Alien histocompatibility determinants on the cell surface of sarcomas induced by methylcholanthrene. I. In vivo studies. Groups of BALB/c mice were immunized to normal tissues (skin and/or liver plus kidney) of C3Hf, C57Bl/6, DBA/2 and AKR strains and challenged with either of two syngeneic 3-methylcholanthrene-induced immunogenic sarcomas, ST2 and TZ15, or with a "spontaneous" non-immunogenic BALB/c sarcoma, B2. It was found that anti-C3Hf and anti-DBA/2 immune mice were significantly protected against the growth of ST2, whereas anti-AKR immune mice rejected TZ15; no protection was elicited by immunizing with normal tissues of any strain against B2, which lacked individual tumor-associated transplantation antigens (TATA). The reciprocal experiment, i.e. the immunization of BALB/c mice with tumor cells and challenge with skin grafts of different strains, was also carried out with ST2 and TZ15. Accelerated rejection of all the various allogeneic skins was observed in anti-ST2 immune mice and of AKR and C3Hf skin in anti-TZ15 immune animals. In addition the Winn test demonstrated that lymph-node cells of BALB/c mice immune to C3Hf or DBA/2 tissues were specifically inhibitory for ST2, and that lymph-node cells immune to AKR tissues protected against TZ15. In a further experiment both ST2 and TZ15 tumors were left to grow in (C3Hf X BALB/c)F1, (C57Bl/6 X BALB/c)F1, (BALB/c X DBA/2)F1 and (BALB/c X AKR)F1 mice; the tumors were then excised and the "immune" mice challenged with the related tumor to measure their immune response in comparison with that elicited by the same procedure in BALB/c mice. ST2 was highly immunogenic in syngeneic BALB/c mice and in all the hybrid combinations except (C3Hf X BALB/c)F1 mice, where it completely lost its immunogenicity; TZ15 showed a certain loss of immunogenic strength in (BALB/c X AKR)F1 hybrids. It was concluded that TATA of ST2 contain antigenic determinants expressed on the normal cells of C3Hf and DBA/2 strains, and that TATA of TZ15 are likely to share antigens with AKR normal tissues.


Antigenic variation among parainfluenza type 1 (Sendai) viruses: analysis of 6/94 virus. 6/94 virus, isolated originally from a multiple sclerosis (MS) patient, was compared antigenically with related parainfluenza type 1 strains. These included two Sendai strains of mouse and two Sendai strains of reported human origin as well as the HA2 strain. By standard hemagglutination inhibition (HI) or hemadsorption neutralization (HAD-N) tests or by the complement-fixation (CF) cross-block titration test for detecting surface antigens, 6/94 virus and the Sendai virus strains were indistinguishable from each other but distinct from the HA2 strain. By the kinetic HI test, however, 6/94 virus could be readily differentiated from the Sendai viruses isolated from mice and more closely resembled those recovered from man.


An antigenically related tryptic polypeptide from several mammalian type C RNA virus p30s. Limited tryptic digestion of p30 antigen (the major internal viral protein) of type C viruses isolated from hamster, rat, and gibbon ape yielded a polypeptide fragment with a molecular weight of approximately 10,000 daltons. Some antigenic determinants with interspecies specificity were retained on these polypeptides, which in serological tests cross-reacted with antibody produced previously against a similar fragment obtained from mouse p30. Exhaustive trypsinization resulted in further fragmentation with concomitant loss of serologic activity. The results suggest that all mammalian type C virus p30s have an antigenically related polypeptide core which is not readily digested by trypsin.


Uranyl-EDTA-hematoxylin: a new selective staining technique for nucleolar material. This paper deals with the visualization of nuclear structures in glutaraldehyde fixed, acetic acid flattened preparations from Chironomus salivary glands, by means of an uranyl mordanting followed by hematoxylin staining. Under these conditions all the nuclear structures (bands, Balbiani rings, and nucleoli) were deeply stained. Treatment with 0.1 M EDTA for at least 30 sec after uranyl mordanting completely prevents the following hematoxylin staining in all the structures but the nucleolus. With increased EDTA extraction times (60-90 sec) the central region (composed of pars fibrosa) in spontaneously or experimentally segregated nucleoli showed the highest capacity for retaining uranyl ions. This selective staining of the nucleolar (possibly proteinic) material proved also efficient in cells from Drosophila testes and Allium roots.


Aldehyde bisulfite-toluidine blue (ABT) staining as a topo-optical reaction for demonstration of linear order of vicinal OH groups in biological structures. The aldehyde-bisulfite-toluidine blue reaction followed by poststaining stabilization with potassium ferricyanide (ABT) is described as a topo-optical, oriented staining reaction of the vicinal OH groups of complex carbohydrates in biological structures such as polysaccharides, glycoproteins and glycolipids. The birefringence as induced by the oriented dye binding as a result of ABT is indicative of linear order of the vicinal OH groups and, in turn, provides information on the ultrastructural pattern of carbohydrate moieties in biological substances, which pattern is often not demonstrable by other ultrastructural methods. The possibilities of this new approach to the ultrastructural analysis of complex carbohydrates with ABT in a great number of biological substances is demonstrated and its practical value in histopathology discussed.


Suitability of the azocoupling reaction with 1-naphthyl-beta-D-glucoside for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) in human enterobiopsies. The suitability of the simultaneous azocoupling reaction with 1-naphthyl-beta-D-glucoside and hexazonium-p-rosanilin in the detection of the activity of lactase (or lactase-beta-glucosidase complex) in jejunal biopsies of patients with various forms of the malabsorption syndrome was tested. Results were compared with those obtained with the indigogenic method using 4-Cl-5-Br-3-indolyl-beta-D-fucoside which is the method of choice. Both methods gave identical results as far as the relative intensity of the brush border staining was concerned. The azocoupling method applied in unfixed cold microtome sections can be recommended for the routine diagnostics of the malabsorption syndrome when the indolyl substrate is not available.


The histochemical demonstration of aminopeptidase with bromoindolyl leucinamide. The indigogenic method for aminopeptidase of Pearson et al. (1963) was critically evaluated. The localization obtained with it is not correct due to diffusion artifacts. Ferricyanide cannot be used as an oxidation agent. Based on experiments with other oxidation agents (phenazonium methosulfate, nitro BT, tetranitro BT) a new method was devised. The recommended incubation medium contains 0.9 mM L-N-(5-bromoindol-3-yl) leucinamide hydrobromide (chloride), 0.73 mM tetranitro BT, 0.27 mM phenazonium methosulfate and 0.67 M phosphate buffer pH 7.4. The enzyme activity is indicated by the deposition of tetranitro BT formazan. Results with this method in rat kidney, jejunum, liver, lung, and submaxillary gland, in monkey kidney and jejunum, and in human jejunal biosies are almost identical with those obtained with L-leucyl-4-methoxy-beta-naphthylamide applied in a simultaneous azocoupling procedure. The given principle of the demonstration of aminopeptidase activity with an indolylamine substrate deserves a further exploration in the demonstration of peptidases "in situ" both on optical as well as electronmicroscopical levels.


[Histochemical and electron microscopical changes in the kidney of pregnant rats at the time of partus (author's transl)]. In the convoluted proximal tubules of the kidney of pregnant rats alkaline phosphatase activity decreases from 21 days gestation till the 2nd day after birth. During the same time the reaction product of the histochemical succinate dehydrogenase assay exhibits an atypical granular character in some of the proximal tubules, which sometimes additionally contain sudanophilic structures. Electron microscopy reveals dilated mitochondria, an increased number of autophagosomes and big lipid vacuoles of low density.


Localization of alpha-D-glucosyl and alpha-D-mannosyl groups of mucosubstances with concanavalin A and horseradish peroxidase. Concanavalin A is a lectin which is known to bind specifically to alpha-D-glucosyl and alpha-D-mannosyl groups in the mucosubstances of mammalian tissues. The lectin molecule is bivalent; after its attachment to mucosubstances present in a histological specimen it can also bind horseradish peroxidase, a mannose-containing glycoprotein. The attached peroxidase may then be visualized by virtue of its histochemically demonstrable enzymatic activity. Other investigators have utilized this principle in the electron microscopic localization of cell-surface carbohydrates. A histochemical technique for light microscopy is described here, along with three control procedures which establish the specificity of the method. The technique is somewhat more sensitive than earlier ones in which fluorescent-labelled concanavalin A was used, and has the additional advantages that all the required reagents are commercially available and that sacilities for fluorescence microscopy are not needed.


Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions. The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.


The interaction of apurinic acid aldehyde groups with pararosaniline in the Feulgen-Schiff and related staining procedures. The equilibrium reactions involved in the formation of the apurinic acid (APA)-Schiff chromophores in the staining phase of the Feulgen-Schiff reaction do not allow a quantitative conversion of APA to these chromophores. By modification of the sulfite and dye concentrations and the pH of the staining reagents, or by using better solvents for pararosaniline like acetic acid or dimethylsulfoxide (DMSO) a shift of these equilibria was attempted in order to obtain a higher amount of APA-bound dye. A 40% higher absorbance, when compared with the normal Schiff-staining, was obtained in model films by staining with a saturated solution of pararosaniline in a 1:1 v/v mixture of DMSO and SO2-water, followed by rinsing in SO2-water. A doubling of the absorbance resulted in the same objects when a saturated solution of pararosaniline in a 2 M acetic acid/acetate buffer of pH 4.45 was used for staining, followed by a short rinse in SO2-water. Amino groups (as found in histones) are shown to compete with the amino groups of pararosaniline for the APA aldehydes. This effect, although causing lower staining intensities, is shown not to be the explanation for the differences in stain content found between more and less compact forms of chromatin. Depending on the pH, and dye and sulfite concentrations of the staining reagents, the following components are considered as possible contributors to the mixture of chromophores (Duijndam et al., 1973 b) formed between APA and Schiff's reagent or its modifications: 1. An acid labile component with a wavelength of maximal absorbance (lambda max) near 510 nm; its structure is probably the azomethine--CH=N--; 2. A relatively acid stable component with a high value of molecular absorbance (epsilon), an lambda max near 570 nm and possibly having an enamine structure--CH=CH--NH--; 3. A component with intermediate acid stability, low epsilon, and lambda max near 540 nm, and which is probably an alkylsulfonic acid --CH(SO3H)--NH--compound. Small differences in the staining conditions in the histochemical application of the Feulgen-Schiff reaction may cause a shift in the ratio between especially components 2 and 3, resulting in variations in stain content and in lambda max.


