Inhibition of leukocyte migration by human breast-cancer-associated antigens. The existence of CMI to tumor-associated antigens present in 3 M KCl extracts of breast carcinomas was demonstrated in a group of breast cancer patients by the leukocyte migration inhibition (LMI) assay. When crude KCl extracts were tested, 3 of 5 breast cancer patients gave a positive response to autologous tumor extracts. Eleven of 20 gave a positive response to allogeneic extracts as compared to 3 of 22 controls (including 6 patients with benign breast disease, 7 with non-mammary cancers and 9 normal controls). Extracts of fibrocystic disease tissue gave positive LMI tests in 2 of 5 breast cancer patients, suggesting the presence of antigenic cross-reactivity between benign and malignant breast disease. An extract of a medullary carcinoma of breast was fractionated on Sephadex G-200 and the effluent pooled into three fractions. The high molecular weight fraction produced LMI in 11 of 22 breast cancer patients and in 1 of 19 controls, including patients with benign breast disease, other cancers and normal individuals. The low molecular weight fraction produced LMI in both the benign (4 of 6) and the malignant breast disease (6 of 20) patients, but not in the controls (0 of 12). A simple fractionation technique has thus separated "cancer-specific" from "organ-specific" activity. Sephadex G-200 fractions were active at a much lower protein concentration than the crude 3 M KCl extracts.


Demonstration of a phase (stage)-specific embryonic brain antigen in human meningioma. The water-soluble antigens of human embryonic brain were studied using a rabbit antiserum to human 8- to 10-week embryonic brain (non-absorbed and absorbed with extracts of human embryonic and adult organs). A phase (stage)-specific brain antigen(s) characteristic for the human 8- to 10-week-old embryo was detected by double immunodiffusion and immunoelectrophoresis in agarose gel. The testing of this anti-embryonic brain serum against saline extracts of human brain tumours (meningiomas, glioblastomas, astrocytomas, neurinomas and brain metastasis of carcinoma) revealed the presence of an "embryonic" antigen in the human meningioma only. Immunochemically this meningioma-associated antigen is identical to a phase (stage)-specific antigen of the 8- to 10-week embryo.


A plasma membrane antigen highly associated with oat-cell carcinoma of the lung and undetectable in normal adult tissue. The plasma membrane antigens of an oat-cell carcinoma of the lung were studied to determine if any antigens absent from normal adult tissue could be identified. Rabbit and monkey antisera were prepared to a highly purified plasma membrane fraction of an oat-cell carcinoma of the lung. The specificities of the antisera were studied by the indirect immunofluorescence method on frozen section substrate. The rabbit antiserum, after absorption with normal lung, liver, colon and peripheral nerve homogenates and extracts, failed to react with any detectable normal adult tissue. The absorbed anti-serum did react with 7 of 7 oat-cell carcinomas of the lung, but failed to react with any of 7 adeno-carcinomas of the lung, 6 epidermoid carcinomas of the lung, 7 colon carcinomas, 8 breast carcinomas, 4 kidney carcinomas, and 1 pancreatic carcinoma. The unabsorbed monkey antiserum failed to react with any detectable normal adult tissue, and had a tumor reactivity pattern nearly identical to that of the absorbed rabbit antiserum. Thus similar results were obtained with antisera from two different species. It is concluded that oat-cell carcinomas of the lung express a plasma membrane antigen(s) undetectable in normal adult tissue and highly associated with this tumor type.


Inducibility of the Epstein-Barr virus (EBV) cycle and surface marker properties of EBV-negative lymphoma lines and their in vitro EBV-converted sublines. Two EBV-negative lymphoma lines of human B-cell origin, BJAB and Ramos, were compared with altogether six of their in vitro EBV-converted, EBNA- and EBV-DNA-carrying sublines (four of Ramos and two of BJAB derivation). All converted lines closely resembled the parental line with regard to karyotype and HL-A and B antigen typing. Induction of EBV antigens (EA and VCA) by P3HR-1 virus superinfection was either similar in the converted and the negative lines, or somewhat increased in certain converted lines. These findings argue against a simple, virally determined repressor model and emphasize the role or cellular controls in restricting the EBV cycle in virus-carrying B-lymphocyte lines of human origin. IUdR inducibility varied in the different converted lines. There was a possible relationship between average number of EBV-genome equivalents per cell and inducibility. Converted sublines did not differ from the original negative lines with regard to surface immunoglobulin and Fc receptors. There was a dramatic increase in complement-consuming ability, however, following EBV conversion. Among the EBV-positive lines, there was a linear relationship between complement-consuming and EBV-receptor activity, the latter measured by a quantitative absorption test.


Mast cells and tumor angiogenesis.

Mast cells and tumor angiogenesis. Tumor angiogenesis factor (TAF) elicits a strong vasoproliferative response when implanted upon the chorioallantoic membrane (CAM) of the chick embryo. This response is first observed stereomicroscopically 2-3 days after implantation. A 40-fold increase in mast cell density is observed within the vicinity of this implant by 24 h. Mast cells that have been isolated from retired breeder Sprague-Dawley rats fail to evoke a vascular reaction when implanted on the CAM. An intermediate role for the mast cell in tumor angiogenesis is suggested.


Membrane-associated antigen from the SV40-induced hamster fibrosarcoma, para-7. I. Role in immune complex formation and effector cell blockade. PARA-7 membrane-associated antigen was prepared by treatment of the tumor cells with pH 9.4 glycine buffer, or by concentration of spent cell culture medium. When admixed with sensitized effector cells, both preparations could specifically block cellular cytotoxicity for PARA-7 target cells. Pretreatment of target cells with antigen did not result in blocking. Incubation of antigen extract with simian virus 40 (SV40) anti-serum caused neutralization of antibody-dependent cellular cytotoxicity (ADCC) with concomitant formation of a factor which blocked at the target cell level but not at the effector cell level. Serum from tumor-bearing hamsters exhibited blocking characteristics comparable to those of the antigen-SV40 teristics comparable to those of the antigen-SV40 antiserum preparation. Washing experiments indicated that PARA-7 antigen was more efficient than PARA-7 antigen-antibody complexes in blocking cell-mediated immunity in vitro. Material extracted from untransformed hamster embryo fibroblasts either by itself or when admixed with SV40 antiserum exhibited no significant blocking activity. These observations support the concept that loss of serum ADCC during progressive tumor growth is due to immune complex formation.


