and fine-granular diffuse marked peripheral blood lymphocytes can be distinguished. Each of these populations seems to have morphologically discernible subpopulations.

Treatment of osteolytic myelomatosis with mithramycin.

The treatment of rapidly progressive skeletal demineralisation in myelomatosis has been studied with the help of metabolic calcium balance in two patients; In one, osteoporosis accelerated during treatment with melphalan and prednisolone, although he remained normocalcaemic throughout, suggesting that osteoporosis was aggravated by corticosteroid therapy. In the other patient, who was initially hypercalcaemic, conventional treatment produced clinical remission before eventual relapse with more hypercalcaemia and skeletal dissolution. Both patients were then treated with mithramycin alone, and, although neither obtained haematological remission, bone pain was relieved, hypercalciuria and hypercalcaemia were abolished, and calcium balances proved that mithramycin was effective in restoring calcium equilibrium. The results indicate that mithramycin may abolish excessive bone resorption in myelomatosis and that severe bone dissolution may occur in the absence of hypercalcaemia. Regular determination of 24-hour urinary calcium excretion as well as of plasma-calcium is important in monitoring process. Mithramycin should be considered in the early treatment not only of hypercalcaemia but also of severe hypercalciuria, if these complications do not rapidly remit during the first course of conventional myeloma therapy, with or without steroids. Finally, these results add to evidence that a humoral factor may be responsible for osteoclast stimulation in myelomatosis.

Potential effect on coronary-heart-disease morbidity of lowering the blood-cholesterol.

As a practical aid to physicians and patients facing the decision whether or not to embark upon a life-long lipid-lowering regimen in the hope of preventing coronary heart-disease, estimates have been made of the potential benefits to be gained from lowering the plasma-cholesterol over a period of 20 years. The estimates have been derived from analyses of experiences in the Framingham population study. The results suggest that if 100 men who are non-smokers, with normal blood-pressure and electrocardiogram, lower their plasma-cholesterol from 310 to 260 mg. per 100 ml. starting at 35 years of age, 6 could potentially benefit by avoiding a coronary incident, 94 would be likely to follow the regimen without apparent benefit, and 8 of these would have an attack within 20 years despite adherence to the regimen. The potential benefit is less for women and for those who start the regimen at an older age. It is greater if cholesterol is lowered further and if other risk factors are present: for instance, 29 of 100 men starting at age 35 who are ciagrette smokers with moderate hypertension and left-ventricular hypertrophy and who reduce their plasma-cholesterol concentration from 310 to 210 mg. per 100 ml. would benefit.

Ovulation induction: ovarian response to human gonadotropins and synthetic gonadotropin-releasing hormone.

Human ovarian responses to FSH- and LH-releasing hormone (FSH/LH-RH) were observed at laparotomy and studies by histologic and histochemical examination of ovarian biopsy specimens. The responses were compared to those induced by human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG) singly and together. The subjects were healthy, fertile, young women rendered anovulatory by injections of depomedroxyprogesterone acetate (DMPA) or depochlormadinone acetate (CA). Supplementary studies included measurement of urinary pregnanediol, examination of the cervix and vagina for estrogenic and progestational responses, and endometrial biopsy, Both HMG and HCG induced follicular growth and proliferation of granulosa and theca cells, but neither, when given alone, induced ovulation or corpus luteum formation. When given in conjunction they induced single or multiple ovulations and corpora lutea in 11 of 18 women treated. FSH/LH-RH CONSISTENTLY STIMULATED FOLLICULAR DEVELOPMENT AND INDUCED OVULATION IN 2 OF 16 WOMEN TREATED. Preovulatory mature follicles were found in 3 more. FSH/LH-RH may prove to be useful in the treatment of some cases of anovulatory sterility of hypothalamic origin.

Physiological mechanisms for cardiac control by nutritional intake after early maternal separation in the young rat.

Series of analytic experiments are presented that explore possible physiological mechanisms for the control of cardiac rate by nutritional intake in the pre-weanling rat. The essential properties of the nutrient and the first site of action were studied by using fluids of different pH, osmolality and chemical composition administered intravenously as well as intragastrically. Several probable effector pathways were explored: neuroendocrine (adrenal medullary and adrenocortical, thyroid), cholinergic and adrenergic. Pharmacological blocking agents, surgical removal of glands, replacement hormones and spinal cord trasaction were utilized. Afferent pathways such as vagus and splanchnic systems were approached surgically and the gastrointestinal hormones, histamine, insulin and glucagon, were studied by administration and pharmacological blockade. The evidence tended to rule out a number of possible mechanisms and pathways and to make it appear likely that nutrient acts initially at the gut wall, that the CNS then responds by increasing tone in the classical spinal cardioacceleratory pathways to the beta-adrenergic synapses of myocardium.

[Behavior of cerebrospinal fluid proteins in degenerative diseases].

132 cerebrospinals fluids from patients with degenerative diseases of the central nervous system have been analyzed for protein distribution in agar gel electrophoresis. After subdivision into diagnostically well defined groups these patients were compared with 48 with metabolic and psychiatric diseases and with 79 normal controls. The majority of diagnostic groups showed a tendency to permeability impairment. Other outstanding deviations were not found, except for single cases which were not statistically typical of the groups as a whole. However, the "degenerative type" of proteinogram emphasized in the literature predominated not only in the degenerative groups but also in certain other diseases with destruction of central nervous tissue which do not belong to the degenerative diseases in the strict sense. On the other hand, there are some degenerative subgroups without this "typical" electrophoretic pattern. There would thus appear to be grounds for amending the term "degenerative" into "tissue destroying" or "atrophic type", to avoid misinterpretation.

Soluble proteins of bronchopulmonary secretions from patients with cystic fibrosis, asthma, and bronchitis.

The concentrations of nine plasma proteins were determined by quantitative immunoelectrophoresis in sputum specimens from 29 patients with cystic fibrosis (CF) and from 24 patients with severe asthma and chronic bronchitis. The results suggested that the population of CF patients could be divided into two groups in spite of an absence of difference in clinical status between the groups. Average concentrations of seven plasma proteins in sputum of group I CF patients were identical with those in sputum of patients with bronchitis, but the average concentrations of six of these proteins in sputum from group II CF patients were higher than those in specimens from the bronchitic patients and were similar to corresponding concentrations in sputum from patients with asthma, all of whom were examined while in status asthmaticus. The average concentrations of 14 secretory proteins were the same in all sputum specimens whether or not they were produced by patients with cystic fibrosis, asthma or bronchitis. It was concluded that the concentrations in the bronchopulmonary secretions of proteins associated with host defence were not diminished in patients with cystic fibrosis, and failure to produce adequate concentrations of proteins with antimicrobial activity was unlikely to be responsible for the above average susceptibility to chest infection in cystic fibrosis. It is suggested that there exists a group of CF patients in whom a pulmonary allergic reaction generates an inflammatory response as severe as that characterizing status asthmaticus and that this response could be detrimental.

Computer analysis of defined populations of lymphocytes irradiated in vitro: I. Evaluation of murine thoracic duct lymphocytes.

Computer-assisted morphometric analysis of murine lymphocytes obtained by thoracic duct cannulation demonstrates two populations of cells; the larger population (73 percent) appears to be thymus-derived and the remaining 27 percent is of bone marrow origin. Following exposure to varying amounts of x-radiation, morphologic alterations in both populations are evident. The smaller cell populations are evident. The smaller cell population exhibits some of these changes at lower dose levels than does the larger population. In addition, the character of the radiation-induced changes appears to be different for the two populations of lymphocytes. After 500 rad, the nuclei of the larger population appears unchanged; the nuclei of the population representing 27 percent of the cells have become enlarged and vacuolated and are thought to be edematous. After 2000 rad, the nuclei of the larger population appear pyknotic with coarsely clumped chromatin. In the examined set of cells, the smaller population could no longer be detected after 2000 rad. Such disparate responses to radiation-induced injury may correlate with known differences in immunologic function which serve to distinguish thymic-dependent and bone marrow-derived small lymphocytes.

Centrifugal Cytology, IV. The Prearation of fixed stained dispersions of gynecological cells.

The Centrifugal Cytology technique has been utilized to produce glutaraldehyde fixed stained dispersions of both conventional Ayre scrapes and Davis pipet (PAPette) samples. Light microscope studies of dispersions of both types of cells on conventional microscope slides indicated that both the tinctorial and morphological appearance of the cells after Papanicolaou staining was very similar to that observed with conventional smears and that the same criteria could be utilized with the Centrifugal Cytology dispersions to screen the cells for cancer as had previously been used with the smears. A preliminary study indicated that six out of six positives with no false negatives or false positives were found. The Centrifugal Cytology technique appears to have promise as a method for preparing suspension samples such as pipets of gynecologic cells. Scanning electron microscope studies reveal that the squamous epithelial cells are very thin and at least some of them are covered by a network structure.

Immune-indian ink method for detection of hepatitis A associated antigen and antibody.

TIndian-ink grains coated with commercial gamma globulin (immune-Indian-ink) were agglutinated by 3 percent of sera from healthy volunteer blood donors; by 4 percent of those from hospital staff in contact with patients suffering from hepatitis; and by 10 percent of those from patients with viral diseases other than hepatitis, In contrast, the rate of positive reactions was 86 percent in the case of sera taken from patients in the acute phase of an illness diagnosed as hepatitis A on the basis of epidemiological and clinical data. Investigation of serum samples taken serially from patients positive in the acute phase of illness revealed that the immune-Indian-ink agglutinating factor does not persist for long in majority of cases. Two months after discharge from the hospital it was present in 18 percent of the patients only. The reaction proved negative when a limited number of cases diagnosed as hepatitis B were investigated. The immune-Indian-ink agglutinating factor was inhibited by all but one of 36 sera taken in the convalescent phase from patients with a diagnosis of hepatitis A. Some sera displaying agglutination with immune-Indian-ink gave a reaction with uncoated Indian-ink, too. Efforts to free the sera from non-specific agglutinating factor by starch-block electrophoresis have led to partial success. Fractionation on Sephadex G-200 columns suggested that in molecular weight (or particle size) the immune-Indian-ink agglutinating factor is smaller than HBsAg and larger than the non-specific agglutinating factor. On the basis of these results it is assumed that the immune-tindian-ink reaction is suitable for detecting an antigen tentatively called IH chi Ag and its antibody (IH chi Ab) specific to hepatitis A.

Alterations in the upper facial growth of Macaca mulatta resulting from high-pull headgear.