Microphotometry of rat liver nucleoproteins during the cell cycle, and comparison of diploid nuclei in the G2 period with tetraploid nuclei. The following facts were established with a microphotometric investigation of isolated nuclei from rat liver in different stages of the cell cycle. During the mitotic wave occurring in the liver of newborn animals after injection of casein it was found that the naphtol yellow S (NYS) protein content of the nuclei increases about 30% during the G1-period. A second increase of around 70% was established during the S-phase whereas no increase could be observed during the G2-phase. An indication for the existence of a "critical protein mass" of the nuclei before the onset of the S-phase could be observed. The protein content of diploid nuclei in the G1-phase of adult animals is about 50% higher than in newborns. This makes it probable that there is no significant difference in the NYS-protein content of diploid nuclei in the G2-period and tetraploid nuclei of adult rats. No differences were observed between diploid nuclei in the G2-period of newborn rats and tetraploid nuclei of adult rats in their Fastgreen histon, RNA and SH plus SS content. The only criterion to distinguish between G2 nuclei and tetraploid nuclei seems to be the number of nucleoli, but this is rather unreliable.


[Histochemical investigations on sialic acid containing compounds in the CNS of teleosts (author's transl)]. By means of histochemical experiments the qualitative (microphotographs) and quantitative (cytophotometry) distribution of sialic acid containing compounds was investigated in the mesencephalon and cerebellum of Carassius carassius. 1. The perikarya of nerve cells are especially stained by means of Azur A. 2. Nerve fibres, on the other hand, showed positive reactions with colloidal ironhydroxyd (CIH). 3. In different structures of the CNS the intensity of CIH-staining is decreased up to about 55% following treatment with neuraminidase. 4. There are no differences in the reactions of 7 degrees C as compared to 20 degrees C adapted animals.


An ultrastructural and cytochemical study of neuromuscular junctions in echinoderms. The ultrastructure of neuromuscular junctions in various organs of a starfish and a holothurian was studied. All neurons were found to contain large (100-250 nm), dense-core vesicles. Cytochemical tests for acetylcholinesterase were negative for these neurons. The presence of proteinaceous neurosecretory material was contested by enzymatic digestion (pepsin) which did not attack the dense-core vesicles, as well as by incubation in phosphotungstic acid (PTA) which did not stain these structures. Staining with dichromate produced a positive reaction for 5-hydroxytryptamine. In the absence of synaptic modifications even after specific staining with PTA, the transmission of nervous impulses is effectuated through exocytosis of 5-hydroxytryptamine at nerve endings in the muscle tissue.


Distribution of acid phosphatase, beta-glucuronidase, n-acetyl-beta-d-glucosaminidase and beta-galactosidase in cornea of albino rabbit. Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase.


Effect of stabilizers on diaminobenzidine reactions of mitochondria. The effect of various stabilizers--0.9% NaCl, 7.5% sucrose and 7.5% polyvinyl pyrrolidone (PVP) on reactions of mitochondria with fresh and photooxidized diaminobenzidine (DAB) was investigated. NaCl and PVP abolished the DAB staining of mitochondria incubated without exogenous cytochrome c; NaCl, however, was ineffective when exogenous cytochrome c or oxidized DAB were present in the incubation medium. PVP considerably decreased the intensity of all reactions performed in the presence of exogenous cytochrome c or oxidized DAB. The mechanism of the observed effect is not likely to involve osmotic protection of mitochondria; extraction of endogenous cytochrome c from fresh frozen tissue by NaCl and greatly increased viscosity of PVP-containing media seem to be most probable explanations.


Feline infectious peritonitis with spinal cord involvement in two cats. Two cats from the same household had posterior paresis and hypergammaglobulinemia. Histologic evaluation of the spinal cords revealed a pyogranulomatous reaction consistent with that reported for feline infectious peritonitis.


Revistin found by screening for inhibitors of reverse transcriptase of an oncogenic virus. Revistin, a substance that strongly inhibits the reverse transcriptase activity of murine leukemia virus in our screening system, was obtained from a cultured broth of a soil streptomyces which was closely related to Streptomyces filipinensis. The assay method for the activity was based on the inhibition by a test material of the incorporation of 3H-dTMP into DNA synthesized by the reverse transcriptase of an oncogenic RNA virus. Crude revistin was isolated by serial procedures of salting out with ammonium sulfate and precipitation with cetylpyridinium chloride. The crude material showed neither antibacterial nor antifungal activity. It exhibited against splenomegaly in mice caused by Rauscher leukemia virus infection.


Platomycins A and B II. Physicochemical properties. Platomycins A and B, two new antibacterial and antitumor antibiotics were found to belong to the phleomycin-bleomycin family, being closely related to bleomycins B. The two ahtibiotics have been differentiated from all of the reported phleomycins and bleomycins.


Action of bleomycin on the bacteriophate T7 infection. A decrease in production of bacteriophage T7 was observed in bleomycin-treated Escherichia coli B cells. Bleomycin was found to shorten the eclipse in phage growth. A T7 early gene product, the T7-specific RNA polymerase which catalyzed the transcription of late gene appeared more rapidly in the bleomycin-treated cells than in the non-treated cells. The rate of phage adsorption increased to some extent in drug-treated cells. A possible mechanism to explain the mode of action of bleomycin is discussed.


Effect of preparatory techniques on the gross morphology of Mycoplasma hominis. A method is presented whereby cells of Mycoplasma hominis can be prepared with minimal distortion for electron microscopy. After the addition of glutaraldehyde to broth cultures, incubation is continued for 1 h. The cells are then collected by centrifugation, washed in distilled water, and used for negative-contrast preparations.


Energetics of the Na+-dependent transport of D-glucose in renal brush border membrane vesicles. The energetics of the Na+-dependent transport of D-glucose into osmotically active membrane vesicles, derived from the brush borders of the rabbit renal proximal tubule, was studied by determining how alterations in the electrochemical potential of the membrane induced by anions, ionophores, and a proton conductor affect the uptake of the sugar. The imposition of a large NaCl gradient (medium is greater than vesicle) resulted in the transient uptake of D-glucose into brush border membranes against its concentration gradient. In the presence of Na+ salts of isethionate or sulfate, both relatively impermeable anions, there was no accumulation of D-glucose above the equilibrium value. With Na+ salts of two highly permeable lipophilic anions, NO3- and SCN-, the transient overshoot was enhanced relative to that with Cl-. With Na+ salts whose mode of membrane translocation is electroneutral, i.e. acetate, bicarbonate, and phosphate, no overshoot was found. These findings suggest that only anions which penetrate the brush border membrane and generate an electrochemical potential, negative on the inside, permit the uphill Na+-dependent transport of D-glucose.


Evidence for a specific dihydrotestosterone-binding cytosol receptor in the human prostate. A technique for the demonstration of a human steroid and organ-specific prostate cytosol receptor is described. Tissue from 17 patients with benign prostatic hypertrophy was incubated in Eagle's medium with 3H-testosterone (T) .06 nM following pre-incubation with an anti-androgen. The minces were homogenized and the cytosol fraction obtained. The cytosol was then fractionated on a duo-gel column of G-50 Sephadex over Bio-gel A 1.5m and fractions counted for 3H, assayed for protein and plotted graphically. Three main peaks were seen. Only the second peak (approximately 150,000 mol wt), which contained predominantly dihydrotestosterone (DHT), was significantly and reproducibly inhibited by 2.1 muM cyproterone acetate (Cyp A) pre-incubation. This inhibiton was considered a specific indicator for receptor since Cyp A at muM had little effect (less than 10%) on 3H-DHT binding to plasma. Similar results were noted for other anti-androgens tested, but cortisol and etiocholanolone had no effects on 3H-5alpha DHT binding to prostate cytosol. Steroid-protein peaks for fractionated human thyroid, muscle, and spleen cytosol were not inhibited by Cyp A. Fractionated kidney cytosol contained a 3H-T-binding protein peak which was significantly decreased by Cyp A. Pronase incubation and heating at 50 C for 30 min both resulted in either a significant decrease or complete loss of receptor. Gel filtration analysis of 3H-cytosol derived from human prostate minces pre-incubated with and without Cyp A, provides a relatively rapid technique for the demonstration of cytosol receptor in approximately 80% of prostates from patients with benign prostatic hypertrophy.