A new technique for the vital staining of the corneal endothelium. A new technique for the staining of the corneal endothelium combines the actions of the vital stain trypan blue and the intercellular stain alizarin red S. The technique is an improvement over former staining methods, because it defines both viable and nonviable cells and thereby permits an investigator to accurately quantify endothelial cell damage.


Niemann-Pick disease-like inclusions caused by a hypocholesteremic agent. AY9944, an inhibitor of cholesterol biosynthesis, was injected into albino rats and the ocular tissue was studied by light and electron microscopy. Abundant lamellar inclusion bodies accumulated in various cells of the eye, especially in the ganglion cells of the retina and glial cells of the optic nerve. Prolonged administration of this drug resulted in degeneration of retinal ganglion cells and oligodendroglial cells of the optic nerve. Micro-organelles of the inclusion body-laden cells were otherwise normal in their appearance. The electron microscopic appearance of these inclusion bodies and their distribution in the ocular tissues closely resembled those of Niemann-Pick diseasees.


Prospects for microscopical cytochemistry. This review is devoted mainly to an evaluation of the status of microscopical cytochemistry seen as a discipline aiming at both the localization and the quantification of molecular processes in cells. Its relationships to ultramicrochemistry, as well as, in a broader sense, to biochemistry and cell biology, are discussed from both the historical and the methodological points of view. Recent developments in quantitative cytophysical techniques, such as automated cytophotometry using microscopes fitted with flying spot systems, TV cameras, or scanning stages, and the development of rapid flow cytometers are discussed. Analytical electron microscopy is touched upon too. The main part of the review is devoted to recent trends that strengthen the analytical basis of cytochemical staining methods. The special character of staining procedures as a kind of matrix chemistry is discussed and the potentialities of the use of matrix-incorporated compounds for the fundamental study and calibration of microscopical staining procedures are elaborated. Parallel developments in the theory and practice of matrix chemistry in biochemistry are stressed. Growing interrelations between microscopical cytochemistry and related fields of investigation, such as the controlled fragmentation of cells, and methods like ultramicroanalysis of individual cells are indicated.


Cytochemistry of colloidal iron binding to the surface of Hela cells and human erythrocytes. It seems from the literature that colloidal iron (C.I.) binding sites on cell surfaces cannot be completely removed by treatment with Vibrio Colerae alpha-neuraminidase. We wondered if C.I. particles bind to negative groups other than the carboxyl groups of sialic acids. Using HeLa cells from suspension cultures and fresh human erythrocytes, we examined, with the transmission electronmicroscope, the influence of the following enzymatic and histochemical treatments on C.I. staining: alpha-neuraminidase; hyaluronidase; ribonuclease; alpha-amylase; mild methylation (MM); MM + saponification (Sap.); MM + Sap +MM; MM + Sap + alpha-neuraminidase; active methylation (AM); AM + Sap; AM + Sap + AM; AM + Sap + alpha-neuraminiadase; CH3OH (80%); Sap. It seemed from these experiments that the carboxyl groups of alpha-neuraminidase sensitive sialic acids constitute the majority of binding sites for C.I. to these particular cells. The most interesting candidates for the residual binding of C.I. are carboxyl groups of alpha-neuraminidase resistant molecules, sulfon, sulfin, and sulfate groups.


Impairment of the M-protein and unmasking of a superficial type-specific antigen by proteolytic treatment of influenza A virions with preservation of host-specific antigenicity. Influenza PR8 particles resulting from strong treatment with caseinase C are spikeless, devoid of neuraminidase and hemagglutinin 1 and 2 glycopeptides, and contain a Schiff-negative polypeptide of about 13,000 molecular weight which exists as traces in intact virions. Their M-protein polypeptide content is reduced to 50% of its original value, but there is no evidence of particle disruption nor of lipid release. They fix complement in the presence of both anti-M-protein antiserum and antiserum raised against a host polysaccharide. During exposure to caseinase C, an antigen is unmasked. It is type-specific and its identity with the M-protein is discussed.


Light microscopic distinction between elastin, pseudo-elastica (type III collagen?) AND INTERSTITIAL COLLAGEN. Distinction between elastin and collagen in arteriosclerotic lesions is difficult because the so-called elastica stains are bound also by collagen fibers which resemble collagen of premature infants. Investigations of effects of organic solvents on dye binding led to the development of methods for selective demonstration of pseudo-elastica, and for simultaneous visualization of elastin and pseudo-elastica in contrasting colors. Paraffin sections of human autopsy material were stained with solutions of resorcin-fuchsin, orcein or aldehyde fuchsin in absolute ethanol. In other series, sections pretreated with this resorcin-fuchsin solution were counter-stained with tannic acid-phosphomolybdic acid (TP)-dye technics. Solutions of these "elastica stains" in absolute ethanol colored only pseudo-elastica; elastin, e.g. elastic membranes of aorta, remained unstained. In sections counterstained with TP-dye technics elastin was colored red; pseudo-elastica retained the purplish blue coloration imparted by resorcin-fuchsin. Other collagens were stained yellow. A review of the literature showed that until the 1920's elastin was classified as a gelatinoid of the collagen group. Elastic fibers were identified by mechanical properties, not a particular chemical composition. Hence, the elastic fibers of classical histology cannot be equated with the elastin of modern chemistry. Correlation of histochemical observations with chemical data indicates that the collagenous pseudo-elastica corresponds to [alpha1(III)]3 collagen.


Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.


Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein, DNA and cytoplasmic protein in rat liver. From rats fed ad libitum and kept under a 12 + 12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined avery four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphthol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max = 0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear non-histone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.