Four prepubertal Macaca mulatta monkeys, ranging in age from 13 to 24 months, were used in an investigation of the effects of high-pull headgear (to a face-bow) therapy on the growth of the upper facial skeleton. Amalgam bone implants were placed across the frontomaxillary, frontozygomatic, zygomaticomaxillary, and zygomaticotemporal sutures in each animal. Three of the monkeys wore appliances consisting of a maxillary dental spling, a face-bow, two coil springs, and an acrylic helmet. The fourth monkey (control) wore only a dental splint and a face-bow. A continuous high-pull headgear force of 300 grams per side was applied to the three monkeys for 81, 87 and 89 days, respectively, before death. Procion brilliant red 8-HBS vital stain was administered to all four animals at the start of and 3 days before the end of the treatment period. The facial growth patterns were determined from lateral cephalograms taken before and after treatment, from direct measurement of implant separation at the sutures, and from histologic sections of the four mentioned facial sutures.

Potential for polyvalent infectious bronchitis vaccines.

Numerous antigenic types and subtypes of infectious bronchitis virus (IBV) have been isolated and characterized from field epornitics of infectious bronchitis (IB). Thus obvious failures in obtaining the necessary protection from vaccination have been documented and they underscore the urgent need for vaccines which stimulate a broader spectrum of protection from heterologous IBV challenge than is presently obtained. If polyvalent IB vaccines are to be employed, assurance must be provided that variant IBV serotypes are not indiscriminately spread throughout the country or globe. Furthermore, possible interference, alteration, and reduction in immune response with the component antigens should be critically evaluated. Differences and comparative levels of postvaccinal respiratory signs with the different antigens, used singly and in combination, should be determined. As an alternative to polyvalent IB vaccines, certain Massachusetts-type strains, e.g., a Holland isolate IBV, at different passage levels in the authors' laboratory, have induced heterologous and homologous protection to some serotypes causing vexing field problems. Advantages are present in using a single antigenic type IB vaccine, if it is safe and effective, when compared to polyvalent vaccines. The absence of a serologic relationship to the results of immunity challenge is frequent with IBV isolates, which emphasizes that challenge results are considerably more important. In some IB problems, on a local or regional basis, autogenous vaccines may be indicated. Because of the multiplicity of IBV serotypes, however, it is doubtful that completely effective and safe IB vaccines will be forthcoming in the near future to satisfy the diverse needs of a dynamic poultry industry.

Comparison of the specificity of human and bovine tuberculin PPF for testing cattle. 3. National trial in Great Britain.

A field trial on a country-wide basis was undertaken to compare the specificity for bovine tuberculosis of single and comparative tuberculin tests in cattle using either Weybridge human or Weybridge bovine PPD. The tests were made on 10,305 cattle in 179 herds distributed throughout all regions of England, Scotland and Wales. Results showed that a comparative tuberculin test using avian PPD with either human or bovine PPD had a much higher efficiency than a single injection of mammalian tuberculin in the neck of cattle, and confirmed that a comparative test is still essential in the British environment. Weybridge bovine PPD gave significantly better discrimination between tuberculous and non-tuberculous cattle than Weybridge human PPD when used together with avian PPD in a comparative tuberculin test. The diameter of induration gave an absolute measure of the extent of oedema, if present, and induration diameter used in conjunction with skin thickening increased the sensitivity and specificity of the test. Rules of interpretation were developed and are presented for an intradermal comparative tuberculin test in cattle using Weybridge avian and bovine PPDs.

Mitochondria from human term placenta. II. Characterization of respiratory pathways and coupling mechanisms.

Pathways of electron transport utilized for respiration in human term placental mitochondrial preparations were differentiated and characterized through the use of classical respiratory chain inhibitors and multiple sources of reducing equivalents. Mechanisms of associated energy conservation and utilization were examined in the preparations with uncouplers and inhibitors of phosphorylation. Inhibition by rotenone, antimycin A and cyanide established the classical electron transport chain as the major pathway of respiration with glutamate and succinate as substrates. Approximately 20% of glutamate-supported respiration was insensitive to inhibitors and may proceed by the cytochrome P-450 linked pathway of electron transport. Approximately 50% of ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine supported respiration was insensitive to 10-3 M cycanide and must utilize an undefined by-pass of cytochrome oxidase. A rotenone- and antimycin-insensitive, exterior pathway for NADH oxidation was demonstrated which could be artificially linked by exogenous cytochrome c to the cytochrome oxidase region of the classical electron transport system. Glycerol 3-phosphate also supported oxidative phosphorylation yielding ADP/O ratios of 2. Respiration of placental mitochondria was stimulated by 2,4-dinitrophenol and gramicidin. With succinate, dinitrophenol-stimulated respiration exceeded that obtained in the presence of ADP. Oligomycin and atractyloside prevented the stimulation of respiration by ADP. Thus, respiration appeared coupled through normal mechanisms to ATP formation and ion transport. A preferential coupling of respiration to the energy-utilizing processes of steroid hormone biosynthesis may exist.

Axoplasmic transport of a brain-specific soluble protein.

The rate and extent of axoplasmic transport of the brain-specific soluble protein (14-3-2 protein) has been investigated in the avian visual system. 1-day-old chicks were injected monocularly with tritiated proline, Incorporation of the isotope into the 14-3-2 protein synthesized within the retina of the injected eye, as well as the appearance of the labeled protein in the optic lobes was determined at 6 h and 6 days. These time periods were chosen to distinguish between the rapid and slow phases of axophlasmic flowmfollowing preparation of high-speed supernatant fractions, dialysis, chromatography on Sephadex G-150 and immunoprecipitation with specific antiserum, identification of the labeled 14-3-2 protein was carried out by sodium dodecylsulfate-polyacrylamide gel analysis of the radioactive immunoprecipitates; 6 days after isotope administration, approxo% of the 14-3-2 protein synthesized in the chick retina had been transported to the contralateral optic lobe. By contrast, at 6 h no labeled 14-3-2 protein was detectablemthus, transport of this neuronal protein appears to be relatively slow process with little or no rapid component.

Coagulation, hemostasis, and plasma expanders:a quarter century enigma.

Despite more than 2 decades of research, the explanation of the long-known hemostatic failure consequent to the use of some natural and synthetic macromolecular agents as plasma substitutes remains obscure. Conventional clotting parameters are not significantly affected in vivo or in vitro. Dextran, hydroxyethyl starch, and many other colloid macromolecules precipitate Factors I and VIII, fibrin monomer, and perhaps v. W. (von Willebrand) factor(s) from plasma, rendering at least the first three insoluble, in relation to the molecule size and concentration of the colloid, and for dextran, its intrinsic viscosity. The precipitate, rich in Factors VIII and I, redissolves on warming, and reprecipitates on cooling, behaving as a cryo-Factor I. In composition it closely resembles the cryoprecipitate obtained by slow-thawing of plasma. Both clot faster with thrombin than the parent plasma. The amount precipitated from plasma by dextran or hydroxyethyl starch varies very widely from individual to individual. Cryo- of dextran-precipitable material can be obtained by interacting purified Factor I with a miniscule amount of thrombin. Dextran, hydroxyethyl starch, polyvinyl pyrrolidone, some forms of gelatin, and several polyamino acids accelerate thrombin clotting of normal plasma, several dysfibrinogenemic plasmas, or Factor I. Albumin, hemoglobin, some modified gelatins do not. Poor platelet thromboplastic function appears some hours after dextran infusion, associated with morphologic capillary abnormalities that strikingly resemble those in v. W. disease. We postulate that the hemostatic defect associated with the use of plasma substitutes is a form of induced v. W. disease or disseminated intravascular clotting, ensuing from precipitation and removal of v. W. factor(s), Factors VIII and I, microcirculatory abnormality, and platelet malfunction. The latter two supervene some time after administration of dextran. It reported antithrombotic activity is perhaps referable to the same action.

Testis antigens of man and some other primates.

Rabbit antisera raised aginst testis preparations of human, chimpanzee, rhesus monkey, and baboon origin were used to study testis-specific antigens within and among the four primate species. Antisera were absorbed with serum, liver, kidney, and spleen preparations of the respective species against which they had been produced. Immunoelectrophoretic analysis of testis extracts, using the absorbed antisera, indicated the following minimum numbers of testis-specific antigens for each species: man, 10; chimpanzee, 8; rhesus monkey, 10; and baboon, 8. Most of the testis antigens were cross-reactive among species. The results suggest that human spermatozoa possess at least four to five specific antigens which originate in the testis. The antigen that induces sperm-immobilizing antibody cross-reacted among the four species of primates. Human and rhesus monkey sperm reacted equally in the immobilization system. Testis extracts from each primate species were capable of removing the sperm-immobilizing activity of human immune sera by absorption. Testis proteinase activity, as determined by using a gelatin membrane substrate, was inhibited by the gamma-glogulin fractions of rabbit and rhesus monkey antisera and also appeared to be cross-reactive among the species.

Immunity to influenza.

Immunity to influenza virus may be considered from the standpoints of viral and hostfactors. Amonst viral factors the phenomena of antigenic 'shift' and 'drift' of the surface antigens. Hemagglutinin HA and neuraminidase NA, are of utmost importance in enabling the virus to combat host immunity and to produce recurrent pandemics and epidemics of disease. 'Shift' involves major changes in the antigenic character of the HA and NA antigens, and serological studies reveal little or no crossreactions between antigens of different subtypes. However, immunological 'memory' may exist between the surface antigens of viruses of different subtypes, for example between the hemagglutinins of Asian (H2) and Hong Kong (H3) subtypes. Antigenic 'drift' occurs more frequently than 'shift' and involves subtle changes in the antigenic configuration of HA and NA within a subtype. It is clear that the hemagglutinin antigen contains a multiplicity of antigenic determinants, one or more of which remain stable (CR determinants) whilst others (strain-specific determinants) change completely during antigenic 'drift'. Amongst host factors humoral antibody to HA and NA appears to be a reasonable index of natural or vaccine induced immunity to infection. As far as the specificity of such antibodies is concerned, it is likely that future studies will place considerable emphasis on the distinction between antibody for the stable (CR) antigenic determinants and antibody to strain-specific determinants. It seems likely that antibody to strain-specific determinants is of more relevance to immunity than CR antibody. The studies of Virelizier et al. described elsewhere in this Symposium employing single-radial-diffusion tests give hope of simple methods for the investigation of such antibody specificity. There is no evidence that antibody to the internal, antigenically stable ribonucleoprotein is related to immunitywhilst the role of the other internal antigen, the membrane protein, in immunity is at present unknown. Numerous studies have been carried out to establish the importance of secretory antibody in the respiratory tract. Although secretory IgA specific for the influenza virus has been clearly demonstrated in the respiratory tract, the levels are low and its significance is not yet established. Whilst the role of cellular aspects of immunity to influenza in man remains to be established there is no doubt from the recent studies of Tyrrell and his colleagues that delayed hypersensitivity to influenza antigens can be demonstrated. In considering the host aspects of immunity it is important to attempt to distinguish factors which are merely an index of past exposure and those which are involved with the actual mechanism of immunity. It is likely that animal experimental models, in particular with inbred mice, will provide an important contribution in this field of investigation.