Localization of T and B cells and alpha fetoprotein in hepatic biopsies from patients with liver disease. Peripheral blood and hepatic tissue T- and B-lymphocyte distributions, serum alpha fetoprotein (AFP) concentrations, and hepatic AFP were studied in 46 patients undergoing diagnostic percutaneous liver biopsy. The patients included 26 with alcoholic liver disease, 13 with nonalcoholic hepatitis or cirrhosis, and 7 with either normal histology or minor nonspecific changes. Serum AFP was determined by radioimmunoassay and hepatic tissue AFP by indirect immunofluorescence. Peripheral blood T lymphocytes were identified by the sheep red-cell rosette technique; and B lymphocytes by fluoresceinated anti-immunoglobulin antisera and IgG aggregates. Tissue identification of T lymphocytes was accomplished using an extensively absorbed rabbit antihuman thymocyte antiserum and indirect immunofluorescence; tissue B lymphocytes were identified using pepsin F (ab')2 fragments of rabbit IgG antibodies to human immunoglobulins. T lymphocytes predominanted in hepatic lymphoid infiltrates from patients with alcoholic liver disease (91+/-4%), whereas in patients with chronic active or chronic persistant hepatitis, viral hepatitis, or cryoptogenic cirrhosis proportions of T and B lymphocytic infiltrates were similar (50+/-15%). Hepatic tissue AFP was detected in 9 of 18 patients with alcoholic hepatitis; serum AFP concentration was increased in only 1 of these 9 patients. Tissue AFP was not observed in the remaining biopsy material nor were serum AFP concentrations increased. Peripheral blood T-cell numbers were significantly decreased in patients with alcoholic liver disease (P less than 0.01) and in nonalcoholic hepatitis or cirrhosis (P less than 0.025). A close relationship between peripheral blood T-lymphocytopenia and hepatic T-cell infiltrates was observed in patients with alcoholic liver disease; this relationship was less apparent in patients with nonalcoholic hepatitis or cirrhosis.


Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue. An 8-15S fraction of RNA isolated from hyperplastic human parathyroid tissue (primary chief-cell hyperplasia) and translated in a cell-free extract of wheat germ directs the synthesis of a protein that shares antigenic determinants and tryptic peptides with parathyroid hormone and its previously recognized immediated precursor, proparathyroid hormone. In addition, the protein contains tryptic peptides not found in proparathyroid hormone and migrates more slowly than does proparathyroid hormone on both urea-acid and urea-sodium dodecyl sulfate polyacrylamide gels, indicating that it is more acidic and larger than proparathyroid hormone. Sequential Edman degradation of the cell-free protein, radiolabeled with [35S]methionine, for 25 cycles released [35S]methionine at cycles 1, 7, 11, and 14, indicating that the NH2-terminal peptide sequence of the protein differs from that of both proparathyroid hormone and parathyroid hormone. We propose that this protein is an early biosynthetic precursor of human parathyroid hormone, pre-proparathyroid hormone, analogous to that identified recently by in vitro translation of bovine parathyroid mRNA.


Quantitation of G-cells in fibreoptic biopsy specimens and serum gastrin levels in healthy normal subjects. The G-cells of the gastric antrum were accurately quantitated in preparations stained with an immunoperoxidase method. In 15 normal young adults the mean G-cell count per unit area was 200 +/- 15-9. Serum gastrin levels were measured in 12, and all fell within the normal range (mean 59-7 +/- 5-4 pg/ml).


An evaluation of some commerical Romanowsky stains. The staining properties of 43 commerical Romanowsky-type stains have been studied. Considerable differences in the appearance of stained blood films were observed with different batches of these stains, the staining of red cells being particularly variable. Attempts have been made to correlate staining patterns with stain composition as revealed by thin-layer chromatography and sulphated ash analyses. In this way it has been possible to define some essential requirements for satisfactory staining.


Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat. The organization of the brainstem serotonin neuron projection to the hippocampal formation was analyzed in the rat. This projection arises in the raphe nuclei of the midbrain. Following destruction of the midbrain raphe nuclei, chiefly nucleus centralis superior, there is a 72% decrease in hippocampal serotonin content. Injection of tritiated amino acid into the midbrain raphe nuclei results in transport of tritiated protein to the hippocampal formation and this transport is blocked in animals pretreated by intraventricular administration of 5,6-dihydroxytryptamine (5,6-DHT). Autoradiographic analysis indicates that the transport reaches the hippocampal formation primarily via two major pathways, the cingulum and the fornix. Cingulum fibers terminate predominantly in the dorsal hippocampus whereas the fornix distributes throughout the entire hippocampal formation. Some fibers reach the ventral hippocampus from the entorhinal area. Within the hippocampus there is dense labeling in a restricted lamina of the CA1 stratum lacunosum-moleculare with moderate labeling in stratum radiatum. Stratum oriens is sparsely labeled in CA1 and moderately so in CA2 and CA3. Stratum radiatum and stratum lacunosum-moleculare are moderately densely labeled in CA2 and Ca3. The area dentata is sparsely to moderately labeled in the molecular layer and heavily labeled in a thin lamina of the hilar zone immediately beneath the granule cell layer. The remaining hilar zone is moderately labeled. All of the discrete labeling of the hippocampus and area dentata described above is absent in animals pretreated with 5,6-DHT. These observations indicate that serotonin neurons of the midbrain raphe provide a highly organized innervation of the hippocampal formation in the rat.


Examination of cottage cheese whey proteins by scanning electron microscopy: relationship to membrane fouling during ultrafiltration. Deposits formed on the membrane during ultrafiltration of aqueous solutions of individual whey proteins were examined under a scanning electron microscope. Gamma-globulin formed granules which agglomerated and stacked into layers to form a porous matrix on the membrane. Beta-lactoglobulin and bovine serum albumin both formed sheets on the membrane and formed multimers which were identified by sodium dodecyl sulfate gel electrophoresis. Beta-lactoglobulin also formed strands where deposits were not thick. Alpha-lactalbumin formed smooth spherical particles which did not hinder water permeation greatly. Studies of ultrafiltration rate indicated that beta-lactoglobulin and gamma-globulin were most significant in causing permeation flux decline by surface fouling.


Demonstration of an alloimmune response to embryonic enamel matrix proteins. Young adult rabbits were immunized with enamel matrix proteins from embryonic tooth organs of the same strain of rabbit. These proteins elicited an alloimmune response as demonstrated by the specific binding of antiserum to enamel matrix which was visualized by indirect immunofluorescent microscopy. The labial and lingual surfaces of embryonic incisor tooth organs were found to share common antigenic determinants. The observations suggest that enamel proteins could possibly be autoantigens.


Quantitative inhalation bronchial challenge in ragweed hay fevery patients: a comparison with ragweed-allergic asthmatics. Fourteen ragweed hay fever nonasthmatic patients comparably sensitive to a group of ragweed-allergic asthmatics by skin test and leukocyte histamine release were tested by quantitative inhalation bronchial challenge with ragweed extract. The provocation dose of ragweed extract producing 35% decrease in airway conductance was determined and designated PD35. PD35 values in the hay fever patients were not significantly different from PD35 values in the asthmatic group. These data suggest that carefully performed skin tests may be as diagnostically useful as bronchial challenge in routinely confirming the allergic etiology of seasonal asthma.


Binding properties of the anti-HBA Ag antibody. The properties of ideal HABb are defined on the basis of the results of box titrations of a random set of HBAg-positive sera with diluted human HBAb in microimmunodiffuse reaction. Ideal HBAb makes it possible to determine relative strength of antibody under test. Relative strength of antibody, together with antigenic determinants, is another criterion conditioning the determination of a HBAb standard.


Problems of housefly (Musca domestica) control due to multiresistance to insesticides. The development of chemical control of Musca domestica on Danish farms 1945--72 is outlined. It has been strongly influenced by successive development of resistance and failure of control by one insecticide after another. The chlorinated hydrocarbons used as residual sprays failed 1947--51. Organophosphorus compounds (OPC) were widely used from 1953, first as strips impregnated with parathion and residual sprays with diazinon. Resistance to OPC was first found in 1955, diazinon was given up in 1957--59 and parathion strips failed in the early '60's. Trichlorfon paint-on baits were widelyused 1958--64 and serious resistance did not appear until 1967, induced by selective pressure of fenthion and dimethoate used as residual sprays. High resistance to the contact effect of trichlorfon now occurs everywhere in Denmark. However, trichlorfon baits are still able to kill many flies. Residual sprays with fenthion, ronnel and fenitrothion were used to some extent 1960--70, but increased resistance reducing the residual effect developed in 2--3 years. Dimethoate was used on the majority of farms 1965--72. It was very effective the first years and resistance increased slowly until 1971--72, when high to extreme dimethoate-resistance became general on Danish farms. This was associated with high resistance to other OPC for fly control, e.g. fenthion, fenitrothion, bromophos, and tetrachlorvinphos, and to carbamates, with the result that no generally effective residual sprays were available. In 1971--72 frequent treatments with synergized pyrethroids have been tried. However, the method is often expensive, and serious resistance problems have appeared on a few farms. In this situation preventive, sanitary measures to eliminate or reduce fly breeding in manure are becoming decisive again, but difficult to practise due to lack of farm labour. The extreme Danish situation is compared with those in other areas, and probable reasons for differences in resistance and control problems are discussed, as well as possibilities for strategies to reduce resistance development.


Immunogenic properties of modified antigen E. III. Effect of repeated injections of modified antigen on immunocompetent cells specific for native antigen. It has been shown that ragweed antigen E loses its major antigenic determinants after denaturation in 8 M urea, but urea-denatured (UD) antigen and an alpha-polypeptide chain isolated from the denatured molecules are capable of priming mouse T cells specific for native antigen. Weekly injections of 10mug UD antigen or alpha-chain into antigen E-primed animals depressed the ongoing IgE antibody response, whereas injections of the same dose of antigen E failed to depress the antibody response. It was found by adoptive transfer experiments that helper activity of antigen E-primed splenic T cells was depressed by the treatment of the donors with either modified antigen or native antigen E. The same treatment of antigen E-primed animals depressed the DNA synthetic response of their splenic T cells to antigen E. The treatment of antigen E-primed animals with UD antigen resulted in a decrease of antigen E-specific IgE-B cells and IgG-B cells in their spleen, whereas the treatment with native antigen expanded the B cell populations. In view of the results obtained in the mouse, cellular basis for the immunologic effects of hyposensitization treatment is discussed.