Uneven distribution of alkaline phosphatase in individual layers of rabbit and ox cornea. Histochemical and biochemical study. In the rabbit and bovine cornea the activity of alkaline phosphatase using histochemical as well as biochemical methods was investigated. Biochemically the enzyme activity was studied in separated corneal layers. In the histochemical investigation the best results were obtained in cryostat sections using the azocoupling method with naphthol AS-MX phosphate and Variamine Blue RT Salt. The enzyme activity was found not only in the epithelium and endothelium (as was described previously) but even in keratocytes. The mutual relation of activities in the epithelium and in keratocytes differed in both species. The overall activity found by histochemical methods is in good agreement with the biochemical determination of alkaline phosphatase (p-nitrophenyl phosphate as the substrate). Besides the histochemical approach shows an uneven distribution of alkaline phosphatase activity in individual cells which cannot be assessed by the biochemical determination.


Histochemical evidence for sulfomucins at the surface of Hela cells. The nature of the negatively charged groups present at the surface of HeLa cells was further investigated. Therefore we applied a series of light microscopic staining techniques, widely used for the demonstration of epithelial mucosubstances on tissue sections, to HeLa cells from suspension cultures. Our histochemical findings confirmed the presence of carboxylated substances at the surface of these cells. Furthermore we obtained conclusive evidence for the presence of sulfated molecules. Both substances seem to be closely related to epithelial sialomucins and sulfomucins.


On using cis-Pt (II)-uracil as a stain for nucleic acids in brain slices and subcellular fractions. Chick brain cortical slices and crude mitochondrial fractions were fixed with glutaraldehyde, stained only with cis-Pt (II)-uracil and processed for electron microscopy. The optimal time of staining was determined to be 10 min. Results show that this platinum-pyrimidine complex is a relatively specific stain for the nucleic acids of brain slices. However, staining of crude mitochondrial fractions apparently resulted in some protein staining and other artifacts. The method should be helpful identifying ribosomal contamination of subcellular preparations and if its specificity can be increased it may prove a useful addition to staining methods of the electron microscopist.


Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds. Several compounds of osmiumVIII, including potassium osmiamate and coordination complexes of OsO4 with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO4 in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation. The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO4, has a negligible vapor pressure of OsO4. It has the molecular formula C6H12N4.2OsO4 and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO4) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO4, and the odor of OsO4 becomes apparent. Such solutions of osmeth are (approximately 0.25%) considerably less concentrated with respect to OsO4 than solutions (1-2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO4 appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO4 solutions of equivalent concentrations. Our findings indicate that OsO4 solutions of less than 1% may be satisfactorily utilized in many cytochemical studies. Osmeth is safer and more convenient to handle than OsO4 because small amounts may be solubilized as needed. It should be considered as a substitute for OsO4 in ultrastructural cytochemistry. These results suggest that the effectiveness of OsO4 as a fixative may, in part, be related to its nonpolarity. The infrared spectra indicate that the OsO4 molecule is tetrahedral, perfectly symmetrical and, therefore, as a whole nonpolar. As a consequence, it could be expected to readily penetrate charged surfaces of tissues, cells, and organelles. The spectral studies show that osmeth is much less symmetrical and, to that extent, polar; thus, it penetrates biomembranes less readily.


Feulgen-Naphthol Yellow S cytophotometry of liver cells. The effect of formaldehyde induced shrinkage on nuclear Naphthol Yellow S binding. 1. In isolated liver cells, fixed in 4 per cent formaldehyde (NFS) for Feulgen-Naphthol Yellow S (F-NYS) staining of DNA and protein, nuclear shrinkage increases the nuclear concentration of solids to 46 per cent (w/v) before the start of the NYS staining. 2. When a fixative mixture of methanol:acetic acid:formalin (85:5:10 by volume; MAF) is used, the concentration of nuclear solids during NYS staining remain at a physiological level of 19 per cent. 3. By exposing liver cells to NFS for 10 to 120 seconds before fixation in MAF, increasing nuclear shrinkage can be induced with increasing pretreatment in NFS. Nuclear NYS binding decreases in parallel with the decreasing nuclear volume in cells thus treated. As the shrinkage induced reduction in NYS binding may vary with the net charge of nuclear non-histone proteins, MAF fixation must be preferred for quantitative determinations of nuclear non-histone protein in F-NYS stained, isolated cells. 4. Fixation in MAF offers the same advantages as NFS fixation as regards the small loss of proteins during the Feulgen staining procedure and the excellent reproducibility of the F-NYS staining. Storage of MAF fixed cells in the fixative for a few days does not alter their F-NYS staining properties. 5. In MAF fixed, F-NYS stained cells there is no NYS binding to histone basic amino acid residues.


Comparison of dieldrin, lindane, and DDT extractions from serum, and gas-liquid chromatography using glass capillary columns. Rats were given an oral dose of 14C-labeled chlorinated pesticides to obtain serum containing p,p'-DDT, dieldrin, or lindane. Simple hexane and formic acid-hexane extraction methods, involving pretreatment of the serum with formic acid, were compared by radiometric and by paper chromatographic and gas chromatographic analysis. In vivo binding of chlorinated pesticides to constituents of the serum does not necessarily prohibit their isolation by simple hexane extractions, provided that the extraction is very vigorous and at least 5 min long. Stable emulsions were broken by cooling in liquid nitrogen or Dry Ice-acetone. The hexane extraction method described yields quantitative recovery of the pesticides studied, whereas the formic acid-hexane method is quantitative for p,p'-DDT, 93% for dieldrin, and 89% for lindane. Gas chromatographic comparison of both methods, using human serum, shows that the hexane method extracts 16% more beta-BHC, 7% more dieldrin and HCB, and 4% more p,p'-DDE from serum than does the formic acid-hexane method. The difference for p,p'-DDT is not significant. Gas chromatography with glass capillary columns and an all-glass solids injection system yielded detection limits as low as 15 fg. Data show that the use of an internal standard considerably improves the precision of quantitation.