The effects of fast neutrons on inoperable carcinoma of the stomach.

Thirty-nine unselected patients suffering from inoperable, recurrent, or residual adenocarcinoma of the stomach were referred for palliation with fast neutrons from the Medical Research Council's cyclotron at Hammersmith Hospital. A full course of 1440 rads given in 12 treatments over 26 days was administered to the patients. Because of the relatively low energy (7-5 MeV) of the beam from this particular machine, it was not possible to deliver the full dose uniformly throughout the tumour except in extremely thin patients. Pain, dysphagia, vomiting, and bleeding were relieved in the majority of cases. The side effects were minimal and easily controlled. Palpable masses disappeared. Five patients required surgery after neutron therapy. All the incisions were made through irradioated tissue and all except one healed normally. Tumour was present outside the treated area, but the absence of any palpable mass within the treated area was a consistent finding. Radiologically, the stomachs remained abnormal and later changes included gross mucosal abnormality and shrinkage. Fourteen patients came to necropsy and in 10 no tumour was present macroscopocally. Tumour cells were seen in all except two cases but these were few, surrounded by dense fibrous tissue, and may not have been viable. The remaining stomach was abnormal with a thickened wall and destruction of mucosa. Three of the four cases in which macroscopic tumour was present received less than the standard dose because of the inadequate penetration of the beam. Excellent regression of tumors was achieved by the neutrons, but the stomachs did not recover from this satisfactorily. Gastrectomy four to six months after treatment is therefore suggested. This operation and other surgical procedures in other patients were successfully carried out. There is a need for higher energy neutrons to improve treatment and extend it to patients of thick-set build.

Epstein-Barr virus-induced transformation of human leukocytes after cell fractionation.

The efficiency of transformation of human lymphocytes after infection with Epstein-Barr virus (EBV) was determined in fractionated and non-fractionated preparations derived from 16 human cord blood samples and two blood samples from adult donors. The transformation efficiency of macrophage-depleted leukocytes was consistently lower as compared to non-fractionated leukocytes. Additional depletion of B-cells resulted in a further decrease. Reduction of T-cells, however, did not influence significantly the transformation rate. In non-fractionated leukocyte cultures, as well as in macrophage-depleted and B-cell enriched cultures, colonies of transformed cells were regularly observed within the first week of cultivation. All cell lines established after EBV-infection revealed membrane-bound immunoglobulin. Reconstruction of macrophages-depleted, B-cell enriched or B-cell depleted cultures with autologous macrophages resulted in an increase of the transfromation efficiency up to the values of non-fractionated leukocyte preparations. Addition of heterologous human embryonic lung fibroblasts resulted in a similar increase. The results support the interpretation that EBV transforms only those cells of the hematopoetic system which are derived from the bone-marrow entity. The transformation efficiency is considerably increased by co-cultivation of lymphocytes with macrophages and heterologous human fibroblasts which seem to excert a feeder-layer effect by enhancing survival of lymphocytes in vitro.

Histochemical conditions influencing metachromatic staining. A comparative study by means of a model system of polyacrylamide films.

The influence of different histochemical conditions on some metachromatic staining reactions has been studied using polyacrylamide films containing pure glycostaminoglycans. The films were incubated in fixatives without staining, and in glycerol, diethylene glycol and other glycols, formamide, N,N-dimethylformamide, dimethyl sulphoxide and ethanol (of several concentrations) after staining and their absorption (metachromatic) spectra recorded. In the case of heparin and heparan sulphate the metachromasy was disturbed when the films were immersed before staining in some fixative solutions containing formaldehyde and acid. After equilibration of stained films in organic solvents, changes in the absorption peaks were found to depend on the type and concentration of solvent, the type of glycosaminoglycan and the type of dye. Films containing glycosaminoglycan plus protein were used to investigate the blocking of the metachromatic reaction as the result of ionic interactions with proteins. The parameters that influence this phenomenon (e.g type of protein, glycosaminoglycan and dye, pH of staining) are discussed and a three-dimensional picture is introduced which can explain some of the results obtained in these experiments.

Localization and characterization of carbohydrates in adrenal medullary cells.

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.

Adenylate deaminase. A method for its localization in skeletal muscle.

A method for light microscopic localization of adenylate deaminase in sections of frozen rat quadriceps muscle is described. The method depends on the hydrolysis of 6-chloropurine ribonucleotide (the 6-chloroanalogue of adenylate) and the trapping of Cl minus by Ag plus. The resulting AgCl precipitate was made visible by exposing the sections to light. After this treatment black deposits about 1 mu in diameter were seen in muscle cells. These observations indicate that adenylate deaminase of rat quadriceps muscle is located at discrete sites within the muscle cells.

Biologic significance of disulfide bonds in human IgE molecules.

E myeloma protein, PS, was reduced in different concentrations of dithiothreitol (DTT) for 1 hr followed by alkylation with 14C-iodoacetamide. The affinity of the reduced-alkylated molecules for target cells was evaluated by their ability 1) to sensitize primate skin in a reversed P-K reaction, 2) to sensitize human basophils in a reversed-type histamine release and 3) to block passive sensitization with reaginic antibody. Antibody-epsilon0 antibody was employed for reversed type reactions to avoid participation of cell-bound normal IgE in the reactions. The sensitizing activity of IgE did not change following reduction in 1 mM DTT, which split inter-heavy-light chain disulfide bond. The activity of IgE significantly diminished after reduction in 2 mM DTT followed by alkylation. This treatment resulted in the cleavage of two intra-epsilon-chain disulfide bonds, which are present between the hinge and the Fd portion of the molecules. The reduced-alkylated protein was capable of sensitizing primate skin and human basophils, however, a much higher concentration of the reduced-alkylated protein than the native protein was required for passive sensitization. The optimal sensitization period for the reversed P-K reaction was 3 hr with the reduced-alkylated protein. The protein had the ability to block passive sensitization with reaginic antibody. The reduced-alkylated protein and the native protein were labeled with 125I, and binding of these proteins with human basophils was examined by autoradiography. The results showed that affinity of the reduced-alkylated protein for basophils was less than that of native protein. Since the disulfide bonds split by 2 mM DTT were not included in the Fc portion of the molecules, the Fc fragment was obtained from the reduced-alkylated protein and was tested for affinity for basophils. It was found that the Fc fragment had higher affinity than the reduced-alkylated protein. Recovery of the affinity by papain digestion strongly suggested that cleavage of disulfide bonds in the Fab portion of the molecules induced conformational changes in the Fc portion which is involved in binding to the target cells. Reduction of IgE with 10 mM DTT followed by alkylation resulted in cleavage of 5 disulfide bonds, which is accompanied by a loss of both sensitizing and blocking activities. The fifth disulfide bond which was cleaved by 10 mM DTT, but not by 2 mM DTT, appears to be an inter-heavy chain disulfide bond in the Fc portion of the epsilon-chains. Neither epsilon1 nor epsilon2 determinants in the Fc portion of epsilon-chains were degraded by this treatment.

Inheritance of antibody specificity. II. Anti-(4-hydroxy-5-bromo-3-nitrophenyl) acetyl in the mouse.

Mice of 17 inbred strains produced anti-(4-hydroxy-5-bromo-3-nitrophenyl)-acetyl (NBrP) of three different fine specificity types. Anti-NBrP antibodies of all allotype b mice (five strains tested) had a high relative affinity for (4-hydroxy-3.5-dinitrophenyl) acetyl (NNP) but low for (4-hydroxy-5-cloro-3-nitrophenyl) acetyl (NCP). Another category was characterized by high relative affinity for NCP but low for NNP. This category included most of the tested strains. The third category (CBA and C3H strains) had an intermediate fine specificity. Associated with fine specificity characteristics were anti-NBrP titers, mice of allotype b had lower titers than the other mice. Studies of congenic, recombinant inbred, F1 and backcross mice showed that both fine specificity and the magnitude of the anti-NBrP response of tbalb/C MICE WERE CONTROLLED BY AN ALLOTYPE-LINKED GENE. This gene was dominant over the C57BL/6 ALLELE. Lack of recombinant mice in the backcross generatioterns on the other suggest close linkage between the two genes.

Genetic control of immune response. The dose of antigen given in aqueous solution is critical in determining which mouse strain is high responder to poly(LTyr, LGlu)-poly(LPro)--poly(LLys).

Antibody response to different doses of (T,G)-Pro--L, given in aqueous solution, was investigated in the high responder SJL and low responder DBA/1 strains by measuring hemolytic plaque-forming cells (PFC) in the spleens as well as hemagglutination titers in the sera. The gene responsible for the difference between the two strains in the response to this antigen, given in complete Freund's adjuvant, has been previously denoted Ir-3. This gene is not linked to the major histocompatibility locus. In the response to the optimal dose (1 mug) of antigen, no difference could be shown between the strains. The peak of the response and the numbers of direct and indirect PFC were similar in both strains in the primary and secondary response. After injection of higher doses (10-100 mug) of antigen, both the direct and indirect PFC responses were lower in the low responder than in the high responder strain. Moreover, the peak of the response occurred earlier in the high responder strain in the primary response to the 10 mu dose of antigen. After administration of a suboptimal dose (0.02 mug) of antigen, the low responder strain produced in the primary response 4-20 times more indirect plaques than the high responder strain. Also the number of direct plaques was higher in the low responder than in the high responder strain. The serum antibody responses to the optimal and higher doses of antigen were parallel to the PFC responses. From inhibition of PFC with free antigen, it was concluded that a similar proportion of cells was producing high and low affinity antibodies to (T,G)-Pro--L in both strains. High and low zone tolerance could be induced in the two strains with (T,G)-Pro--L, but no difference could be shown between the strains. It is suggested that the Ir-3 gene plays a role in the regulation of the balance stimulation and suppression according to the dose of antigen given.

Tolerance induction in B lymphocytes but thymus-dependent antigens. T cells may abrogate B-cell tolerance induction by prevent an antibody response.