The effect of alloantisera on antigen-induced T cell proliferation. In the guinea pig, alloantisera raised by cross-immunization of strain 2 and strain 13 animals are capable of specifically inhibiting the in vitro proliferative response of (2 X 13)F1 T lymphocytes to those antigens the response to which is controlled by Ir genes linked to the genes controlling the alloantigens against which the serum is directed. However, in similar studies performed in the two parental strains, the responses to antigens not known to be under unigenic control were also markedly inhibited by the appropriate alloantisera. We have extended our studies of this "nonspecific" inhibitory effect of alloantisera on T cell proliferation and have demonstrated that the proliferative response of strain 2 and strain 13 T cells to a large number of antigens is markedly inhibited by anti-2 and anti-13 sera, respectively. Antisera to the B alloantigen, the product of a linked but distinct histocompatibility locus, which is present in both strain 2 and strain 13 animals, also produced a marked inhibition of T-lymphocyte proliferation. A number of possible explanations for the generalized inhibitory effect of alloantisera on T cell proliferation are discussed.


Determinants of the hierarchy of humoral immune responsiveness during ontogeny. A model system of ontogeny was utilized to investigate the development of humoral immunity in both AKR and BALB/c mice. Lethally irradiated adult mice were reconstituted with syngeneic fetal or neonatal liver. These mice were immunized at various times after reconstitution with a series of eight antigens: the bacteriophages F2, phiX-174, and T4; the hapten carrier complexes 2,4 dinitrophenyl-bovine serum albumin and fluorescein-bovine serum albumin; and the small proteins: hen egg lysozyme, sperm whale myoglobin, and bovine pancreatic ribonuclease. Subsequent antibody production to the antigens was assayed with either a direct or a modified bacteriophage neutralization technique. Individual mice responded to the various antigens in a sequential pattern which was basically the same for all mice within each strain. However, there was a marked difference between the two strains in the time at which they developed responsiveness to myoglobin. In order to begin to delineate the separate roles played by B and T cells in the generation of this hierarchical response pattern during ontogeny, the development of anti-DNP and anti-FTC activity was examined in carrier-primed mice. Results of this experiment indicated that functional B cell specificities for the two haptens arise at different times during ontogeny. Further studies are needed to determine whether the hierarchical pattern of immune responsiveness observed for the other antigens is a function of sequential appearance of B cell specificities, T cell specificities, or both.


Histamine release from human leukocytes: relationships between cyclic nucleotide, calcium, and antigen concentrations. The antigen-induced IgE-mediated release of histamine from human basophils has previously been shown to require calcium, to be inhibited by agents which raise cyclic AMP levels and by high antigen levels, and to be unaffected by cyclic GMP. The interrelationship between these phenomena has been studied. The major findings are: 1) in the region of antigen-excess inhibition dibutyryl cyclic AMP potentiates release; 2) antigen-excess inhibition is seen at lower antigen concentrations when the calcium concentration is reduced from 0.6 to 0.1 mM; and 3) cyclic GMP modestly potentiates release when the calcium concentration is 0.1 mM.


Measurement of absolute amounts of antigen-specific human IgE by a radioallergosorbent test (RAST) elution technique. A technique for the absolute quantification of antigen-specific human IgE is described. It employs elution of a calculable amount of antigen-specific IgE from an allergosorbent-antibody complex by means of alkaline pH treatment, followed by measurement of the IgE content of the eluate with a modified radioimmunosorbent test (RIST). With this method IgE antibody directed against the benzylpenicilloyl determinant of penicillin (BPO) was measured quantitatively in sera from seven penicillin allergic patients. IgE specific for ragweed antigen E was measured in sera from 33 ragweed allergic patients. Values obtained for IgE anti-BPO ranged from 19 to 1806 ng/ml and comprised from 1.3 to 27.5% of total serum IgE. Values of IgE anti-antigen E ranged from 9 to 1807 ng/ml, comprising from 3 to 84% of total serum IgE. Excellent correlation (r = 0.99; p less than 0.001) was obtained for both antigen systems between values determined by the RAST elution technique and by simple RAST assay with interpolation from a reference serum of known specific IgE content as determined by the elution technique may be needed only for primary standardization of reference sera.


Ionophore A-23187 induced histamine release from rat mast cells and rat basophil leukemia (RBL-1) cells. Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of greater than 0.2 mug/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 mug/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained approximately 700 ng histamine/10(6) cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give non-cytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.


IgE-induced release of a platelet-activating factor from rabbit lung. Sensitized rabbit lung fragments release a platelet-activating factor (PAFL) after challenge with specific antigen or monospecific antibody to rabbit IgE. This release requires calcium and is less evident in lungs from rabbits producing IgG as well as IgE antibody. The PAFL released from lung stimulates the secretion of serotonin from washed rabbit platelets. PAFL is distinguishable from ADP or thrombin and has properties similar to PAF derived from basophils (PAFB). It is not, however, identical to PAFB since rabbit platelets specifically desenitized to PAF still respond by releasing serotonin if stimulated with PAFL.


The major histocompatibility complex of the guinea pig. I. Serologic and genetic studies. Serologic and genetic studies of the antigens which comprise the guinea pig MHC have demonstrated three distinct but linked genetic regions. Antisera to the B region were raised by cross-immunization of random-bred animals; this region controls antigens B.1, B.2, B.3, and B.4 which behave as alleles at a single locus and which resemble the products of the murine D or K region genes in their tissue distribution and molecular characteristics. Cross-immunization of inbred strain 2 and strain 13 animals, both of which bear the B.1 antigen, leads to sera which identify antigens which resemble the products of the I region of the murine MHC. Specific absorption experiments have demonstrated four distinct I region antigens. In addition to the B and I regions, inbred strain 2, strain 13, and some outbred animals bear an antigen (S.1) which is the product of a third genetic region and which also resembles the murine D or K region gene products in molecular size. The results of these studies should facilitate the use of the guinea pig as an experimental model for studies of genetic control of the immune response and the function of the histocompatibility-linked Ir genes.


Specific tumor immunity induced with soluble materials: restricted range of antigen dose and of challenge tumor load for immunoprotection. Immunogenic materials were solubilized from two methylcholanthrene-induced fibrosarcomas using 3 M KCl and were assessed for antigen activity in an immunoprotection assay. Mice which had been pretreated with a single injection of extract (0.1 to 2.0 mg protein) were challenged 10 days later with 10(3), 10(4), or 10(5) tumor cells. Optimal protection, as evidenced by survival, tumor incidence, and prolonged latent period before neoplastic outgrowth, was found after pretreatment with 0.5 mg of crude KCl extract and challenge with 10(4) tumor cells. These results further reinforce the weak nature of tumor-specific antigens as immunogens and show that only in very restricted circumstances can solubilized preparations administered without adjuvant induce immunoprotection in syngeneic hosts.


In vitro selection and extended culture of antigen-specific T lymphocytes. II. Mechanisms of selection. Functional selection of antigen-specific T lymphocytes can be achieved by culturing thymus-dependent (T) lymphocytes from immunized guinea pigs on "monolayers" of antigen-pulsed adherent peritoneal exudate cells (PEC) from nonimmune syngeneic donors. Several aspects of the in vitro selection of T lymphocyte-rich peritoneal exudate lymphocytes (PEL) were studied. It was shown that irradiated adherent PEC were equivalent to nonirradiated adherent PEC in supporting selection cultures, indicating that the lymphocytes harvested at the end of the selection culture derive from the immune donors of the PEL and not from the nonimmune donor of the adherent PEC. The relative importance of specific adherence and specific proliferation for achieving selection was determined by comparing the degree of selection obtained when nonadherent cells were discarded at 24 hr with that noted when the discard step was omitted. It was found that omitting the discard step markedly diminished the degree of selection. On the other hand, blocking proliferation with specific alloantisera after the discard step did not diminish the degree of selection, although it did diminish the cell yield. Thus, specific adherence to antigen-pulsed PEC appeared to be critical in the selection culture procedure. An estimate of the degree of enrichment obtained by the selection culture procedure was obtained by culturing selected cells in an excess of nonprimed PEL, so that auxiliary cells would not be limiting. Under these conditions, it appeared that selected cells were enrichied from 4- to 10-fold in antigen-responsive cells with respect to the initial cell population.


A sandwich method of enzymoimmunoassay. I. Application to rat and human alpha-fetoprotein. An enzymoimmunoassay (EIA) for the quantitation of soluble antigens having at least two antibody-combining sites is descirbed. This non-competitive sandwich method comprises three steps: 1) the antigen to be assayed is reacted with the antibody-coated cellulose immunosorbent, 2) the enzyme-labelled antibody is then incubated with the antigen bound to the solid phase, 3) the enzymatic activity of the immunosorbent is then measured. This activity increases with the quantity of antigen to be assayed. Examples of the application of this method to the assay of rat and human alpha-fetoprotein (AFP) are given. When applied to rat and human AFP, this assay gives reproducible results in the range of 10--1000 ng/ml and 3-1000 ng/ml respectively. AFP sera concentrations of normal and pregnant rats were assayed by EIA, radioimmunoassay RIA and rocket-immunoelectrophoresis (RIE). In all the cases good agreement was noted among these three techniques. Rheumatoid factor, whenever present, may interfere with the assay. However, the effect of this interaction can be eliminated. The advantages of the EIA method can be listed as follows: a) no pure antigen is required, b) the final color reaction is developed from the solid phase. This feature eliminates most non-specifically interfering factors, c) the range of the assay covers a 2 log-scale, d) only inexpensive equipment is used, e) results are obtained within 24 hr, f) sensitivity and reproducibility lie within a range comparable to that of RIA.


Antigen-coupled beads adherent to slides: a simplified method for immunological studies. Small amounts of antigen-coupled beads adherent to object slides provide a simple, quick, economical and sensitive immunohistochemical means of detecting antibodies in serum by both immunofluorescence and immunohistoperoxidase procedures. Sensitivity increases with decreasing quantities of antigen-coupled beads, as was demonstrated in the fluorescence procedure.