Bacterial cis-dihydrodiol dehydrogenases: comparison of physicochemical and immunological protperties. Cells of Pseudomonas putida NP, Pseudomonas species (NCIB 9816), and a Nocardia species, after growth on naphthalene as sole source of carbon and energy, contain a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that oxidizes cis-dihydrodiols of mono- and polycyclic aromatic compounds. Similarly, cells of a strain of P. putida biotype A, when grown either on toluene or benzene vapors, were found to contain a dehydrogenase that oxidized dihydrodiols of aromatic hydrocarbons with cis stereochemistry and required NAD+ as an electron acceptor. In all these cases, no enzymatic activity was detected when trans-naphthalene dihydrodiol was used as a substrate. Purified cis-naphthalene dihydrodiol dehydrogenase was injected into rabbits to obtain antibodies. Physiocochemical and immunological properties of cis-dihydrodiol:NAD+ oxidoreductases from four different organisms were examined. Kinetic analysis showed that, in all the cases, enzymes exhibited higher affinity for cis-dihydrodiols than for NAD+ and had pH optima between 8.8 and 9.0. except in the case of the enzyme from Nocarida sp., which showed maximum activity at pH 8.4. Molecular-weight determination of the dehydrogenases from the four different organisms by gel filtration on a Sephadex G-200 column gave values ranging from 92,000 for the enzyme from Nocardia sp. to 160,000 for that from P. putida biotype A. All the dehydrogenases, except the one from Nocardia sp., exhibited immunological cross-reaction with the antibodies prepared against the enzyme purified from P. putida NP.


House staff training in cardiopulmonary resuscitation. An audiovisual instructional program has been developed to teach cardiopulmonary resuscitation (CPR) to new interns and house staff. The entire program conformed to standards established in this subject and was devised so that it could be used for either large or small group instruction. Special projection equipment and a computerized student response system made it possible to use the audiovisual material with large groups and yet maintain most advantages of individual instruction. Following the instructional sessions small group workshops gave students an opportunity to demonstrate CPR and to receive guidance from trained personnel. Small group instruction was also enhanced by use of the audiovisual materials. After participation in the program, all interns were judged competent in performing required CPR tasks; 98% rated the program "good" or "excellent".


Distribution and use problems of the institutional developer arising from copyrights. Institutional developers of mediated instruction for the health sciences in higher education must take whatever steps are reasonable and necessary to obtain copyright protection for their original works and avoid liability for infringement. Twelve questiona frequently asked by such developers in these two areas are discussed. Special requirements are set forth pertaining to material copyrightable by the developer, copyrighted by others, and in the public domain (not copyrightable by anyone). Unique requirements for writings, sound recordings and visual products are summarized. Relevant aspects of fair use, pre-publication copyright, post-publication copyright, and marketing and distribution through the private sector are set forth together with the elements of proof in infringement actions.


Regulation of folate reductase synthesis in sensitive and methotrexate-resistant sarcoma 180 cells. In vitro translation and characterization of folate reductase mRNA. A highly specific assay for folate reductase mRNA activity from Sarcoma 180 cells was developed using the rabbit reticulocyte lysate protein synthesizing system. Quantitation of in vitro folate reductase synthesis was accomplished by direct immunoprecipitation from lysate reactions. The in vitro labeled folate reductase was synthesized in a linear response to a wide range of RNA concentrations, migrated as a single prominent radioactive species upon polyacrylamide gel electrophoresis, and was indistinguishable from authentic 14C-labeled folate reductase on the basis of molecular weight and immunotitration with anti-folate reductase gamma-globulin. The assay was used to quantitate folate reductase mRNA activity in various cell lines and under several conditions known to affect folate reductase synthesis. These included (a) sensitive and methotrexate-resistant Sarcoma 180 cells, (b) two lines of resistant cells having different relative rates of folate reductase synthesis, (c) growth of methotrexate-resistant cells in the absence of methotrexate, and (d) growth phase. The results indicate that the relative rate of folate reductase synthesis in each case can be explained solely by the level of translatable folate reductase mRNA. The use of poly(U)-Sepharose and sucrose gradient fractionation procedures indicated that folate reductase mRNA contains poly(A) and has a sedimentation coefficient of approximately 14 S. These two fractionation steps were combined to achieve an approximately 90-fold purification of folate reductase mRNA over total cytoplasmic RNA.


Reconstitution of Biological Molecular generators of electric current. Bacteriorhodopsin. 1. Photoinduced generation of electric current by bacteriorhodopsin, incorporated into the planar phospholipid membrane, has been directly measured with conventional electrometer techniques. 2. Two methods for bacteriorhodopsin incorporation have been developed: (a) formation of planar membrane from a mixture of decane solution of phospholipids and of the fraction of violet fragments of the Halobacterium halobium membrane (bacteriorhodopsin sheets), and (b) adhesion of bacteriorhodopsin-containing reconstituted spherical membranes (proteoliposomes) to the planar membrane in the presence of Ca2+ or some other cations. In both cases, illumination was found to induce electric current generation directed across the planar membrane, an effect which was measured by macroelectrodes immersed into electrolyte solutions on both sides of the membrane. 3. The maximal values of the transmembrane electric potential were of about 150 mV at a current of about 10(-11) A. The electromotive force measured by means of counterbalancing the photoeffect by an external battery, was found to reach the value of 300 mV. 4. The action spectrum of the photoeffect coincides with the bacteriorhodopsin absorption spectrum (maximum about 570 nm). 5. Both components of the electrochemical potential of H+ ions (electric potential and delta pH) across the planar membrane affect the bacteriorhodopsin photoelectric response in a fashion which could be expected if bacteriorhodopsin were a light-dependent electrogenic proton pump. 6. La3+ ions were shown to inhibit operation of those bacteriorhodopsin which pump out H+ ions from the La3+-containing compartment. 7. The photoeffect, mediated by proteoliposomes associated with thick planar membrane, is decreased by gramicidin A at concentrations which do not influence the planar membrane resistance in the light. On the contrary, a protonophorous uncoupler, trichlorocarbonylcyanidephenylhydrazone, decreases the photoeffect only if it is added at a concentration lowering the light resistance. The dark resistance is shown to be higher than the light one, and decreases to the light level by gramicidin. 8. A simple equivalent electric scheme consistent with the above results has been proposed.


Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin-G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells.