Thymus-dependent protein antigens such as fowl gamma globulin (FGG) and dinitrophenylated-human gamma globulin (DNP-HGG), readily induced tolerance of the B cell in the absence of T cells even when these antigens were not deaggregated. However, when the same doses of antigen were given in the presence of T cells, the B-cell population was shown to be protected from tolerance induction, especially when the antigen was not in a deaggregated form. In this case, there was in fact evidence of a priming effect, manifest in both the B-cell and T-cell populations. The priming effect on the B-cell population was demonstrated by an increased response of mice pretreated with DNP-HGG, upon challenge with DNP conjugated to a heterologous carrier. The priming effect on the T-cell population was evident in a helper effect demonstrated in vitro. However, when euthymic mice which had been pretreated with large doses of FGG or DNP-HGG were challenged with the homologous carrier, the results were different. In this case, there was a profound suppression of the response against the carrier or the hapten on that carrier. Suppressor activity was also demonstrated in vitro and was shown to be sensitive to treatment with anti-theta-serum plus complement. Additionally it was shown that the effector phase of the suppression had a definite nonantigen-specific component. Thus, in pretreated euthymic mice, provided the homologous carrier was present, the response to a heterologous carrier was also suppressed. To account for the observation that nondeaggregated antigens can induce B-cell tolerance in athymic mice, but B-cell priming and T-cell-mediated suppression in euthymic mice, it is proposed that B-cell tolerance occurs when antigen at some critical dose interacts with the B cell in the absence of some second signal. This second signal is normally provided by the macrophage, probably with the assistance of the T cell, and its effect is to divert the result of the interaction of the B cell with antigen towards immunization and away from tolerance induction. When a large dose of an antigen that tends to form aggregates is given to an animal possessing functional T cells, both T-dependent helper and T-dependent suppressor activities are generated, thus accounting for a situation where the B-cell population is immunized, but B-cell activation is suppressed in the presence of the original carrier.

Spasmolytic constituents of Cedrus deodara (Roxb.) Loud: pharmacological evaluation of himachalol.

Himachalol has been identified as the major antispasmodic constituent in the wood of Cedrus deodara. The pharmacological studies of himachalol on various isolated smooth muscles (guinea pig ileum, rabbit jejunum, rat uterus, and guinea pig seminal vesicle) and against different agonists (acetylcholine, histamine, serotonin, nicotine, and barium chloride) indicated spasmolytic activity similar to that of papaverine. It was a more potent antagonist of barium chloride-induced spasm of guinea pig ileum than papaverine but less effective in reverting a similar spasm of rabbit jejunum and had no relaxing effect alone. In the conscious immobilized cat, intragastric administration of himachalol or papaverine (100 mg/kg) produced equal inhibition of carbachol-induced spasm of the intestine, lasting about 2 hr, but himachalol had a faster onset of action. Himachalol was devoid of spasmolytic effect on the bronchial musculature of guinea pig but was 3.3 times more potent than papaverine in antagonizing epinephrine-induced contraction of the guinea pig seminal vesicle. Intravenous injection of himachalol (3-10 mg/kg) in the cat produced a dose-dependent fall in blood pressure and an increased femoral blood flow.

Effects of intravenous hyperalimentation of plasma-lipoproteins in severe familial hypercholesterolaemia.

The plasma-lipoprotein response to intravenous hyperalimentation was studied in three patients with severe familial hypercholesterolaemia. Hyperalimentation substantially lowered plasma concentrations of cholesterol, low-density lipoproteins (L.D.L.P.), and high-density lipoproteins. The chemical composition of L.D.L.P. did not change. Triglyceride levels increased slightly in two patients and decreased in the third. Turnover of radiolabelled L.D.L.P. was disturbed as L.D.L.P. concentration fell but then returned to normal. The mechanism by which intravenous hyperalimentation rapidly lowers plasma-cholesterol in severe hypercholesterolaemia is unknown.

Correlation of the blood-level of a pregnancy-associated alpha-macroglobulin with the clinical course of cancer patients.

The variations in serum level of a pregnancy-associated alpha-macroglobulin (P.A.M.) were studied during the treatment of breast-cancer patients. Good correlation was found between P.A.M. concentrations and the course of the disease--levels rose prior to the clinical recognition of metastatic disease and decreased significantly on successful treatment. Periodic P.A.M. determinations may allow detection of tumour recurrence while it is still at a treatable stage.

Thioguanine as primary treatment for chronic granulocytic leukaemia.

In an attempt to convert the bone-marrow population from Philadelphia-chromosome (Ph-1) positivt to partially or wholly Ph-1 negative, thioguanine was used as primary therapy in seven patients with chronic granulocytic leukaemia (C.G.L.). Although eight episodes of neutropenia were induced, prolonged remission or conversion to Ph-1-negativity was not achieved in any patient. However, thioguanine was at least as effective as busulphan for initial therapy in C.G.L., and had the advantage of rapid reversibility of haemopoietic depression when discontinued. Thioguanine merits further evaluation as an agent for the management of C.G.L. in its chronic phase.

Second malignancies complicating Hodgkin's disease in remission.

The incidence of second tumours occurring in the course of Hodgkin's disease has been investigated in a series of 452 patients treated with standard chemotherapy or radiotherapy, combination chemotherapy alone, intensive radiotherapy alone, or both intensive radiotherapy and combination chemotherapy administered in sequence. 16 tumours were noted. When analysed according to mode of treatment, 6 cases occurred in a group of 62 patients who received both modalities. When analysed for age, sex, and man-years of follow-up, this group appears to have 14-5 times the risk of developing a second tumour. However, that subgroup which had a complete remission after intensive radiotherapy followed by a relapse of disease, prior to receiving combination chemotherapy, had the highest risk with 18-5 times greater incidence of second tumour than expected. It is noteworthy that, of the 16 second tumours, 2 were acute myeloid leukaemia; in both cases a similar chromosomal abnormality (45 chromosomes, C-group deletion) was noted. The mechanism of oncogenesis may represent a combination of the immunosuppressive effects and cellular effects of those forms of treatment.

Unique and repetitive sequences in multiple genes for feather keratin.

Embryonic chick feather keratins are a family of homologous polypeptide chains. The mRNA coding for these has been obtained in a pure state and transcribed into complementary DNA (cDNA) using the reverse transcriptase from avian myeloblastosis virus. Studies on the kinetics of hybridisation and reannealing of cDNA indicate that there are 25-35 different keratin mRNA species in the embryonic chick feather, and a total of 100-240 keratin genes in the chick genome. Each keratin gene contains both a unique and a repetitive sequence. It is proposed that the repetitive sequences are the keratin coding sequences and that the unique sequences correspond to untranslated regions.

Antibodies reactive with cell surface carbohydrates.

Normal and immune sera from various animal species were fractionated on columns of Sepharose covalently coupled with the glycoprotein fetuin. Elution of the material bound to fetuin yielded low but reproducible amounts of protein, ranging from 0.02 to 0.2% of the protein mass of the input sera. This material has been identified by immunoelectrophoresis in agar and by zone electrophoresis on cellulose acetate as immunoglobulin. The Ig fractions bound and agglutinated erythrocytes of various species, and also bound to cells from various mouse tissues including heart, kidney, thymus, and spleen. In all cases, the binding was inhibited by glycoproteins such as fetuin and thyroglobulin, by a glycopeptide isolated from fetuin, and by some bacterial lipopolysaccharides. When the binding of these Ig fractions to mouse splenocytes was tested in the presence of 17 saccharides, no inhibition of binding was observed except by sialic acid, D-galactose, N-acetyl-D-glucosamine, and D-mannose, all of which showed partial inhibition. Inasmuch as these four saccharides are present on the carbohydrate moiety of fetuin, the results suggest that the isolated material is a carbohydrate-specific Ig (CS-Ig) fraction of serum capable of binding to the carbohydrate portion of cell surface receptors and glycoproteins. When bound to lymphocytes, these CS-Ig molecules induced redistribution (patching and capping) of cell surface receptors. Moreover, the CS-Ig fractions from chicken and rabbit sera were weakly mitogenic for mouse splenic lymphocytes. CS-Ig fractions are useful new reagents for studying glycoproteins and the interactions and activities of cell surface carbohydrates.

Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

The carbohydrate moiety of carcinoembryonic antigen could be sequentially degraded by repeated cycles of periodate oxidation, reduction, and mild acid hydrolysis (Smith degradation). After three complete degradations, all fucose and sialic acid, 80% of the galactose, 65% of the mannose, and about 40% of the N-acetylglucosamine were eliminated without impairing the ability of degraded carcinoembryonic antigen to react with specific antisera against the antigen. Inhibition studies in a carcinoembryonic antigen/rabbit anti-carcinoembryonic antigen precipitating system with oligosaccharides covering previously known internal structures of glycoproteins and presumably corresponding to the internal carbohydrate region of the antigen, demonstrated that none of the compounds tested was inhibitory. Nor could any inhibitory effect on the binding of carcinoembryonic antigen to antibody against the antigen in a radioimmunoassay system be domonstrated for the carbohydrate moiety prepared by hydrazinolysis or the glyco peptide fraction isolated after papain degradation of the antigen. However, if carcinoembryonic antigen is completely reduced and alkylated, with three intrachain disulfide bonds cleaved per 10-5 g, the immunological activity is reduced to 3-5% of untreated antigen. Furthermore, treatment of the antigen with 0.5 NaOH at 20 degrees for 2 hr completely abolished its ability to react with antiserum, whereas its ability to precipitate with a series of lectins was unchanged. No release of low-molecular-weight carbohydrate orchange in sugar composition of alkali-treated antigen was observed. Our tentative conclusion is that the carbohydrate moiety of carcinoembryonic antigen does not contain the tumor-associated determinant(s).

A comparison of three educational techniques used in a venereal disease clinic.

The communications methods that could be used in educational programs for venereal disease patients were examined as to their relative effectiveness: a programed learning guide, an audiovisual (cinematographic) technique, and an interview method. An experimental design was used to study three groups of patients at a venereal disease clinic; (a) persons who were pretested, exposed to an educational method, and tested again, (b) a control group whose members were pretested and post-tested but not exposed to an educational method, and (c) another control group whose members were exposed to an educational method and then took a post-test. Each of those groups exposed to the educational techniques was further subdivided according to the technique applied. Analysis of the data collected from 443 subjects led to the following tentative conclusions: 1. Representation in the nine groups was demographically uniform as measured by age, sex, marital status, and ethnic origin. 2. All three educational techniques significantly raised the subjects' level of knowledge about venereal disease, as measured by their test scores. 3. All three techniques were favorably received by the subjects. The majority reported that the techniques were the right length (10 to 15 minutes), interesting, informative, useful, and anxiety-reducing. The three techniques apparently accounted for an increase of more than 20 percent in subjects' scores on tests about venereal disease, and the subjects perceived all three techniques as interesting and beneficial. The interview method proved significantly more effective than the other two techniques in raising the knowledge level. It was also the technique most favorably received by the subjects. As expected, those persons who entered the clinic with a low level of knowledge learned much more when exposed to an educational techniques than persons entering the clinic with a high level of knowledge. Reaction to the three methods did not differ significantly by the subjects' age or sex.

The effect of estrogen on the vascular endothelium and its possible relation to thrombosis.

The effect of estrogen on the permeability of the endothelium of the aorta has been studied in the rat. The endothelium was subjected to different forms of chemical injury, and its permeability to silver ions was estimated in rats in a control group and in rats after estrogen treatment. In rats subjected to estrogen treatment, the degree of silver penetration through the endothelium was significantly higher than in untreated rats. This was interpreted as an increased vulnerability of the endothelium after estrogen treatment. Thrombosis in females using oral contraceptives often has an aberrant localization. These findings lead to a theory that an endothelial factor may be involved in the pathogenesis of this form of thrombosis.