Peritoneal macrophages as target cells for measuring virus-specific T cell mediated cytotoxicity in vitro. In inbred or outbred mice lysis of virally modified cells by thymus-derived lymphocytes occurs only within a H-2 compatible system. Peritoneal macrophages provide a convenient source of syngeneic target cells. They are readily infected in vitro with LCM virus, and show high levels of specific 51Cr release when exposed to T cells from LCM immune mice.


Increased reactivity of rat alpha-foetoprotein with corresponding antiserum after 125I labelling. Two rat alpha-foetoproteins (AFP) of different origin and purified according to two different techniques were labelled with 125I using an identical procedure. After labelling the reactivity of one of these AFPs was not modified whereas that of the other was markedly increased; this in spite of a greater purity made the latter unsuitable for the radioimmunoassay of rat AFP. The observed difference may be due to the technique of purification and brings additional evidence for the necessity of checking the reactivity of tracers.


Hepatitis B antibody in conventional gamma-globulin. Recent lots of conventional gamma-globulin prepared from the plasma of outdated blood collected from voluntary donors in Massachusetts contain substantially more antibody (anti-HBs) to the hepatitis B surface antigen (HBs Ag) than do lots manufactured from plasma collected before 1970. Variations in titer of anti-HBs may have been related in part to local variations in the incidence of hepatitis B. However, data available since 1949 suggest that one must consider whether antibody excess (anti-HBs) or antigen excess (HBs Ag) prevailed among individuals contributing to the plasma pools. The titers of anti-HBs have continued to rise since 1971, when screening of blood donors for HBs Ag became mandatory, a step which presumably resulted in the removal of much of the HBs Ag that would otherwise adsorb anti-HBs. The anti-HBs titer in gamma-globulin routinely produced in Massachusetts now equals or exceeds the titer in other lots of gamma-globulin found to be effective in recent studies of preexposure prophylaxis of hepatitis B.


Change of hepatitis B surface antigen in patients with hepatitis during a five-year period. Between January 1970 and December 1974, 122 cases of acute type B hepatitis were subtyped; 66 (54%) were of the ad type and 56 (46%) were of the ay type. "Cluster" cases (from a dialysis unit) were not considered. During the first period, subtype ad predominated, whereas in the second year there was a clear predominance of the ay subtype; the difference was statistically significant (P less than 0.02). In yearly periods the differences were significant between the years 1970 and 1974 (P less than 0.05), 1972 and 1974 (P less than 0.05), and 1972 and 1973 (P less than 0.05). If only patients without parenteral exposure are considered, there was clearly a shift between 1970-1972 and 1973-1974 in favor of the ay subtype (P less than 0.01). Since epidemiological factors such as injections and transfusions seem not to be responsible, it is suggested that a change of virus strain may be responsible for the different distribution of subtypes of hepatitis B surface antigen in the last year.


Bifunctional major histocompatibility-linked genetic regulation of cell-mediated lympholysis to trinitrophenyl-modified autologous lymphocytes. Murine thymus-derived lymphocytes can be sensitized in vitro to trinitrophenyl (TNP)-modified autologous spleen cells (1, 2). Cytotoxic effector cells were generated which were specific for TNP-modified target cells expressing the same H-2K and H-2D serological regions as the modified stimulator cells (3, 7). Spleen cells from two C57BL/10 congenic strains of mice sharing common I-C, S, and D regions, but differing at K, I-A, and I-B regions, generated different levels of lytic responses to the shared modified H-2Dd products upon sensitization with auto logous TNP-modified cells. Lymphocytes from an F1 between responder and nonresponder strain generated a level of cytolysis toward the H-2Dd modified specificity which was of the same order of magnitude as that obtained with the high responder, irrespective of whether F 1 or either parental strain of modified stimulator cell was used. These results suggest that the modification of H-2Dd products resulted in formation of new antigenic determinants in both parental strains. However, the difference observed in responsiveness appeared to be due to a gene or genes mapping in the K, I-A, or I-B region which influenced the ability of the responding lymphocytes to react to these modified H-2Dd products. Responsiveness was expressed as a dominant trait in the F1.


Collaboration of allogeneic T and B lymphocytes in the primary antibody response to sheep erythrocytes in vitro. This study provides a direct quantitative comparison of the helper effects of allogeneic and syngeneic rat T cells in the production of direct SRBC plaque-forming cell (PFC) responses by B cells in culture. In syngeneic T-B combinations, log-log plots of the number of PFC generated after 5.5 days in culture vs. the number of T cells employed as helpers showed a linear response between 10(4) and 2.5 times 10(5) T cells added. Allogeneic T-B combinations, in which the T cells possess the capacity for reactivity to major alloantigens of the B-cell donor, showed a different dose/response relationship in which PFC responses were decreased at high T/B ratios and augmented at low T/B rations. In this system responses were detected with as few as 10(3) allogeneic T cells. Use of negatively selected allogeneic T populations, specifically depleted of mixed lymphocyte interaction (MLI) and graft-vs-host reactivity for B-cell alloantigens, as helpers gave dose/response curves quantitatively identical to responses with syngeneic T-B combinations and also with F1 T-cell parental B-cell combinations. These data indicate that rat T and B cells need not share a major histocompatibility complex haplotype in order to collaborate effectively in a primary direct PFC response to SRBC in culture. In addition, the PFC response required the combinaed presence of T and B cells as well as antigen in the cultures, a finding consistent with the two signal model of B-cell activation. Finally, the dose/response data obtained suggest the possibility that although SRBC antigen is required in the cultures helper activity with low numbers of normal allogeneic T cells may not depend on T cells having specificity for this antigen.


Light-sensitive swelling of isolated frog rod outer segments as an in vitro assay for visual transduction and dark adaptation. Frog rod outer segments swell slowly after being shaken from an excised retina into a modified Ringer's solution. The swelling has the following characteristics: (a) It is suppressed by illumination which bleaches only 500 rhodopsin molecules per outer segment per second. This is approximately the level required to saturate the in vivo receptor potential. (b) Light suppression is seen in  NaCl but not in KCl solutions. (c) Dark swelling is labile and is enhanced by calf serum, low calcium concentrations, dithiothreitol, and cyclic nucleotide phosphodiesterase inhibitors. (d) Lowering the pH to 5.5 or removing magnesium reversibly reduces dark swelling to the same extent as illumination. (e) The amount of light required for maximal suppression of dark-swelling increases approximately 10-fold if the calcium concentrations is lowered by EGTA addition. (f) The effect of illumination is irreversibly abolished by antimycin and other inhibitors of mitochondrial electron transport. (g) A process analogous to dark adaptation in vivo can be observed: If 10-50% of the rhodopsin present is bleached and the outer segments are then kept dark, rapid dark swelling returns after a period of 15-45 min. This swelling is again sensitive to light. We tentatively ascribe the light suppression of swelling to the same decrease in sodium permeability which is observed on illuminating living receptor cells. The experiments suggest that outer segments retain their competence to perform both transduction and dark adaptation after their separation from the retina.


Low temperature slowing and cold-block of fast axoplasmic transport in mammalian nerves in vitro. 1) Fast axoplasmic transport in mammalian nerve in vitro was studied using an isotope labeling technique. The rate of outflow in cat sciatic nerve fibers of 410 mm/day in vitro was reduced at temperatures below 38 degrees C with a Q10 of 2.0 in the range 38-18 degrees C and a Q10 of 2.3 at 38-13 degrees C. 2) At a temperature of 11 degrees C a partial failure of transport occurred. At temperatures below 11 degrees C a complete block of fast axoplasmic transport occurred, a phenomenon termed "cold-block." No transport at all was seen over the temperature range of 10-0 degrees C for times lasting up to 48 hr. 3) Transport was resumed after a period of cold-block lasting up to 22 hr when the nerves were brought back to a temperature of 38 degrees C. Some deleterious effects due to cold-block were seen in the recovery phase as indicated by a reduction in crest amplitude, change in its form, and slowed rate. 4) The approximately P level (combined ATP and creatine phosphate) remained near control level in nerves kept at low or cold-block temperatures for times as long as 64 hr. The reduction in fast axoplasmic transport rate seen at low temperatures for times up to 22 hr was therefore considered due to a decrease in the utilization of ATP, a concept in accord with the "transport filament" model proposed to account for fast axoplasmic transport. 5) The sloping of the front of the crest over the temperature range of 18-13 degrees C suggests an additional factor at the lower temperatures. A disassembly of microtubules is discussed as a possible explanation of the cold-block phenomenon.


A quantitative analysis of isotope concentration profiles and rapid transport velocities in the C-fibers of the garfish olfactory nerve. In the olfactory nerve of the long-nosed garfish (Lepisosteus osseus), unusually well-defined isotope concentration distributions can be established with the rapid transport process. Transport velocities of two profile loci can be accurately described and a quantitative profile analysis is possible after profile normalization. Results from such studies indicate that: (1) peak amplitudes decrease exponentially as a function of distance from the olfactory mucosa according to the equation p = 2130 exp (-0.109chi); (2) the wavefront base and the peak apex loci move at rates of 221 +/- 2 and 201 +/- 4 mm/day, respectively (at 23 degrees C), revealing a peak dispersion or broadening during transport; (3) the broadening is asymmetric with material shifting to the rear of the peak; (4) plateau regions are established behind the peak with material deposited by the peak; (5) only 20% of the total radioactivity in a cut nerve reaches the nerve terminals in the rapid transport peak while 80% is deposited along the axon; (6) profile areas from cut nerves decrease and lose 15% of their activity in 20 hr, while intact nerve profiles increase 10% in 16 hr due to continued somal contribution to the profile; (7) the displacement of the wavefront base (WFB) and peak apex (PA) profile loci can be described by the functions s(WFB) = (0.055T - 0.345)t - 1.43 s(PA) = (0.053T - 0.391)t - 2.71 (8) transport velocities are linear functions of temperature between 10 and 25 degrees C and increase 370% in that range. A linear extrapolation of the WFB and PA functions to 37 degrees C yields 410 and 377 mm/day, respectively.


Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-beta-hydroxylase. An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-beta-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3 degrees C, while keeping the remainder at 37 degrees C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37 degrees C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 +/- 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.


Axonal transport of RNA during regeneration of the optic nerves of goldfish. The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of 3H-uridine has been studied during various stages of optic nerve regeneration. 3H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Five were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, 14C-proline was also injected into the right eye as a marked of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of 3H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microsocpic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be 3H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of 3H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikaprya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.


Effect of hypocholesterolemic drug AY9944 on cultured nervous tissue: morphologic and biochemical studies. The effects of different concentrations of the hypocholesterolemic drug AY9944, an inhibitor of delta7-reductase, on organotypic cultures of fetal mouse spinal cord, were studied by light and electron microscopy. Exposure to 10(-6)M produced no observable changes. After 6 hours exposure to 10(-4)M, dense membrane-bound inclusions were occasionally observed in neurons. After 24 hours exposure to 10(-4)M, numerous cytoplasmic inclusions occurred in neurons, glia and macrophages. The form of these inclusions varied but were predominantly of two types; concentric, loosely-packed lamellae resembling membranous cytoplasmic bodies (MCB) of Tay-Sachs disease and irregular dense bodies. They were identical to those observed in our previous in vivo study. Prolonged exposure to the drug at 10(-4)M caused an increased number of inclusions in all cell types. Eventually the cultures degenerated. The number of inclusions increased for at least 38 days following a 2-5 day exposure to AY9944 at 10(-4)M. However, by 70 days, although inclusions persisted, the cultures were mostly astrocytic. In long-term cultures, in addition to these inclusions, curved or straight electron-dense paired profiles were seen in some cells, presumably macrophages. Biochemical analysis of cultures exposed to 10(-4)M revealed the continuous presence of delta7, 24-cholesta-diene-3beta-ol and 7-dehydrocholesterol even after the drug wa removed from the cultures. In our previous animal experiments, intracytoplasmic inclusions and abnormal sterols with a double bond at the 7 position disappeared quickly after discontinuation of the drug. Therefore, the results obtained in our present in vitro experiments are different in this regard from the in vivo studies of AY9944.


[Blood flow and oxidative metabolism of the brain in patients with dementia (author's transl)]. The purpose of this retrospective study was to investigate how the blood flow and oxidative metabolism of the brain was changed in dementia and the influence of the age factor. Cerebral blood flow (CBF) was measured in 115 patients aged from 40 to 83 years by means of the Kety-Schmidt technique with the modification of Bernsmeier and Siemons. The cerebral metabolic rates of oxygen and CO2 were determined by the van Slyke method and by gaschromatography respectively and of glucose and lactate by standard enzymatic methods. All cases of dementia due to head injuries, cerebral infections, cerebral infarctions, exogenous or endogenous intoxications or circulatory diseases were excluded from this study, but no classification of the dementias was made. Statistical calculations were carried out by means of the analysis of variance for a two-way design. Cerebral blood flow did not show a normal distribution curve but was at least triphasic; CBF in demented patients was either lower than normal, normal or higher than normal. The distribution curves showed further that a low cerebral blood flow of mean 32.5 ml/100 g min coincided with a low CMR oxygen of 2.50 ml/100 g min; however, CMR glucose was either low (2.50 mg/100 g min), or nearly normal (4.50 mg/100 g min) or elevated (7.50 mg/100 g min). A normal (45.0 ml/100 g min) or enhanced (62.5 ml/100 g min) CBF correlated with a CMR oxygen which was either decreased to 2.75 ml/100 g min or increased to 4.75 ml/100 g min; CMR glucose was either decreased to 1.50 mg/100 g min, or nearly normal (4.50 mg/100 g min), or was elevated to 6.50 and 10.50 mg/100 g min with respect to the peaks of the distribution curves. It is assumed that the variability of the findings with respect to the blood flow and oxidative metabolism of the brain in dementia is due to different pathophysiological and pathobiochemical disturbances in the brain. A significant influence of age on CBF and metabolism in patients with dementia was not found.


[Indications for stereotaxic neurosurgery of patients with multiple sclerosis (author's transl)]. The long-term results of 12 stereotaxic operations on 11 multiple sclerosis patients with incapacitating intention tremor were evaluated and compared with the experiences of other authors. The selection of the patients, the criteria applied for the success and the length of the follow-up period influenced the reported results. Considering not only the relief of the intention tremor but the overall performance after the operation only a certain group of patients seemed to profit by neurosurgical treatment. Applying this criterion of overall performance and evaluation only 3 out of 11 patients in our series had real benefit from the operation. The reasons for this small number of good or moderate results are given with brief discussion of some of the cases. History, course and fatal outcome of one patient are presented in detail together with the neuropathological findings. According to the literature and the limited number of our own cases the following indications for stereotaxic operations on MS patients can be established: 1. Tremor and hyperkinetic movements should be the dominant features of the symptomatology. 2. The overall performance should be essentially improved by the operation. 3. Patients in the terminal stage of the disease gain little from the procedure, whereas patients with longstanding more benign course are the best candidates.


[Migraine and the vestibular apparatus (author's transl)]. Thirty patients with simple or ophthalmic migraine had an otoneurological examination. 13% complained of vertigo during attacks of migraine, 17% of giddiness and 6.7% of tinnitus. The usual tests for equilibrium were normal. On electronystagmographical examination 17% of the patients showed only pathological spontaneous nystagmus, 33% only disturbances of caloric response and 7% a combination of both. Audiological testing was normal. The incidence of vestibular disturbances, particularly sensitivity differences of the labyrinths, was significantly greater in the group of patients with migraine than in a control group of 30 healthy persons (P less than or equal to 0.025). On the average, the sensitivity differences, in terms of duration of reactive nystagmus, were greater for these than for the healthy persons (P less than or equal to 0.01). Patients with vestibular disturbances did not show a greater frequency of pathological EEG readings than patients with normal vestibular function. Some hypotheses concerning the pathogenesis of vestibular symptoms as found in migrainous patients are mentioned.


[Orbicularis oculi reflexes and evoked response of orbicularis oris muscle in Bell's palsy. A prognostic study (author's transl)]. In 84 patients with idiopathic, clinically complete Bell's palsy the electrically induced blink reflexes with their two components (OOR I and II) were electromyographically recorded on both sides using skin electrodes. In 67 of these patients the evoked responses of the orbicularis oris muscle were also studied. The latencies and amplitudes were measured and related to the clinical outcome of the facial paralysis. The patients were divided into two groups, one with good recovery of the palsy (46 patients), the other with significant residual paresis and/or strong associated movements of the facial musculature (38 patients). In the group with good recovery the following results were obtained: 1. the OOR I remained elicitable or reappeated during the first 12 days after the onset of palsy; 2. the OOR II began to rise during the first 10 days of palsy; 3. the amplitude of the orbicularis oris response did not decrease to below 10%. In the group with poor recovery: 1. both components of the OOR were absent or diminished to below 4% for more than 12 days after the onset of palsy; 2. the latency difference of the OOR I exceeded 8 msec; 3. the amplitude of the orbicularis oris responses decreased to below 10%. Using these criteria it appears to be possible in about 85% of patients to make a prognosis between the 3rd to 5th and the 10th to 12th day after the onset of Bell's palsy.


Polyradicular neuritis and Hashimoto's thyroiditis. The case of a patient with a pluripotential immuneproliferative syndrome is reported. She suffered from Hashimoto's thyroiditis with secondary cold agglutination disease and from polyradicular neuritis. Polyradicular neuritis with albumino-cytological dissociation in the CSF had developed chronically and improved only when corticosteroid therapy was begun. Based on this case report fundamental considerations concerning the pathogenesis and therapy of Landry-Guillain-Barre-tstrohl syndrome are discussed.


Late infantile metachromatic leucodystrophy (MLD). Clinical and diagnostic evaluation in a typical case. A typical case of late infantile MLD is presented with all available clinical, morphological and biochemical results. The diagnostical value of the different parameters is evaluated and the pathogenesis of the disorder discussed. In spite of successful experimental enzyme substitution in cultured MLD fibroblasts with restitution of function by added Arylsulfatase, the therapeutic possibilities for the fatal disease in the patients are extremely limited.


Chemical and biologic properties of isolated radiolabeled bleomycin preparations. Cobalt-57-bleomycin is a diagnostically useful radiopharmaceutical, but little is known about the nature of its individual fractions in regard to their metal-binding capacity and their in vivo distribution. Bleomycin was separated by high performance liquid chromatography (HPLC) into four major components. These were labeled and the distribution studied in tumor-bearing rats at 2 and 24 hr. In vivo radiochemical purity was also determined. Of the nine HPLC systems studied, Porasil A eluted with a mobile phase of 0.3% ammonium formate in methanol gave the best separation of the fractions. These fractions were copper free and retained their biologic activity and purity. An in vitro competitive binding study of 57Co-bleomycin with either 57Co-human serum albumin (HSA) or 57Co-ethylenediaminetetraacetic acid (EDTA) showed the labeled bleomycin to be a strong chelate. The biologic distribution in tumor-bearing rats showed significantly higher concentration in tumors at 2 hr for fractions A2 and B2 as compared to the bleomycin mixture. The other fractions, A1 and demethylA2, gave lower tumor concentrations than the bleomycin mixture. The tumor-to-blood ratios for A2 and B2 were not significantly different from the bleomycin mixture, suggesting that the concentration of the bleomycin in the tumor was related to blood concentration. Tumor-to-blood ratios of greater than 10:1 at 2 hr were achieved for A2, B2, and the mixture; ratios of greater than 31:1 were achieved at 24 hr for all three. From these data it appears that the major components A2 and B2 are the most useful for diagnostic tumor imaging.