Redistribution of surface macromolecules in dissociated epithelial cells. A number of ultrastructural and cytochemical techniques were used to study intact epithelial cells lining the frog urinary bladder: high resolution autoradiography after administration of [3H]glucosamine or [3H]fucose; 125I iodination of external protein; concanavalin A-peroxidase, periodic acid-chromic acid silver methenamine; and colloidal thorium. Results indicate that the material (probably glycoprotein) coating the apical surface differs from that which lines the lateral and basal surfaces. After dissociation and isolation of the epithelial cells, the material previously confined to the apical surface invaded progressively the opened "tight junctions" (about 5 min), then the lateral membranes (about 40 min), and finally the basal membrane (about 80 min): at that time, the whole cell surface was entirely enveloped by the apical material. Since, on the one hand, the reacting material was confined to the apical surface when the tight junctions were closed (in intact epithelial cells) and since, on the other hand, the apical material was sliding down the laterobasal membranes when the tight junctions were opened (in dissociated cells), it may be concluded that tight junctions contribute to maintain the cell surface specialization in epithelia.


Effect of megestrol acetate (Megace) on steroid metabolism and steroid-protein binding in the human prostate. Megestrol acetate (Megace), an antiandrogen, was administered in a dosage of 80 mg daily to 6 patients with benign prostatic hypertrophy (BPH) for 4 to 25 days prior to transurethral resection of the prostate (TURP). Surgical tissue from drug-treated patients was compared to untreated controls in regard to: 1) the enzymatic reduction of testosterone (T) and dihydrotestosterone (DHT); 2) DHT binding to a cytosol receptor protein; 3) tissue levels of endogenous dihydrotestosterone and androstanediols (diols). When minced prostate was incubated with 3H-T and 14C-androstenedione for 1 h at 37 C, prostate 5alpha-reductase activity, measured as reduced products formed from substrate, decreased to 31% and 39%, respectively, of the control values. Prostate 3-oxido-reductase enzyme activity, measured as diols formed from 3H-DHT, was decreased to neglible values in Megace-treated patients compared to an 8.7% conversion to diols in controls. No 3H-DHT binding to a cytosol receptor protein could be demonstrated in 4 out of 5 prostates from Megace-treated patients, whereas the presence of such a receptor was noted in 14 out of 17 untreated controls. Endogenous DHT levels in Megace-treated patients averaged 1.1 ng/g (SE = 0.26), significantly less than the average of 3.9 ng/g (SE = 0.49) found in controls (P less than 0.001). No significant difference was noted in endogenous diols. In addition to these effects on tissue, Megace significantly decreased plasma levels of T, LH, and FSH at the end of the 4- to 25-day period; plasma prolactin levels did not change. Continued studies of Megace for the possible treatment of benign prostatic hypertrophy may be warranted since the drug appears to block several important biochemical steps which mediate the effects of androgen on the human prostate.


Pathogenic mechanisms in pulmonary fibrosis: collagen-induced migration inhibition factor production and cytotoxicity mediated by lymphocytes. The universal features of the histopathology of fibrotic lung disease are derangement of parenchymal collagen and infiltration of the parenchyma with chronic inflammatory cells. To determine if this cellular reaction might be associated with autoimmunity to a consitituent of the alveolar interstitium, peripheral blood lymphocytes were exposed to human type I collagen in vitro and evaluated for the production of migration inhibition factor and cytotoxicity. Data from 18 patients with idiopathic pulmonary fibrosis, 8 patients with pulmonary fibrosis other than idiopathic pulmonary fibrosis, 12 patients with nonfibrotic lung disease, and 9 normals demonstrated that circulating lymphocytes from more than 94% of patients with fibrotic lung disease take part in processes where the recognition of collagen results in migration inhibition factor production and lysis of collagen-coated sheep red blood cells. These collagen-induced cell-mediated phenomena are obviated with human T-lymphocyte antiserum. Collagen-induced migration inhibition factor production and cytotoxicity were found in less than 20% of patients with nonfibrotic disease and were not found in normals. Qualitatively, there was no organ (lung, skin) or species (human, rabbit) collagen specificity in these assays, but human lung alpha 2 chains were recognized more often than alpha 1(I) chains. Circulating lymphocytes from patients with fibrotic disease are present in a normal T to B ratio. These lymphocytes did not incorporate [3H]thymidine when exposed to collagen but did when exposed to T-cell mitogens. These in vitro observations suggest that circulating T-lymphocytes and lung collagen may be intimately associated in the pathogenesis of human fibrotic lung disease.


Assessment of group B streptococcal opsonins in human and rabbit serum by neutrophil chemiluminescence. The factors important in host defense against group B streptococci are not well understood. The role of antibody and complement in the prevention of serious infection by these organisms is not known because, to date, a reliable measure of functional opsonic activity has not been developed. Recently, it has been shown that neutrophils produce a chemiluminescence after ingestion of particulate matter, and that this event can be detected and quantitated in a liquid scintillation system. We have adapted the chemiluminescence procedure to examine rabbit hyperimmune and human serum for the presence of group B streptococcal opsonins. Group B streptococci of types Ia, II, and III that were opsonized in homologous but not heterologous type serum produced a peak in chemiluminescence when added to normal human neutrophils. Such activity was correlated, in each instance, with ingestion of bacteria by neutrophils and deposition of immunoglobulin and C3 on the bacterial surface as detected by indirect immunofluorescence. With this assay, we have examined sera from colonized and diseased patients for the presence of opsonins to types Ia, II, and III group B streptococci. Maternal sera often contained type-specific opsonins which resided in the IgG fraction and which crossed the placenta to appear in paired cord specimens. 63% of patients colonized with group B streptococci had serum opsonins to their colonizing type of organism. In contrast, none of the 15 patients with sepsis or meningitis had opsonins directed against their infecting strain. These data suggest that the lack of type-specific opsonins to group B streptococci may be one of the important factors in determining host susceptibility to systemic infection with strains of this group.


Use of food colourants as plaque disclosing agents. The effectiveness of plaque disclosure by several liquid food colourants and disclosing agents was compared in a group of eight subjects. The subjects refrained from all forms of oral hygiene for a 48-hour period prior to rinsing with 5 ml of each dye in turn at weekly intervals. Kodachrome film records were taken and projected for the assessment at weekly intervals. Kodachrome film records were taken and projected for the assessment of plaque staining efficacy by a panel of 38 assessors. Acceptability with respect to taste, extent and duration of mucosal staining and any side effects was also evaluated. The food colourants were as effective as the disclosing agents. Ability to stain plaque appears to be related not only to the constituents of each dye, but also to their concentrations and relative proportions. Other desirable properties of an ideal disclosing agent tended to be fulfilled to a level equivalent to, or better than, that reached by the proprietery disclosing agents. Difficulty in obtaining proprietary disclosing agents should not act as a handicap to achieving better levels of oral cleanliness as inexpensive food colourants of equal effectiveness to the best proprietary agent are readily available.