Pituitary chromophobe adenomas consisting of prolactin cells: a histologic, immunocytological and electron microscopic study.

Morphologic studies of pituitary neoplasms removed by srugery from 36 human patients revealed 8 chromophobe adenomas which differed clearly from the remaining tumors. The cytoplasm of the adenoma cells failed to stain with PAS, aniline blue, adehyde fuchsin, aldehyde thionin, orange G or light green, but positively stained granules were found by using erythrosine or carmosine. Immunoperoxidase technique disclosed the presence of prolactin in the cytoplasm of some adenoma cells. The adenoma cells exhibited distinct ultrastructural features such as well developed rough surfaced endoplasmic reticulum with Nebenkern formation, prominence of Golgi apparatus, presence of misplaced exocytosis as well as pleomorphism of secretory granules with a considerable variation of size ranging from 130 to 500 nm in diameter. Thus, by electron nicroscopy the adenoma cells showed a close resemblance to prolactin cells of the non-tumouous pituitary glands except for the reduced size and number of secretory granules. Thes chromophobe adenomas are regarded as representing a distinct pathological entity clearly distinguishable from other forms of pituitary neoplasms. In view of the morphologic findings and the elevation of blood prolactin level (measured in 3 patients) the term, "sparsely granulated prolactin producing pituitary adenoma", appears to be the most appropriate one to designate these tumors.

Diagnosis and quantification of arrhythmias in ambulatory patients using an improved R-R interval plotting system.

An improved technique for identification, diagnosis and quantification of arrhythmias during rest or ambulatory electrocardiographic recording is described. With simultaneous plotting of the R-R interval and the QRS duration and QRS vector measurement of each beat versus time, all periods of arrhythmias or abnormal complexes can be identified and characterized. Analog electrocardiographic samplings are used to confirm the diagnosis of the arrhythmia and to exclude artifact. The availability of a permanent record for the characterization of each QRS complex enables the physician to check the technician's analysis of the recording and to relate all events to the patient's heart rate and clinical symptoms. This technique also provides data for quantification of ventricular arrhythmias.

Prenatal detection of neural tube defects. Comparison between alpha-fetoprotein and beta-trace protein assays.

The prenatal diagnosis of neural tube defects is now possible in about 90 per cent of cases by assaying the amniotic fluid for alpha fetoprotein. The accuracy of beta-trace protein assays on amniotic fluid samples from defective fetuses was compared to alpha-fetoprotein studies. At present, alpha-fetoprotein studies provide more reliable results.

Quinidine hepatitis.

Long-term administration of quinidine was associated with persistent elevation of serum concentrations of SGOT, lactic acid dehydrogenase, and alkaline phosphatase. Liver biopsy showed active hepatitis. Discontinuance of quinidine therapy led to normalization of liver function tests. A challenge dose of quinidine caused clinical symptoms and abrupt elevation of SGOT, alkaline phosphatase, and lactic acid dehydrogenase values. We concluded that this patient had quinidine hepatotoxicity and believe that this is the first case reported with liver biopsy documentation. This report also suggests that, even after long-term administration, the hepatic toxicity is reversible.

Frequency potentiation and postextrasystolic potentiation in patients with and without coronary arterial disease.

Frequency potentiation and postextrasystolic potentiation of myocardial contractility were induced in 17 patients found not to have cardiac disease (group 1) and in 10 patients with coronary arterial disease (group 2). Atrial stimulation was performed starting at a rate of 110/min and going up to 200/min (frequency potentiation). Single, premature ventricular beats with decreasing coupling intervals were induced every fifteenth beat during basal atrial stimulation at 125/min, after which compensatory pauses were provided (posts used an an index of contractility. With increasing heart rate dp/dt max was augmented equally, in both groups of patients, by frequency increases and premature beats (the coupling interval of the extrasystole being expressed as heart rate). dp/dt min and left ventricular systolic pressure remained unchanged while left ventricular end-diastolic pressure decreased in both groups of patients with the two forms of potentiation It was concluded that both these forms of potentiation have the same augmenting effect on myocardial contractility. Shortening the coupling intervals of premature beats caused a decreased in left ventricular end-diastolic pressure, suggesting that the Frank-Starling mechanism was not involved in postextrasystolic potentiation. Patients with coronary arterial disease had lower values of dp/dt max, dp/dt min, and higher values of left ventricular end-diastolic pressure during rest and stimulation procedures, while the systolic pressures equalled those in the control group. Though individual case values from the healthy and diseased hearts might be similar, it was only under the stress of potentiation that the true state of contractility was made apparent. Impairment of dp/dt min was not found without an impairment of dp/dt max in the presence of myocardial ischaemia.

Microscope fluorometric investigations on the reticulocytic maturation distribution as diagnostic criterion of disordered erythropoiesis.

A microscope fluorometric technique is described which permits not only the visual identification of reticulocytes under the fluorescence microscope but also the determination of their relative stage of maturation to normocytes. The technique is based on a specific staining procedure which results in a fluorescent complex between the reticulocytic RNA and acridine orange. Thus, the relative mass of RNA in the individual reticulocytes can be measured by means of mciroscope fluorometry. As the reticulocytic RNA content decreases and finally disappears during the final maturation process of reticulocytes after their release into the peripheral blood stream, the fluorescence signal indicates the relative degree of this maturation. A characteristic frequency distribution of this parameter can be obtained for a given blood sample by microscope fluorometry measuring 200 to 300 reticulocytes. The preliminary use of this technique for following up the course of two cases of hemolytic anemia and one of pernicious megaloblastic anemia during their treatment demonstrates the potential diagnostic value of this technique of identifying the change of the reticulocyte maturation distribution in addition to the reticulocyte count. Satisfactory agreement between the microscope fluorometric results and those obtained by counting separately the four reticulocytic maturation stages according to Heilmeyer and Wesbäuser has been achieved. The possibility of obtaining quantitative and comparable results by use of this method may be considered a general advantage and a promising basis for the development of an automated technique.

Changes in brain hydrolytic enzyme activities in rats treated with cholesterol biosynthesis inhibitor, AY9944.

Intraperitoneal administration of AY9944 causes accumulation of 7-dehydrocholesterol, appearance of abnormal neuronal cytoplasmic lamellar inclusions containing acid phosphatase activities, and degeneration of oligodendroglial cells. In the present study, we attempted to correlate appearance and disappearance of abnormal inclusions, oligodendroglial degeneration, activities of lysosomal enzymes, and accumulation of 7-dehydrocholesterol. One group of 5-day-old rats received daily injection of AY9944, 30 mg/kg, for 30 days. Other groups received the same daily dosage but only in 10 consecutive days, starting from 5, 15, 25, and 35 days, respectively. Activities of 13 brain hydrolytic enzymes were determined. In the first group, activities of most enzymes increased over the controls, reaching the peak between the 15th and 25th day, corresponding to the maximum appearance of the neuronal inclusions and degenerating oligodendroglia. Despite the continued administration of AY9944 and the high tissue concentration of 7-dehydrocholesterol, activities of most enzymes declined after 25 days to relatively steady levels somewhat higher than controls. In the group which received 10-day injections, enzyme activities reached the peak at the end of the injections and then rapidly returned to normal within 10 days thereafter, corresponding to the appearance and disappearance of the abnormal inclusions. However, 7-dehydrocholesterol continued to increase for 10 days after the drug administration was discontinued. AY9944 had no direct effect on any of the enzymes in vitro. There appears to be generalized lysosomal activation concomitant with formation of abnormal neuronal inclusions in the experimental conditions.

Properties of prostatic cultures transformed by SV40.

SV-40-transformed hamster prostatic tissue has been previously evaluated as a model for human prostatic carcinoma. Because the original cell line was lost, Syrian golden hamster prostatic tissue has been established in explant culture and infected with a 10-6-cell tissue culture infectious dose (50 percent effective) of SV40. After in vitro transformation, the cells were produced in quantity and 60 times 10-6 cells were injected into adult male Syrian golden hamsters 24 hours after 400 rads of whole-body radiation. After 60-90 days, a small palpable tumor developed. These tumors could be serially transplanted in adult male animals without immunosuppression. The tumor cells were established in tissue culture and the cells were returned to adult animals without immunosuppression where they rapidly produced fast-growing tumors. The solid tumors were composed of sheets of pleomorphic polygonal cells with large nuclei and many nucleoli; they resembled undifferentiated human prostatic carcinoma. In vitro, the cultures contained small, rapidly growing cells with a population doubling time of about 1.3 days. The cells carried the SV 40-specific antigen. The modal chromosome number was 66-68 with a distribution of 47-120. Cells exposed to 2-bromo-5'-deoxyuridine in culture did not release particles with RNA-dependent DNA polymerase activity. Endocrine sensitivity in vivo and in vitro is undertermined to date.

Tumor antigen and acid phosphatase isoenzyme in prostatic cancer.

Plasma and prostatic fluid from man, dog, and baboon were measured for carcinoembryonic antigen (CEA) by a radioimmunoassay technique. No CEA was detected in plasma, prostatic fluid, or seminal fluid in 12 dogs and three baboons. Elevated CEA (less than 2.5 ng/ml) was found in 13 of 20 human prostatic fluids. It was inferred that there was no immunologic cross-reactivity of CEA among man, dog, and baboon. CEA has been isolated and purified from liver tumors. Biochemical studies reveal that CEA consists of 60 percent carbohydrate and 40 percent protein. It contains the following carbohydrates: fucose, mannose, galactose, sialic acid, N-acetylglucosamine, and a small amount of N-acetylgalactosamine. The following amino acids were found in CEA: lysine, histidine, arginine, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, emthionine, isoleucine, leucine, tyrosine, phenylalanine, and cysteine. The amino acid sequence (first 30 amino acids) of the N-terminal has been determined. The N-terminal amino acid was lysine. Using this study as a model, other tumor antigens from prostatic tumor tissues are being investigated. The acid phosphatase isoenzyme from prostatic tissue was also studied. After a series of purifications, two chromatographic fractions were obtained. Treatment with neuraminidase removed the sialic acid content of the molecule, changed the isoelectric focusing patterns, and abolished the chromatographic heterogeneity. Sedimentation studies indicated a molecular weight of about 100,000. Biochemical studies showed that prostatic acid phosphatase isoenzyme is a glycoprotein which consists of 7 percent carbohydrate and 93 percent protein. It contains fucose, galactose, mannose, sialic acid, N-acetylglucosamine, and the following amino acids: aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, tryptophan, and cysteine. An antiserum to this purified prostatic acid phosphatase isoenzyme is being prepared in animals.

Anomalous left coronary artery originating from the pulmonary artery. Report on 15 cases.