Some experience with 57Co-labeled bleomycin as a tumor-seeking agent. Bleomycin labeled with 57Co was used as a tumor-localizing agent in 132 patients. In patients with pulmonary tumors the primary localization concentrated radioactivity in 52 of the 54 appropriate cases; out of the 22 clinically known metastases, 19 were visible on the scan; 40 unknown metastases especially in hilus and mediastinum were found by the method and subsequently confirmed. In 22 patients with malignant lymphomas, 18 out of 22 known pathologic lymph glands above the diaphragm were visible on the scan; below the diaphragm the results of scanning in lymph glands and spleen were disappointing, probably because of the disturbing concentration of radioactivity in the kidneys, the bladder, the liver, and sometimes the gut. In 25 patients with various other tumors, 16 out of 22 known localizations above the diaphragm were visible; 2 were uncertain  and 4 negative. Below the diaphragm the results were usually negative. In 24 patients with benign lesions, uptake of 57Co-bleomycin was visible on the scintigram in 4 patients with cavitating pulmonary tuberculosis, in 2 with pulmonary infections, in 1 with Caplan lesions of rheumatoid arthritis in the lung, and in 1 with sinusitis ethmoidalis. The significance of these results is discussed.


Enzyme histochemical studies on the formation of hyalin bodies in the epithelium of odontogenic cysts. Frozen sections of eight odontogenic cysts, including one keratocyst, were incubated to show the following enzyme activities: NADH2 diaphorase, NADPH2 diaphorase, glucose-6-phosphate dehydrogenase, acid phosphatase and leucine aminopeptidase. The disbribution of lipid was shown by the oil red 0 method. The activities of all three oxidative enzymes were strongest in epithelial cells bordering hyalin bodies and in basal cells in the epithelial lining. Hydrolytic enzyme activity was absent from all but the most superficial epithelial cells but was present in macrophages and, in lesser amounts, in granular material in the same sections. The granular material frequently contained lipid. The lack of hydrolytic enzyme activity in bordering epithelial cells is inconsistent with the theory that hyalin bodies form from degenerating blood vessels. High aerobic oxidative enzyme activity in the same cells also conflicts with the concept that the bodies are a keratinous product. The findings lend support to the theory that hyalin bodies are an epithelial secretion.


Antigens of the Kell blood group system on neutrophils and monocytes: their relation to chronic granulomatous disease. KX, an antigen related to the Kell blood group system, is present in trace amounts on normal red cells and is strongly active on the neutrophils of all of 50 persons thus far tested. Normal circulating monocytes are now shown to also bear KX determinants. Absence of neutrophil KX has been associated with all of three previously tested patients with chronic granulomatous disease. In this study two male siblings with CGD also have been shown to have KX negative leukocytes, and white blood cells from their heterozygous mother were found to have a reduced competency to absorb anti-KX. Five CGD boys are known to lack KX; the probability of this occurring by chance is greater 10(-6).


The effect of endotoxins and enzymes in vitro on the release of gingival histamine. Gingival samples removed from fifteen Beagle dogs were sectioned into small pieces, parts of which served as the uncultured piece; the remaining pieces were organ cultured for four hours at 37 degrees C in MEM control, compound 48/80, endotoxins, protease, collagenase, hyaluronidase, trypsin and chymotrypin media. Uncultured and cultured tissues and spent media were analyzed spectrofluorometrically for histamine content. The uncultured gingiva contained a mean of 2.80 mug histamine/g of tissue and was considered to contain 100% total histamine available for release. The percentages of histamine released into the medium were 5.4% for culture control, 57.3% for compound 48/80, 5.4% for endotoxins, 77.3% for protease, 16.1% for hyaluronidase, 24 for collagenase, 39.3% for trypsin, and 36.5% for chymotrypsin. When compared to the culture control, all test substances showed statistically significant histamine release (P less than 0.005 to P less than 0.0005) except for the endotoxins and for hyaluronidase (P greater than 0.05). The results demonstrate (1) that gingiva contains a potential source or reservoir of histamine, presumably in mast cells, and when appropriately challenged in vitro can release this histamine; (2) no direct effect of endotoxins on histamine release in vitro, (3) that all enzymes tested except hyaluronidase resulted in significant histamine release. The results of this in vitro study support a thesis that enzymes are active in the early events of gingival inflammation.


On the pharmacology of Org 6338 (2beta, 16beta-dipiperidino - 5alpha - androstan - 3alpha - ol acetate dimethobromide), a new steroidal neuromuscular blocking agent. Org 6368 is a homologue of pancuronium bromide. Its interactions with other agents in the cat sciatic nerve-gastrocnemius muscle preparation revealed that paralysis was of the non-depolarizing type. This was confirmed in experiments using avian muscle. Org 6368 is a potent muscle relaxant being 2-4 times as potent as (+)-tubocurarine in the cat. Paralysis in the cat is rapid in onset and of appreciably shorter duration than that of pancuronium and (+)-tubocurarine. Repeated injections of the same dose of Org 6368 show no cumulative effect. Muscle relaxant doses generally cause a slight increase in both blood pressure and heart rate. Although its histamine-releasing capacity is greater than that of pancuronium it is less than that of (+)-tubocurarine. Org 6368 shares with pancuronium a very weak effect on both the muscarinic receptor and ganglionic transmission. Differences in the muscle relaxant profiles of Org 6368 and pancuronium are discussed.


Preliminary investigations of intrinsic and extrinsic optical activity as purity criterion for human serum albumins. Induced circular dichroism measurements were made to follow the binding of four acidic drugs to two lots of crystalline and two lots of fraction V human serum albumins. The magnitude of the induced circular dichroism varied with all lots of albumin, suggesting a strong sensitivity of the phenomenon to small changes in purity or secondary structure of the albumins. The circular dichroism of the albumins themselves showed much less variation. The more classical analytical techniques of UV absorption, measurement of absorption following methyl orange binding, gel electrophoresis, and ultracentrifugation were also performed on the albumins for comparison.


Effect of ascorbic acid on histamine metabolism in scorbutic guinea-pigs. 1. In guinea-pigs fed an ascorbic-acid-free diet, as the ascorbic acid levels decreased the histamine levels in blood and urine rose steadily to maxima in about 10-12 days. The elevated histamine levels persisted in the blood and urine of scorbutic guinea-pigs and the histamine levels in lung, gastric mucosa and spleen also increased. The increased histamine content of the urine, blood and other tissues in the ascorbic-acid-depleted condition could be brought back to normal levels by administration of a single dose of ascorbic acid 5 mg/100 g body wt. guinea-pig. 3. The drop in the elevated histamine level was not due to an indirect effect of ascorbic acid on histamine forming capacity, histaminase activity or histamine release.


Antiglobulins in ankylosing spondylitis. An immunoadsorption technique was used to investigate the presence of antiglobulin factors in 28 patients with ankylosing spondylitis and 30 healthy individuals. Both groups were "sero-negative" by standard agglutination techniques. Whereas the controls were consistently negative, 18 of the 28 patients with ankylosing spondylitis had antiglobulins of the IgG class.


Isolation of an endogenous C-type RNA virus from Mus musculus molossinus. We isolated a type-C RNA virus from the Japanese field mouse, Mus musculus molossinus. M. musculus musculus and M. musculus molossinus are two different subspecies of Mus and thus only distantly related. The virus grew only on cells foreign to the host, was xenotropic, and readily rescued the murine sarcoma (MuSV) genome from a normal rat kidney cell line transformed nonproductively by the Harvey strain of MuSV. The virus banded at a density of 1.16 g/ml and contained an RNA-dependent DNA polymerase.


Oncofetal protein accompanying irradiation-induced small-bowel adenocarcinoma in the rat. A tumor-associated protein was found in tissue derived from an X-irradiation-induced adenocarcinoma in the small bowel of the rat. The protein was associated with the cell membranes of the tumor tissue. It shared common antigenic determinants both with a rat fetal protein and a perchloric acid-soluble protein isolated from the serum of the tumor-bearing rat.


Autogenous immunity to endogenous RNA tumor virus: reactivity of natural immune sera to antigenic determinants of several biologically distinct murine leukemia viruses. Sera from normal (C57BL/6XC3H/Anf)F1(B6C3F1) mice reacted with several biologically distinct murine leukemia virus(es) (MuLV) by radioimmune precipitation assays with the use of purified tritiated leucine-labeled virus. The reactivities of this natural antibody to viral envelope antigens of two laboratory strains (Rauscher and Moloney) and two endogenous mouse C-type viruses (AKR and BALB:virus-2) were further analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar patterns of antibody reactivities to AKR MuLV and the two member viruses of the Friend-Moloney-Rauscher group were found. Three major antigenic determinants of the virus envelope, gp71, gp43, and p15, were recognized by and precipitated natural antibody. In all viruses examined, normal B6C3F1 sera precipitated comparable amounts of gp71 and gp43. However, compared with the other viruses, the amount of p15 (relative to the glycoproteins) precipitating from BALB:virus-2 was significantly lower. This appears to be due to a lesser amount of p15 on the xenotropic virus. While heterologous antisera to purified gp71 and p15 of MuLV reacted to a certain degree with rhabdomyosarcoma virus 114 and rat leukemia virus, natural mouse antibody did not. These results suggest that MuLV have common antigenic determinants recognized by natural antibody, and that the reactivities of natural antibody in an autogenous immune response are restrictive in contrast to immune antibody produced in a heterologous host.