Facilitating sentence formulation: a case study. A training program was designed to facilitate sentence formulation in a Broca's aphasic patient. Two training methods were compared and generalization to untrained stimuli was measured. Training, using either method, resulted in increased production of correct linguistic constituents and appropriate lexical items. Gains tended to occur on the trained items and generalization to untrained items was limited.


Observations on the projections and intrinsic organization of the pigeon optic tectum: an autoradiographic study based on anterograde and retrograde, axonal and dendritic flow. Three aspects of the labelling pattern seen after the injection of 13 different radioactive amino acids into the pigeon optic tectum have been described: The efferent projections of the optic tectum; the specific labelling of two pathways; and the dendritic organisation of tectal layer III neurons based on the retrograde and anterograde movement of label within these dendrites. Discrete injections of tritiated amino acid that involved all or only the superficial tectal layers suggested that layer III gave rise to the massive non-topographically organised and bilateral projections (fibers crossing within the decussato supraoptica ventrlis) upon the nuclei rotundus, subpraetectalis and interstitio-praetecto-subpraetectalis and to the ipsilaterally directed pathways terminating within the nuclei praetectalis, triangularis, subrotundus, dorsolateralis anterior thalami, posteroventralis and ventrolateralis thalami. Layer III neurons may also be the source of efferents to the posterior dorsolateral thalamus (the layer III pathway), the pontine grey and, bilaterally to the reticular formation and of the layer IV or tectal commisural pathway terminating within the contralateral tectal cortex. In contrast projections originating from layer II were generally topographically organised and terminated either within certain of the isthmic nuclei (n. isthmi pars parvocellularis, n. isthmo-opticus and n. semilunaris) or ran within layer I (layer I pathways) to end in the pretectum (griseum tectale) and ventral thalamus (n. ventrolateralis thalami, n. geniculatus, pars ventralis). A small projection from layer II upon the ipsilateral nucleus rotundus may also be present. Triated serine and tyrosine were found to be particularly effective in labeling perikarya as well as axons and terminals. The layer I pathway could be selectively labelled after tectal injections of 3H-GABA while the cell bodies of Ipc neurons were labelled in a retrograde fashion after tectal injections of 3H-glycine, serine or alanine. Intrinsic tectal labelling was found by correlation with Golgi material to reflect both anterograde and retrograde transport of label within dendrites of layer III cells. Anterograde movement of label indicated that the terminal portions of layer III cell dendrites ended in an orderly radial arrangement within sublayers IIb and IId, while the retrograde movement of label resulted in the labelin of layer III perikarya outside the injection field.


Selective uptake and transport of label within three identified neuronal systems after injection of 3H-GABA into the pigeon optic tectum: an autoradiographic and Golgi study. After injection of tritiated gamma-aminobutyric acid (GABA) into the pigeon optic tectum and thalamus it was possible to correlate certain aspects of the autoradiographic labeling pattern with observations made from Golgi material. Three neuronal, GABA specific systems were identified both from the uptake of the amino acid and from the subsequent and bidirectional in tracellular transport of labe. The first system derives from cell bodies within sublayer IIi the axons of which could be selectively labelled throughout their course within layer I and to the areas of termination within the pretectum and ventral thalamus. The radially ascending dendrites and axon collaterals of these neurons arbourised within sublayer IIf, and could be labelled in a retrograde fashion after tectal or thalamic injections. The second system was represented by small perikarya within sublayer IIc with locally and superficially directed dendrites and with a radially and deep directed axon from which an extensive axon collateral system arose. It was found possible to label these perikarya either directly or indirectly after tangential tectal injections which preferentially labelled the axons and terminals of these neurons within the deeper regions of the tectal cortex and resulted in the retrograde axonal movement of label to theoverlying cell bodies. A third system was found within sublayer IId, was horizontally organized and from a correlation with degeneration, other autoradiographic and Golgi preparations thought to be mainly dendritic in nature. The biochemical and anatomical implications of specific GABA uptake and subsequent transport of label are discussed and a model of the tectal cortex, based on the three proposed inhibitory systems and their relation to a number of tectal afferent inputs, considered.


Electron-microscopic identification of Marchi-positive bodies and argyrophilic granules in the spinal cord white matter of the guinea pig. Sections from spinal cord white matter of normal and rhizotomized guinea pigs fixed by glutaraldehyde perfusion were stained with Marchi fluid or according to a suppressive silver technique. With the aid of the section-embedding method thin sections, cut from light-microscopically identified areas containing Marchi-positive bodies or argyrophilic granules, were examined in the electron microscope. The results show that the Marchi-positive bodies and argyrophilic granules, which are present in normal white matter, represent different histochemical expressions of the same entity--the myelinoid body. In view of the similarities between myelinoid bodies and myelin fragments formed during Wallerian degeneration it is suggested that this type of so called artifact staining of normal white matter inherent to both methods should instead be considered as an expression of a specificity of the two methods for degenerating nervous elements.


Retinal projections in the ringtailed possum Pseudocheirus peregrinus. The retinal projections in the ringtailed possum, Pseudocheirus peregrinus were determined using Fink-Heimer material and autoradiography. At least seven regions in the brain receive retinal projections. These are (1) the suprachiasmatic nucleus of the hypothalamus (2) the dorsal lateral geniculate nucleus (3) the ventral lateral geniculate nucleus (4) the lateral posterior nucleus (5) the pretectum (6) the superior colliculus, and (7) the accessory optic system. The accessory optic system and lateral posterior nucleus receive a contralateral retinal projection only and the other five regions receive a bilateral retinal projection. The dorsal lateral geniculate nucleus consists of two parts: an outer alpha division of closely packed cells and an inner beta division containing loosely scattered cells. There are no cell layers apparent within the alpha division in Nissl sections. The autoradiographs and Fink-Heimer material reveal four concealed laminae within the alpha division. Lamina 1, which is adjacent to the optic tract and lamina 3 receive a predominantly contralateral input. Laminae 2 and 4 receive a predominantly ipsilateral input. The beta segment contains a fifth lamina which receives contralateral retinal input.