Fifteen infants and children with the diagnosis of anomalous left coronary artery from the pulmonary trunk have been encountered at the Children's Hospital Medical Center, Boston, Massachusetts from 1958 to 1973. After thorough clinical and laboratory evaluation, they have been treated by anticongestive measures. Nine patients have had ligation of the anomalous left coronary artery at its entrance into the pulmonary artery; one patient has undergone coronary bypass surgery. The lelctrocardiogram proved to be the most helpful diagnostic clinical laboratory test, Vectorcardiograms are valuable not only in diagnosis but also in the follow-up of the patients from the prognostic point of view. The most sensitive tool for the definitive diagnosis is an aortic rool angiogram; we have no false negatives or false positives with this method. The twelve patients with complete cardiac catheterization data could be divided into three groups, according to the pressure and magnitude of the left-to-right shunt at the pulmonary level. All patients with an appreciable le?T-TO-RIGHT SHUNT SURVIVED. Patients in whom no left-to-right shunt could be demonstrated by angiography died. Half of the patients with only small left-to-right shunt survived; The results of surgical and medical treatment, were identical within the three groups. Medical management in infancy, according to coronary care principles, with definitive surgical correction at a later age, is the preferred treatment. Ligation of the anomalous left coronary artery is recommended in severely symptomatic infants with documented left-to-right shunt at the pulmonary artery level, who do not respond to medical management.

Dihydrotachysterol-induced aortic calcification. A histochemical and ultrastructural investigation.

Early dihydrotachysterol-induced calcification of the rat aorta occurs in elastic lamellae. The first deposition of inorganic substance leads to the formation of very thin filament-like structures of low electron density. The characteristic shape of these structures suggests that they could correspond to calcified filamentous components of the elastic tissue. When calcification spreads from the calcified elastic lamellae into the adjacent tissue, the inorganic substance is initially collected in roundish structures, probably of cellular origin, and is successively laid down in the entire matrix of the aortic wall, including collagen fibrils. All the calcified areas contain glycoproteins and acid proteoglycans. A cartilage-like tissue often develops near calcified areas. Its fine structure is very similar to that of the normal hyaline cartilage. It can be calcified, but usually the inorganic substance is not crystalline as it is in normal cartilage. It seems to consist of very small, linearly aggregated inorganic granules which form irregular structures. These seem to develop in close relationship with the fibrillar, probably collagenic, network of the matrix. No ultrastructural findings have been obtained for explaining cartilage induction near calcified areas of the aortic wall. It is possible that cartilage differentiation is regulated by diffusible substances which cannot be recognized under the electron microscope.

Mechanisms of chromosome banding. V. Quinacrine banding.

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quanacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound be intercalation with relatively little sid binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nuclei acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2 times 10-6 to 2 times 10-5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescenc. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCL AND Cs-2SO-4-Ag+ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding. We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.

Interaction of the hinge region of human immunoglobulin G with a murine lymphocyte membrane receptor. Relevance to the problem of antiglobulin induction in rheumatoid arthritis.

Evidence is presented which indicates the presence on murine lymphocytes of a membrane receptor for determinants on the hinge region of human IgG. These determinants are exposed following pepsin scission of IgG molecule, i.e. on the F(ab')2 fragment. The evidence for a hinge receptor derives, in vivo, from splenic localization of F(ab')2 in germinal centres and, in vitro, from immunofluorescent binding studies. The sequential immunofluorescent pattern for the uptake of human F(ab)2 fragments into murine spleen germinal centres was identical with that previously observed for heat-aggregated human IgG, but F(ab')2 fragments appeared to be retained in the germinal centres for a shorter time than aggregated IgG. Experiments with nude mice and T cell-deprived mice showed that the localization of F(ab')2 fragments does not require T cells. Competition experiments suggest that the receptor for F(ab')2 may bear little relation to the receptor for aggregated IgG. The relevance of such a lymphocyte membrane receptor to the immunopathology of rheumatoid arthritis is discussed in the light of previous findings that a proportion of the serum IgG of patients with rheumatoid arthritis has a structural anomaly compared with control IgG, characterized exposure of new determinants at the hinge region.

Antibody formation in the mouse induced by hapten-carrier complexes.

The influence of hapten, carrier and their ratio in a complex on T-cell helper stimulation and antibody formation against the dinitrophenyl (DNP) hapten was studied. Complexes of DNP with bovine serum albumin (BSA), bovine gamma-globulin (BGG), isologous mouse immunoglobulin (MIG) and polyvinylpyrrolidone (PVP) as carriers were used. Optimal antibody formation against DNP was obtained with complexes with an intermediate hapten:carrier ratio (DNP 16-minusBSA, DNP 43-minusBGG and DNP 48-MINUSMIG). DNP-PVP complexes were not active either in the primary or in the secondary response. The anti-BSA titre was independent of the number of DNP groups on the complex used for immunization. Inhibition of DNP-plaque formation by spleen cells of immunized mice shows an increase of the inhibitory capacity of the complex with the increase of the hapten-carrier ratio. DNP 16-minusPVP was the only PVP complex which was inhibitory. These results suggest that helper cells involved in the antibody formation against BSA and DNP are reactive with different parts of the complex. Priming of mice with carrier or complex after cyclophosphamide (Cy) treatment followed by a secondary injection with complex 10 days later gave strong indications that there is a greater involvement in stimulation of helper T cells by determinants of the complex (new antigenic determinants (NAD) or NAP-DNP groups) or DNP, than by true BSA determinants. This holds for both the IgM and IgG responses.

Intraaticular treatment of tarsal degenerative joint disease in cattle.

Tarsal degenerative joint disease (DJD) in 12 cattle was classified as primary or secondary, based on age, evidence of hereditary or congenital joint conformation defects, faulty hindlimb alignment, duration and type of usage joints were subjected to, and history or signs of repeated trauma. Three of the cattle had bilateral primary tarsal DJD, 7 had bilateral secondary tarsal DJD, and 2 had secondary DJD of the left tarsus. Analyses of synovial fluid samples provided a means of characterizing pathologic changes of tarsal DJD, Results of blood and synovial fluid analyses were grouped in compilation of data for cattle affected with either primary or secondary tarsal DJD. Corticosteroids and a long-acting synthetic progestational agent were injected singly or in combination with aqueous antibiotics into affected tarsal joints. Tarsal joints of 5 of the cattle responded favorably to a single intraarticular treatment, as manifested by palliative relief and functionally usable joints. Seven joints of 5 cattle were subjected to repeated intraarticular treatment. Serial synovial fluid analyses in 7 of the cattle provided a means of assessing tarsal joint response to intraarticular treatment or to therapeutic arthrocentesis, exclusive of patient objective response. One cow developed a mild self-limiting bilateral postinjection synovitis that was resolved after the 2nd and final intraarticular injection. Usable function returned to tarsal joints of cattle that responded favorably to intraarticular treatment at different periods after single or repeated injections. Three cattle with advanced tarsal DJD experienced minor temporary relief and were euthanatized at their owner's request. Improvement did not occur in the tarsal joint of 1 cow subjected to therapeutic aspiration only. Intraarticular treatment in all cattle was considered supportive to the animal's well-being rather than curative.

In vivo studies of mediator release in cold urticaria and cholinergic urticaria.

Six patients with cold urticaria were found to possess elevated plasma histamine levels after cold challenge by placing one hand in ice water for 4 minutes. A single patient became hypotensive during the procedure and had a level of 260 ng/ml. histamine in the venous effluent from his hand. No elevation of plasma serotonin or bradykinin was observed. Two patients with cholinergic urticaria possessed elevated plasma histamine levels during and after vigorous exercise for 10 minutes; these patients also gave a positive test for vibration-induced angioedema. A single patient with cholinergic urticaria possessed elevated baseline serotonin levels and elevated levels during and after exercise but no elevation of plasma histamine or bradykinin. The results suggest that histamine is the major mediator of urticaria and hypotension in cold urticaria. Histamine also appears to be released coincident with the development of urticaria in some patients with cholinergic urticaria, while elevated serotonin levels in a single atypical patient suggest that a subpopulation of patients with cholinergic urticaria possess a different pathogenesis.

Occurrence and natural history of chronic lymphocytic thyroiditis in childhood.

In a six-year survey of 5,179 school children in Arizona, Utah, and Nevada 62 cases of chronic lymphocytic thyroiditis were identified giving a prevalence of 1.2%. Thyroids were enlarged in 85%, firm in 60%, and had an irregular or lobulated surface in 75%. Antibodies to thyroglobulin were demonstrable in the serum at some time during the course of the disease in 76% by the tanned red blood cell technique and in 93% by radioimmunoassay. Serum TSH concentrations were elevated in seven of 15 subjects. Many of the cases were early or mild thyroiditis and, in most instances, subjects were asymptomatic and considered clinically euthyroid. Two subjects were hypothyroid, and two appeared clinically hyperthyroid. Spontaneous resolution of thyroiditis occurred in 15 of 32 individuals who received no treatment. Resolution occurred in 14 of 30 children treated with thyroid hormone supplement. The results suggest that lymphocytic thyroiditis in children may be present without symptoms and in many is a self-limiting disorder from which complete recovery occurs spontaneously.

Triglyceride-production rates in patients with type-4 hypertriglyceridaemia.

A new method has been devised to study triglyceride-production rates in man. The clearance of plasma-triglyceride transported on very-low-density lipoproteins (V.L.D.I.) to peripheral tissues is inhibited by about 50 percent with protamine sulphate; and this results in a rise of blood-glycerides over 1 hour. In a group of nine patients with type-IV hypertriglyceridaemia (mean fasting glycerides 252 mg. per 100 ml.) the mean increment of plasma-triglycerides was 29.8 compared with 1.4 mg. per 100 ml. per hour for nine controls whose mean fasting glycerides had been only 105 mg. per 100 ml. Patients with fasting plasma-triglycerides greater than 400 mg. per 100 ml. showed a response to protamine more like controls. It is suggested that patients with type-IV hyperlipaemia and fasting levels of triglycerides less than 400 mg. per 100 ml. are secreting triglycerides into blood at greater rates than controls; whereas patients with sever hypertriglyceridaemia (levels greater than 400 mg. per 100 ml.) may have a defect of triglyceride clearance so that inhibition by protamine produces no further change in level of plasma glycerides.

Pleomorphic virus-like particles in human faeces.

Pleomorphic fringed particles bearing some resemblance to orthomyxoviruses and coronaviruses were seen in 90 percent of stools from south Indian children and adults but not in stools from neonates. This finding may be related to the abnormalities of intestinal structure and function common in this region of India.

Impairment of platelet function by growth-hormone release-inhibiting hormone.