Lymphoid leukosis in chickens chemically bursectomized and subsequently inoculated with bursa cells. Lymphoid leukosis (LL), a neoplasm of the bursa-dependent lymphoid cells of the chicken, was induced by Rous-associated virus-1 in susceptible chickens. Cyclophosphamide (CY), which destroyed the lymphoid elements of the bursa of Fabricius and abrogated humoral immunity, prevented LL. Concomitantly, osteopetorosis and other neoplasms increased. Transfer of bursa cells from chickens into CY-treated hatchmates restored immune competence. Birds whose B-cell functions were reconstituted died of LL and were less likely to die of osteopetrosis and other neoplasms than were CY-treated chicks. These results suggested that the bursa cell transferred into the CY-treated chicks were the target cells for lymphoid leukosis transformation.


Multiple in vitro mechanisms of tumor cytotoxicity demonstrated in the line-1 guinea pig hepatoma model. The line-1 guinea pig hepatoma was used to study in vitro tumor cytotoxicity. Cytotoxicity was determined by measurement of the loss of tritiated thymidine-labeled target cells from culture vessels. With this technique, we demonstrated that significant tumor cytotoxicity was caused by lymphoid cells from tumor-immune guinea pigs, by cells from guinea pigs immunized against an antigen urelated to the tumor target, and by cell-free supernatants rich in lymphocyte mediators. Addition of normal peritoneal exudate cells enhanced the cytotoxic potential of a small number of highly purified immune lymphocytes, which suggested that recruitment of normal cells is an additional mechanism of tumor cell death in this system.


Tumor-associated antigens in isoantigenic variants of a 3-methylcholanthrene-induced sarcoma. A sarcoma was induced in an (A.CA X A.BY)F1 mouse. Two isoantigenic variants were selected by loss of one H-2 antigen. The tumor-associated transplantation antigens (TATA) of these variants were compared as to their specificities in (A.CA X A.BY)F1 mice. Both transplantation and indirect membrane immunfluorescence tests revealed that TATA of both variants did not cross-react. Thus selecting angainst different H-2 antigens also selected different TATA. Karyotype studies suggested that both variants originated from a unique clone.


Temperature-dependent rosette formation by mouse lymphoma cells as a result of viral hemadsorption. Cells from several mouse lymphomas formed rosettes with nonsensitized foreign erythrocytes through C-type virus particles clustered on the cell surface in serum-free medium held at 4 degrees C. This type of rosetting was found most typically in a lymphoma induced by Rauscher leukemia virus in tissue culture (RD-12), but it also occurred in 23 of 61 spontaneous thymic lymphomas in AKR mice. Chemically or X-ray-induced leukemias and spontaneous reticulum cell sarcomas did not form rosettes. The nature of the rosette formation may be interpreted as viral hemadsorption, with a possible relationship to hemagglutination by murine leukemia viruses. The receptor on virus particles was trypsin sensitive and showed high affinity to serum inhibitors (RIF). Serum rosette-inhibiting activity was assessed by a quantitative rosette inhibition test; rosette inhibition proved widely distributed among species. Physicochemical properties of serum RIF and their function both in vivo and in vitro were described. Rosette formation with similar temperature requirements, previously reported in a mouse lymphoma carrying membrane-bound heterophile cold hemagglutinin, was readily distinguished from viral hemadsorption by its insensitivity to mouse serum RIF.


Alloantigen expression of a rat Moloney sarcoma. Cells of a brown Norway (BN) rat Moloney sarcoma (MST) failed to express certain BN alloantigen specificities and bound only about 30-50% the amount of labeled alloantibody bound by normal BN spleen cells. MST cells lacked antigen specificities shared by BN and WF rats, but expressed some of the antigen shared by BN and AUG rats. Further loss of alloantigens that occurred with prolonged in vitro culture was associated with reduced virulence of MST cells for syngeneic hosts and with increased expression of tumor-associated antigens. The LEW rats, which are resistant to MST cells, might have rejected the tumor on the basis of factors other than Ag-B antigens.


Host range studies of FLOPC-1 murine myeloma C particles. The host range of the C particle produced by FLOPC-1 myeloma cells, FLOPC-1 murine myeloma-associated virus (FL-MuMAV), was assessed in terms of its ability to productively infect and/or induce new viral antigens in a variety of different cell lines. Production of C particle-like structures by cells exposed to FL-MuMAV) was determined by incorporation of [3H]uridine into particles with a density of 1.16 g/ml and/or measurement of RNA-dependent DNA polymerase activity in concentrated culture medium. to FL-MuMAV was capable of infecting NIH/3T3, normal rat kidney (NRK) cell, BALB/c 3T3, and the A31 clone of BALB/3T3 cells but not rabbit cell line, SIRC. Thus, it is an N, B-tropic murine virus as replication in NRK cells has been shown not to delineate a group of murine viruses with a separate host range (M. M. Lieber, C. J. Sherr, and G. J. Todero, 1974). Further neoantigens, reactive with anti-FL-MuMAV serum, were detected on infected cells. Production of the MuMAV-like particle and MuMAV-associated cell antigens in infected NIH/3T3 and NRK cells persisted for three subcultures. The limited production could not be explained by the lack of an RNA-dependent DNA polymerase or high-molecular-weight RNA as the particles possessed both of these properties. The particles produced by infected NIH/3T3 or NRK cells were antigenically and physicochemically similar to FL-MuMAV and not K-MuLV. The MuMAV-like particles produced by infected NIH/3T3 were capable of limited replication in NIH/3T3 and and BALB/3T3 cells, whereas NRK-MuMAV replicated for a limited period in NIH/3T3, NRK, and SIRC cells; i.e., they had a different host range than FL-MuMAV. The particles produced by infected BALB/3T3 and A31 cells had the same host range as FL-MuMAV. In certain situations, isotopically labeled particles with a density of 1.16 g/ml were produced which appeared to lack RNA-dependent DNA polymerase.


In vitro transcription of DNA from the 70S RNA of Rous sarcoma virus: identification and characterization of various size classes of DNA transcripts. DNA transcripts, ranging from 1,500 to 4,500 nucleotides in length, can be identified in the DNA product synthesized in vitro by the Rous sarcoma virus-associated RNA-directed DNA polymerase. Although these DNA transcripts are considerably larger than the size classes of the DNA product previously reported, they only represent a minor proportion (less than 5%) of the total DNA synthesized in standard reaction mixtures containing rate-limiting concentration of one of the four deoxynucleoside triphosphates. However, the proportion of these larger transcripts relative to the total DNA product increases substantially when the enzymatic synthesis of DNA is performed in the presence of equimolar concentrations of deoxynucleoside triphosphate precursors. Both rate-zonal sedimentation in alkaline sucrose and nucleic acid hybridization techniques have confirmed the length and genetic complexity of these larger DNA transcripts. The identification of large DNA chains in the DNA product synthesized in vitro by the avian oncornavirus RNA-directed DNA polymerase provides an explanation for the paradox that exists between the limited number of primer sites per 70S RNA genome, the small size of the bulk of the DNA product, and the extent of the Rous sarcoma virus genome represented by the DNA product.


Surface structure of Uukuniemi virus.

Surface structure of Uukuniemi virus. Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques. The spherical virus particle measures about 95 nm in diameter. Its envelope consists of a 5-nm thick membrane covered by 8- to 10-nm long surface projections. These are composed of two polypeptides species of about the same size. Both of them can be removed by digestion with the proteolytic enzyme thermolysin except for a small fragment. The enzyme-treated particles are smooth surfaced and extremely deformable. The glycopolypeptides are clustered to form hollow cylindrical morphological units, 10 to 12 nm in diameter, with a 5-nm central cavity. Both negative staining and freeze-etching suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice. The membrane to which the surface subunits are attached is probably a lipid bilayer as evidenced by its double-track appearance in thin sections and the tendency of the freeze fracturing to occur within it. The strand-like nucleoprotein appears from thin-sectioning results to be to a large part located in a zone underneath the membrane.


Transrectal ultrasonotomography of the prostate. New equipment for transrectal ultrasonomography has been developed. Wth this equipment an excellent cross-section picture of various intrapelvic organs is easily obtained. Diagnostic application of the technique is particularly significant for abnormalities of the prostate because the shape of the prostate cannot be revealed by x-ray. Diagnostic criteria for prostatic disease are described, with the demonstration of typical cases. In a test by well trained physicians the diagnostic accuracy of the new technique was more than 80 per cent. Ultrasonotomography provided for precise measurement of prostatic size. Transrectal ultrasonotomography is a promising new diagnostic technique that yeilds abundant information not available with former methods of examination. The procedure is likely to become one of th most important diagnostic tools in the field of urology.


Radiologic detection and prostatic carcinoma by double contrast retrograde urethrocystography. A method of radiologic visualization of the male urethra especially for detecting prostatic carcinoma is reported. This method is called double contrast retrograde urethrocystography and is similar to that of Flocks in combining air cystography and retrograde urethrography but it differs in the manner of making the exposure. In our method the exposure is made with the patient in an exaggerated Trendelenburgs position to allow the contrast medium injected into the bladder to accumulate at the dome. Thus, in the urethrocystogram obtained by this method, the bladder neck and the prostatic urethra immediately adjacent to it are always clearly visualized unlike that obtained by conventional retrograde urethrography, evacuation cystourethrography and sometimes even the method of Flocks, in which these regions are prone to be covered and masked by the contrast medium injected and accumulated in the bladder. Therefore, with our method the radiological detection and differentiation of the malignant lesion involving these  regions are more accurate and definite. From our clinical experiences the mode of compression and displacement caused by benign prostatic hypertrophy is continuous and regular, while that caused by prostatic carcinoma is interrupted, irregular and with moth-eaten and granular defects. Four cases are reported.


Symptomatic care of patients with amyotrophic lateral sclerosis. Until a definitive treatment is found for amyotrophic lateral sclerosis, there will continue to be a need to provide symptomatic care. Experience with caring for a large number of patients has proved that symptomatic therapy can be based on rational principles. Treatment should be limited to procedures that are simple and low-cost, with low risk of serious morbidity. When all remedies fail, the physician can still provide a comforting presence and foster a dignified death.