Structural and functional changes in an identified cricket neuron after separation from the soma. I. Structural changes. The morphological effects of separation from the soma were examined in isolated arborization and isolated axon segments of an identified motor neuron in the Polynesian field cricket, Teleogryllus oceanicus. The identified neuron, the contralateral dorsal longitudinal motor neuron of the metathoracic ganglion (CDLM), possesses an arborization most of which lies contralateral to its soma within the metathoracic ganglion. Midline surgical lesions in the metathoracic ganglion separated CDLM into a distal segment composed of the axon and most of the arborization, and a proximal segment comprised of the remaining arborization, neuritie, and soma. Isolated axonal segments were produced by cutting the nerve root containing the axon of CDLM close to the ganglion. The normal anatomy of CDLM was determined by axonal dye-fills using cobaltous chloride. Morphological changes in the isolated arborization of CDLM were examined by axonal dye-fills at successive time intervals. Changes in the isolated CDLM axon were examined via dissection and histological cross-sections of the distal nerve at graded time intervals. In one example, a remnant of the isolated CDLM arborization survived to 168 days postoperative, a time comparable to the longest previously-reported physiological and morphological survival times of distal axonal segments of invertebrates. In general the isolated arborization does not survive this long. Normally-occurring branches of the arborization can be preserved about 0 to 50 days. After this period branches of the arborization seem to be lost in progressive fashion from smaller to larger, leading to complete loss of the arborization and axon in most cases at 100 or more postoperative days. There is evidence for the presence of supernumerary fibers in the isoalted CDLM arborization between 0 to 63 days postoperative. Such supernumerary fibers indicate an independent capacity for outgrowth of the isolated arborization without connection to the nucleus. The distal axonal segment of CDLM degenerates physiologically and morphologically within 4 to 15 days after peripheral nerve section. This time course is close to that of Wallerian degeneration of vertebrate peripheral nerve axons.


The accessory optic system of the ferret. The accessory optic system of the ferret has been studied both by silver degeneration staining and by autoradiography after intraocular 3H leucine injections. There is no anterior accessory optic tract but a posterior tract containing only crossed fibres leaves the brachium of the superior colliculus and ends in the two nuclei, the medial and lateral terminal nuclei, lying either side of the cerebral peduncle. Both suprachiasmatic nuclei receive retinal fibres visible only on autoradiographs; most fibres end in the contralateral nucleus. No other area in the hypothalamus appears to receive retinal fibres demonstrated by these methods.


Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations. The axoplasmic retrograde transport of horseradish peroxidase (HRP) from axon terminals to their parent cell bodies and histochemical fluorescence microscopy have been used to study the ipsilateral centrifugal fibers to the olfactory bulbs and anterior olfactory nucleus in the rabbit. Focal injections of peroxidase were placed unilaterally into the main or accessory olfactory bulb or into the anterior olfactory nucleus. In animals with injected HRP confined within the main bulb, perikarya retrogradely labeled with the protein in the ipsilateral forebrain were observed in the anterior prepyriform cortex horizontal limb of the nucleus of the diagonal band, and far lateral preoptic and rostral lateral hypothalamic areas. Brain stem cell groups that contained HRP-positive somata include the locus coeruleus and midbrain dorsal raphe nucleus. Except for the prepyriform cortex, the basal forebrain structures with labeled perikarya correlate well with locations of cell bodies containing acetylcholinesterase and choline acetyltransferase. These somata may represent a cholinergic afferent system to the main olfactory bulb. Peroxidase-labeled cell bodies in the locus coeruleus and midbrain raphe are indicative of noradrenergic and serotonergic innervations respectively of the olfactory bulb. In rabbits in which peroxidase was injected or diffused into the accessory olfactory bulb and anterior alfactory nucleus, HRP-positive somata were identified in the prepyriform cortex bilaterally, the horizontal limb of the diagonal band nucleus, lateral hypothalamic region, nucleus of the lateral olfactory tract, corticomedial complex of the amygdala, mitral and tufted cell layers of the ipsilateral main olfactory bulb, locus coeruleus, and the midbrain raphe. Evidence for centrifugal fibers to the accessory olfactory bulb from the corticomedial complex of the amygdala, locus coeruleus, and possibly the nucleus of the lateral olfactory tract and midbrain raphe is discussed. A similar distribution of labeled perikarya in the forebrain and brain stem was seen in rats in which peroxidase injected into the main olfactory bulb had spread into the accessory bulb and anterior olfactory nucleus. Histochemical fluorescence microscopy of the main and accessory olfactory bulbs in the rabbit and rat revealed fine caliber, green fluorescent fibers and varicosities predominantly in the granule cell layer and less so among cells in the glomerular layer. In sections through the root of the main olfactory bulb, a similar fluorescence was seen in the deep half of the plexiform layer of the pars externa of the anterior alfactory nucleus. These fluorescent fibers likely represent the noradrenergic innervation of the olfactory bulbar and retrobulbar formations. A fluorescent yellow hue was observed in the glomerular layer of the main bulb and may signify a serotonergic innervation of this lamina...


Dental learning resources center.

Dental learning resources center. The University of Kentucky College of Dentistry has been operating the Dental Sciences Study Center since 1971 to support a curriculum with a large percentage of individualized instruction. Three crucial aspects of the Study Center were discussed: the facilities, the instructional materials, and the atmosphere. Administrative attention to and support of each aspect are essential to the successful functioning of a learning resources center in a largely individualized dental curriculum.


Mediator release from basophil granulocytes in chronic myelogenous leukemia. Mediator release from the leukocytes of two patients with chronic myelogenous leukemia and basophilia was studied using rabbit antihuman IgE antibody. The release of histamine, slow reacting substance of anaphylaxis (SRS-A), platelet activating factor (PAF), chemotactic activity for neutrophils and eosinophils, and an inhibitor of eosinophil migration was observed. However, the release of SRS-A from the basophils of one patient and the release of chemotactic activity from both patients displayed unusual properties. During acceleration of the disease process, the basophils of one patient released maximal SRS-A activity at progressively lower concentrations of anti-IgE. Both patients released a high molecular weight factor (M.W. greater than 20,000) which enhanced the migration of neutrophils and eosinophils and a low molecular weight chemotactic factor (M.W. less than 500) which selectively attracted eosinophils. A double peak of eosinophil chemotactic activity was routinely observed for the low molecular weight factor; this was shown to represent the eosinophil chemotactic activity of histamine with relative inhibition of migration at the histamine peak. There was little release of the tetrapeptides, ECF-A, in these patients which facilitated demonstration of this eosinophilotactic activity of histamine. These results suggest that the eosinophil chemotactic activity observed in acute allergic reactions is the net effect of the release of multiple chemotactic factors.