Synthetic cyclic growth-hormone release-inhibiting hormone (G.H.-R.I.H.) impaired platelet aggregation in each of four healthy men given 6-hour infusions. The effects lasted over 24 hours in three of them. There was no consistent change in platelet-counts during the infusions, but 18 hours after the end of the infusions there was a slight but significant increase in platelet-count. There was no change in prothrombin-time, partial thromboplastin-time, fibrinogen titres, and fibrinogen-degradation products. Incubation of G.H.-R.I.H. with blood in vitro did not affect platelet aggregation. Similar impairment of platelet function has been reported by others in baboons given linear G.H.-R.I.H. Infusions in the four healthy men studied also produced abdominal pain, dizziness, and diarrhoea in three, as have been reported in patients similarly infused. Although other side-effects or impairment of platelet-counts or bleeding-tendencies have not been reported in patients infused for up to 72 hours, caution should be exercised when using G.H.-R.I.H. over extended periods until further data on its toxicity are available.

Chronic antigenic stimulation, herpesvirus infection, and cancer in transplant recipients.

An increased incidence of malignancy has been reported in transplant recipients. The pathogenesis of this increase was originally attributed to immunosuppressive therapy. However, not all tumours are increased in proportion to their occurrence in the general population-75% of reported tumours are lymphorproliferative or carcinoma of the skin, lip, or cervix. This cannot be explained by impaired immunosurveillance, and alternative hypotheses must be considered. 90% of transplant recipients develop clinical or serological evidence of herpesvirus infection. Herpesviruses have been implicated in the pathogenesis of lymphorproliferative tumours and carcinoma of the skin and cervix. They can remain in latent form and be reactivated by allogeneic stimulation and/or immunosuppression. These viruses localise to skin, cervix, and neural tissue-i.e., exactly those sites where cancer develops in transplant patients. Herpesvirus infections in association with the presence of an allogeneic graft in an immunosuppressed patient may be responsible for the increased incidence of both lymphoproliferative tumours and carcinoma of the skin, lip, and cervix in the transplant recipient.

Diabetes mellitus: changes in the transport properties of isolated intestinal microvillous membranes.

Isolated, small intestinal microvillous membranes from normal and acutely diabetic rats were compared with respect to D-glucose transport. D-Glucose was accumulated to a greater extent by diabetic membrane vesicles when supplied with energy in the form of a NaC1 or a NaSCN gradient across the vesicle membrane. The difference appeared to be caused by an ability of the diabetic membranes to maintain a higher driving force for active D-glucose transport and not by changes in the glucose "carrier." Increasing the glucose-independent Na-+-conductance of the membrane with monactin or gramicidin D reduced the active accumulation of D-glucose by membrane preparations from both control and diabetic groups. Concentrations of monactin and gramicidin D in the incubation medium of membrane vesicles from diabetic animals could be adjusted so that their D-glucose transport became indistinguishable from that of membranes from normal animals not treated with ionophores. These observatins suggest the microvillous membranes as one site where changes occur in acute diabetes. In addition, the change in the transport properties of the isolated membranes offer a rational explanation for the simultaneous elevation of active intestinal sugar, amino acid, and bile salt transport observed for intact intestinal tissue.

Isolation of Escherichia coli mutants defective in enzymes of membrane lipid synthesis.

A new method has been developed which permits the rapid screening of E. coli colonies for mutants with defective enzymes of phospholipid metabolism. In this procedure, a disc of filter paper is pressed down on an agar plate containing several hundred colonies of mutagen-treated cells, after which the paper is lifted off. In the process the colonies are transferred to the paper, giving rise to a replica print of the master plate. The few cells from each colony left on the master keep growing in the original pattern. The pattern of colonies is also retained on the filter paper, even after the cells are rendered permeable with lysozyme and EDTA. Colonies treated in this manner remain absorbed to the paper, where they can convert sn-(U-14-C)glycero-3-P to phosphatidyl(U-14-C)glycerophosphate, dependent on added CDP-diglyceride. Unrelated reactions of sn-(U-14-C)glycero-3-P that may obscure the synthesis of phosphatidyl-glycerophosphate are inhibited by the addition of reagents poisoning energy generation. The radioactive phospholipid that forms around each colony on the paper is precipitated in situ with trichloroacetic acid, and unreacted sn-(U-14-C)glycero-3-P is washed away. After autoradiography, the colonies on the filter paper are stained with Coomassie blue. When the autoradiogram is superimposed on the strained paper, mutants are identified as blue colonies lacking a black halo. With this method, 20,000 colonies were screened in several days. Four mutants were identified with low levels of CDP-diglyceride:snglycero-3-P phosphatidyl transferase (EC 2.7.8.5, GLYCEROL-PHOSPHATE PHOSPHATIDYLTRANSFERASE, PHOSPHATIDYLGLYCEROPHOSPHATE SYNTHETASE) IN EXTRACTS. With a similar assay, 10,000 additional colonies were screened for mutants with altered CDP-diglyceride:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase), and four strains were found in which the enzyme is thermolabile. The screening technique described here is termed replica printing and should be applicable not only to studies of phospholipid metabolism but also to nucleic acid and protein synthesis.

Mechanism of b-lymphocyte activation: failure to obtain evidence of a direct role of the Ig receptors in the triggering process.

Experiments were designed to test two hypotheses of B-cell activation by antigen: the cross-linking concept, postulating that a suitable degree of antigen-induced cross-linking of the Ig receptors is sufficient for immunocyte triggering, and the two-signal hypothesis, suggesting that a first signal delivered by antigen interacting with the Ig receptors followed by a second signal given by, for example, a polyclonal B-cell activator is necessary for activation. The results did not support either of these hypotheses. Thus, the hapten FITC coupled to human serum albumin and human gammaglobulin in different conjugation ratios failed to activate B cells, whether the hapten-protein conjugates were soluble or precipitated, whether the experiments were carried out in the presence or absence of different concentrations of sera from different species, and irrespective of the day of assay. Furthermore, the same FITC-protein conjugates or FITC itself coupled to Sepharose particles failed to induce a specific anti-FITC response, even though a range of 10-9-fold concentrations of FITC were used. In contrast, FITC coupled to lipopolysaccharide (LPS) regularly induced a primary anti-FITC response in all the above systems, whether FITC-LPS was soluble or coupled to Sepharose particles. The conjugation ratio of FITC to LPS was within the range of epitope densities used with FITC-protein conjugates. Analogous studies were performed with the above compounds and, in addition, NNP-cap and fowl gammaglobulin, added alone or together with LPS to lymphocyte cultures. In no case did the antigen plus LPS give a better specific anti-FITC response than LPS alone, irrespective of the culture conditions, the epitope densities, the physical form of the conjugates, and whether they were bound to Sepharose particles or not, although this would be expected in terms of the two-signal concept. The results are compatible with the one nonspecific signal hypothesis, ascribing a passive role to the Ig receptors and an active triggering function to thymus-independent antigens. Therefore, the ability to trigger B cells directly will depend on the nature of the carrier, triggering being achieved if the carrier is a polyclonal B-cell activator; the epitope density and the degree of cross-linking of Ig receptors are unimportant for delivering the triggering signal, although they can facilitate the binding of the conjugate to the specific B cells.

[Morphological and cytofluorometric study of the giant cells of the trophoblast of the common vole].

The primary and secondary giant cells of trophoblast in placenta Microtus arvalis were studied. The giant polyploid nuclei are formed in result of series of successively proceeding endomitotic polyploidization of chromosomes. Two stages of endomitosis are described: endointerphase with the uniform net of thin chromatin threads and the stage when small round or rod-shaped paired chromosomes gather mostly under the nuclear membrane. Great number of round, oval, and complex-shaped nucleoli may be seen in nuclei during both stages of endomitosis, the number growing during polyploidization. The morphology of the chromosome-nucleolar apparatus involves peculiarities of the polyploidization mechanism in placenta Microtus arvalis trophoblast. Endomitosis occurs both in low and high-polyploid nuclei. Cytofluorometric determination of the DNA amount in nuclei polyploid nature. The degree of polyploidy of the trophoblast giant cells nuclei during terminal differentiation of placenta corresponds to 128c-512c, and some nuclei contain the DNA amount corresponding to 1024 and 2048 chromosomal sets. The cause of origin of the polyploid cells in trophoblast of rodents placenta is discussed.

[T and B lymphocytes in chronic lymphocytic leukemia and Hodgkin's disease. Electron microscopic and immunologic studies].

The number of lymphocytes forming spontaneous rosettes with sheep erythrocytes, a property of thymus-dependent (T) cells, and the number of lymphocytes bearing surface immunoglobulins, a characteristic feature of bone marrow-dependent (B) cells, were determined in the peripheral blood of normals and of patients with chronic lymphocytic leukemia (CLL) and Hodgkin's disease. As compared with normal individuals CLL patients had an increased percentage of lymphocytes with membrane-bound immunoglobulins, whereas the proportion of rosette-forming lymphocytes was reduced. In Hodgkin's disease either normal, diminished, or increased B cell values were obtained; the percentage of T cells was decreased or within the lower range of normals. Lymphocyte transformation by various mitogenic agents in vitro may be regarded as a model of lymphocyte reactivity during immunologic processes in vivo. In order to study the functional capacity of lymphocytes in CLL and Hodgkin's disease in comparison with normal cells, purified peripheral blood lymphocytes from normals and patients with these diseases were incubated in vitro with phytohemagglutinin (PHA) and pokeweed mitogen (PWM) over 7 to 11 days. DNA synthesis was determined by incorporation of 3-H-thymidine. The cyto-architectural features of the cells before and during incubation with these phytomitogens were studied by electron microscopy. Planimetric measurements were performed on micrographs of comparable cell sections (through nucleus and Golgi zone) for the determiniation of cell, nuclear, cytoplasmic, and mitochondrial area. Furthermore, the number of mitochondria and of membrane-bounded acid phosphatase-positive lysosome-like organelles was determined in comparable sections of unstimulated and mitogen transformed lymphocytes.

Values, Myths, and Symbols.

Defining "symbol" as that which draws together and unites experience and "myth" as a cluster of symbols set in dramatic form, the author discusses the lack of both symbols and myths in contemporary sociaty. This lack had led to a disintegration of commonly held values and, ultimately, to a great need for psychotherapy on the part of individuals seeking personal identity. The individual must define his or her own values according to personal myths. One basic function of psychotherapy is to help individuals in their attempt to recover values.

A comparison of different methods of detecting mucin in adenocarcinomas of the lung.

The correct classification of carcinoma of the lung is not only of therapeutic and prognostic importance but is also considered to have epidemiological and aetiological significance. Histological tests for mucin are essential in the classification of lung tumours but there is little information available about the influence of the method of detection used on the results of classification. Five established staining techniques were tested using paraffin blocks from surgical specimens of 81 human lung tumours diagnosed as adenocarcinoma, i.e. tumours of WHO Type III. Mowry's alcian blue-periodic acid-Schiff (AB-PAS) technique gave the highest proportion of positives (93%) slightly fewer (90%) being obtained by the PAS technique alone. Both these methods were influenced by the presence of cytoplasmic hyaline globules, structures which cannot be regarded as mucin. The stain recommended by the World Health Organization was also influenced by the presence of hyaline globules, was less frequently positive than the PAS techniques and was considered to have no special advantages. The aldehyde fuchsin-alcian blue sequence was positive in only 83% of cases but provided some information about the type of mucin present. Southgate's mucicarmine also detected mucin in only 83% of cases. It was concluded that the apparent incidence of adenocarcinomas may be influenced by staining methods used. Some standardization of technique is desirable and the AB-PAS combination appears to be the most satisfactory.

Retrograde transport of horseradish peroxidase in the magnocellular neurosecretory system of the rat.

Horseradish peroxidase (HRP) was injected into the pituitary in adult rats. After 2-3 days, the neurones of the supraoptic nuclei, the magnocellular parts of the paraventricular nuclei, and the various accessory neurosecretory hypothalamic nuclei showed accumulation of HRP. The HRP reaction product consisted of fine, discrete cytoplasmic granules, and in electron micrographys it was seen to be located in the lysosome-like dense bodies of 0.4-0.6 mum diameter which are normally present in the cytoplasm of the neurosecretory neuron.es. Very little reaction product was found in the neurosecretory axons. Reaction product was also found in the hypothalamic arcuate nuclei. This was the result of an endogenous peroxidase-like activity, since it occurred in control animals which had not received HRP. This endogenous reaction product is non-neuronal. Morphologically, it takes the form of distinctive clusters of coarse granules which are seen in electron micrographs to be characteristic angular bodies of 0.7-1.0 mum diameter located in the cytoplasm of astrocytes or their processes.

Kuru.

Kuru is a progressive, invariably fatal cerebellar degeneration which occurs in a limited area of the New Guinea Highlands. The word kuru is derived from a term in the Fore language meaning to shake from fear, and it stems from the trembling which is a conspicuous symptom. Few neurologic diseases have attracted the attention of specialists in so many disciplines. No rare disease can have aroused more interest among the laity. Apart from an extensive scientific and anthropological literature, kuru has been the source of inspiration to journalists and travel writers, and it even occupies a prominent position in the plot of a popular novel. The facts concerning kuru are no less remarkable than the mythology that has grown up around it.

Observations on the T wave of the equine electrocardiogram.

The paper describes changes observed in the T wave and T vectorcardiogram in horse after various periods of exercise. Using radiotelemetry and a bipole lead all horses showed negative T waves immediately after exercise. In some of them this was followed by a markedly positive T deflection. Possible reasons for these changes are briefly discussed.

Immunosuppresive effect of a human hepatic glycoferroprotein, alpha2H globulin. A study on the transformation of normal human lymphocytes.

A macroglycoferroprotein of hepatic orgin, alpah2H globulin, the serum level of which increases a few weeks or months before local recurrence of metastases, has been essayed for its immunosuppressive activity. The study was carried out using the lymphoblastic transformation test and was judged by tritiated thymidine incorporation and microscopic examination. PHA-induced blast transformation of 97 per cent of normal donor lymphocytes is inhibited by 100 mug/ml of alpha2H globulin. This inhibitory effect is proportional to the quantity of added alpha2H globulin. In is obvious with a concentration of 2-5 mug/ml, a frequently observed level in the serum of patient with tumours. Preincubation of lymphocytes with alpha2H globulin renders more effective the inhibitory action on PHA-induced transformation. A mechanism of competition between PHA and alpha2H globulin is suggested by preincubation and the inhibitory effect related to the doses. However, microscopic observation shows that alpha2H globulin acts on the earliest events occurring to the stimulated lymphocytes, by inhibiting cytoplasmic RNA and protein synthesis. The alpha2H globulin effect may not only have an immunosuppresive activity but it may have a more general effect, for example blocking or modifying cellular respiration.

Interaction of hepatitis B surface antigen (Australia antigen) with membrane vesicles of Pseudomonas aeruginosa.

A lysogenic strain of Pseudomonas aeruginosa was cultured from the dialysis fluid of a patient on chronic hemodialysis treatment whose blood contained hepatitis B surface antigen (HB8Ag). When this bacterium was incubated for 4 to 7 days with serum containing HB8Ag or with purified HB8Ag, a loss of the HB8Ag-specific immunological reactivity was observed. Bacteriophages can be induced from the isolated P. aeruginosa with mitomycin C; the phages, after purification on CsCl gradients, also lyse P. aeruginosa strain 25102 (ATCC). Subsequent to gradient centrifugation of the lysate, a fraction was found with a density around 1.40 g/ml that inactivated HB8Ag after a 4-h incubation at 37 C as determined by counterelectrophoresis and hemagglutination inhibition. The activity was not found in appreciable amounts in other gradient fractions. The electron microscope shows that the active fraction contains envelope vesicles of 45 to 60 nm in diameter. In spite of their loss in HB8Ag activity, the HB8Ag particles (22nm) appeared morphologically intact. These findings suggest that an enzyme(s) is present in the vesicle fraction which inactivates antigenic determinants on HB8Ag particles. Thus, the presence of these bacteria in environments such as feces, dialysis tanks, and contaminated drinking water may prevent the detection of HB8Ag.

[Critical study of extra-lysosomal acid phosphatase localizations in thyroid follicular cells by the Gomori reaction (author's transl)].

With the Gömöri technique, lead precipitates have been found in thyroid follicle cells in unusual localizations such as apical hyaloplasm and microvilli; it has been established that they were actually significant for acid phosphatase activity: constant results in spite of repeated controls and several variations from the original cytochemical technique, allow to think that lead precipitates were not merely artefactual, but actually significant of enzymatic activity. However it is pointed to the fact that the origin of the enzyme has to be questioned; it is assumed that most likely acid phosphatase has diffused from its original lysosomal site. Such diffusion implies variations of the selective permeability of lysosomal membranes; inappropriate relation between the quantity of enzyme present in these organelles and the quantity of substrate used might also be considered, though changes in the amount (resp. concentration) of substrate remained ineffective and induced no modification in the localization of observed enzymatic activity. In addition, one point of interest is an obvious relation between the observed enzyme diffusion and the state of activity resp. rest of the cell; in the present state of investigations, this remains unexplained and likely related to factors escaping control during processing; moreover, no explanation can be provided for the fact that it revealed impossible to avoid such diffusion even by means of variations of the numerous parameters involved in the Gömöri technique. So that it finally appears necessary to remain on a critical position regarding the results at the ultrastructural level of this standardized technique, and there is no doubt it would reveal useful that several assumptions in the literature about extra lysosomal acid phosphatase activity should be reinvestigated with a similar critical purpose.

Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. II. Stimulation by a K+ diffusion potential.

Addition of valinomycin, nonactin, or monactin plus KCl in the dark to preilluminated chromatophores induced the synthesis of a large amount of ATP. This stimulation of postillumination ATP synthesis by a dark-imposed K+ diffusion potential was different from the stimulation caused by addition of permeant anions or cations in the light, since it increases when the pH of the light stage decreased from 8.0 to 6.0. It was thus most pronounced when the chromatophores were preloaded with protons but the light-induced proton concentration gradient (deltapH) was low. Imposition of a Kplus diffusion potential resulted however in stimulation of ATP synthesis even when the light-induced deltapH was already above the threshold value required to initiate postillumination ATP synthesis. This situation was realized when valinomycin plus KCl were added in the dark to chromatophores preilluminated above pH 6.7 with thiocyanate as the permeant anion, and the amount of ATP formed was the sum of the yields obtained with each of these affectors by itself. On the other hand addition of thiocyanate together with valinomycin plus KCl in the dark led to inhibition of ATP synthesis. In this case the permeant anion could not affect the light-induced deltapH but it did eliminate the diffusion potential by decreasing the difference between the permeabilities of Kplus and the anion present in the reaction mixture.

Preparation of 125-I-labeled human thyroxine-binding alpha globulin and its turnover in normal and hypothyroid subjects.

A protein with the electrophoretic, immunologic, and hormone-binding properties of thyroxine-binding globulin (TBG) has been prepared from human plasma and labeled with radioiodine (125-I) by an enzymatic method of iodination. The [125-I]TBG retained the electrophoretic and immunologic characteristics of unlabeled TBG but exhibited a partial loss of thyroxine-binding activity, as assessed by affinity chromatography. The in vivo behavior of [125I]TBG was studied in six euthyroid subjects (controls) with normal serum levels of TBG as measured both by radioimmunoassay and by determination of maximal T4-binding capacity and in four male patients with untreated primary hyperthyroidism, three of whom had elevated serum TBG. The half-time of the final slope of the plasma disappearance curve averaged 5.0 days plus or minus 1.2 (SD) in the controls and ranged from 3.9 to 109 days in the hypothyroid patients. The distribution volume was similar in the two groups, 6.7 plus or minus 1.3 vs. 7.1 plus or minus 2.1 liters. The catabolic clearance rate averaged 0.99 plus or minus 0.33 liters plasma/24 h in the controls and 0.92 plus or minus 0.46 in the hypothyroids. The absolute turnover rate of TBG, calculated from the catabolic clearance rate multiplied by the serum concentration of radioimmunoassayable TBG, averaged 17.8 plus or minus 2.1 mg/day in the controls and ranged from 14.8 to 33.2 mg/day in the hypothyroids. Among the entire group of subjects there was no correlation between the serum TBG concentration and the absolute turnover rate of TBG.

Induction of T-lymphocyte responses to a small molecular weight antigen. III. T-T cell interactions to determinants linked together: suppression vs. enhancement.

The experiments presented in this paper demonstrate the existence of T-T cell interactions in responses to azobenzenearsonate (ABA)-protein conjugates, and also make the point that the spectrum of T-cell regulation from facilitation (i.e., help) at one end to suppression at the other, which has been well documented in T-B cell interactions, is also followed in T-cell regulation of other T lymphocytes. The data extend the activity of ABA-specific suppressor cells, which were shown to specifically suppress the development of delayed hypersensitivity to ABA-T, to T cells responsible for delayed hypersensitivity to protein antigens provided immunization is carried out with ABA conjugates of these antigens. Thus, suppressor T cells acting on the development of delayed hypersensitivity are not limited in their effects to T cells bearing the same specificity but can effectively suppress responses on immunologically unrelated T cells if they are specific for carrier antigens covalently linked to the ABA-T determinant. Moreover, these studies demonstrate that, as is true of T-B cell interactions, the most efficient T-T cell interactions occur to determinants linked together on the same molecule thus supporting the concept that development of effector T-cell function may involve participation of at least two distinct precursor cells, each of which may convey independent determinant specificities and/or genetic control.