Inhibition of vaccinia virus replication in skin of tuberculin-sensitized animals challenged with PPD. Induction of a celayed-type hypersensitivity reaction in tuberculin-sensitized animals by tuberculin purified protein derivative (PPD) at the site of dermal vaccinia virus (VV) infection markedly accelerated elimination of VV and led to clinical recovery. Viral titers were depressed by over 99.9% in the skin of animals given PPD as compared to animals given phosphate-buffered saline or nonspecific irritants. Low concentrations of acid labile interferon were found in the skin of uninfected tuberculin-sensitized animals challenged with PPD. High concentrations of acid stable interferon were found in skin of tuberculin-sensitized animals infected with VV and challenged with either PPD or PBS. The time of appearance of the acid stable interferon was markedly accelerated, however, in the animals challenged with PPD as compared with PBS. It is concluded that recovery from dermal vaccinia infection can be enhanced by induction of a local delayed-type hypersensitivity reaction with an antigen untelated to the infecting virus.


Relationship between the C5 peptides chemotactic for leukocytes and tumor cells. A reciprocal relationship has been demonstrated between the generation of the leukotactic and the tumor cell chemotactic peptides from C5. Trypsin-produced C5 leukotactic peptides can be converted into tumor cell chemotactic factors by treatment with tumor cell extracts or by incubation with the alpha-globulin chemotactic factor inactivator (CFI) isolated from human serum. A similar CFI isolated from rat serum can, like the alpha-globulin CFI from human serum, generate the tumor cell chemotactic factor from the whole human serum. These results suggest that the tumor cell chemotactic factor derives from the same portion of the C5 molecule as the leukotactic peptide. Furthermore, it appears that the critical change may be cleavage of the amino-terminal portion of the leukotactic peptide. In addition, it has been shown that most normal tissues contain an enzyme-like material that is able to generate in normal human serum a factor chemotactic for tumor cells. The implications of these findings are discussed.


Effect of alpha-fetoprotein and other serum factors derived from hepatoma-bearing rats on the mixed lymphocyte response. Serum alpha-globulin fractions isolated by physiocochemical techniques from normal adult Buffalo rats suppressed lymphocyte proliferation in vitro. The factors responsible for mixed lymphocyte culture suppression appeared to be strain specific since they were not demonstrable in the same fractions from normal LBN rat serum. Similar fractionation of the serum from Buffalo rats bearing the Morris Hepatoma 7777 obtained from two different sources also yielded suppressive protein fractions that differed both chemically and functionally. Both variants of this hepatoma produced high serum concentrations of alpha-fetoprotein (AFP), providing an opportunity to study the possible immunoregulatory role of their fetal-associated globulins. Fractions rich in AFP that lacked other serum alpha-globulins were obtained by gel filtration chromatography and were devoid of any in vitro immunosuppressive activity. When AFP that was further purified by immunoabsorption was added to mixed lymphocyte cultures, no effect was observed at doses below 400 mug/ml. The MLC response was augmented with higher doses, similar to albumin purified by the same methods.


Purification and partial characterization of a tumor-specific blocking factor from sera of mice with growing chemically induced sarcomas. The purification of a blocking factor from the sera of tumor-bearing mice is described. Whole serum with blocking activity-the ability to inhibit specific cell-mediated anti-tumor immunity in microcytotoxicity tests-was fractionated on immunoadsorbent columns containing Sepharose-bound syngeneic normal mouse immunoglobulins and immunoglobulins from tumor-immune donors. The blocking serum was derived from mice which had carried a transplanted methylocholanthrene-induced sarcoma for 21 to 28 days. Elution of the immunoadsorbents recovered the blocking activity in a single fraction. This fraction was blocking activity in a single fraction. This fraction was radiolabeled and analyzed by SDS gel electrophoresis and Sephadex G-200 column chromatography. The active component of the blocking serum was shown to be a polypeptide of m.w. 56,000. Specificity testing implied that the factor was likely to be either tumor antigen or an antigen-specific suppressor molecule.


Cell interactions between histoincompatible T an B lymphocytes. IX. The failure of histoincompatible cells is not due to suppression and cannot be circumvented by carrier-priming T cells with allogeneic macrophages. Two possible explanations for the failure of primed histoincompatible T and B lymphocytes to cooperate in secondary responses of the IgG antibody class have been investigated in the present study: 1) The possible existence of subtle suppressive mechanisms developing as a consequence of mixing two histoincompatible cell populations; and 2) The possible inability of histoincompatible carrier-primed T cells to recognize and/or be induced to function by  carrier determinants presented to them in association with foreign MHC antigens (i.e., the "altered-self" recognition hypothesis). Absence of suppression has been verified by two different approaches, including: 1) the failure of histoincompatible T cells, even in great excess, to interfere with physiologic cooperation between syngeneic T and B lymphocytes; and 2) the failure of histoincompatible B cells to interfere with physiologic cooperation between semi-syngeneic F1 hybrid T cells and parental B cells. The unlikelihood of the "altered-self" explanation for failure of histoincompatible T and B cells to cooperate has been incicated by an inability to circumvent the requirement for I-region identity by priming T cells with allogeneic macrophagebound antigen even when the latter cells are of identical haploytpe with the allogeneic B cells employed in the final cooperation assay. These results strongly substantiate the existence of true genetic restrictions in T-B cell intractions in secondary responses to hapten-protein conjugates. The validity of other observations indicating an absence of genetic restrictions in certain circumstances, considered in the context of our own data, has suggested the possibility that virgin T and B lymphocytes reciprocally influence one another during the course of cell interactions in response to antigenic and/or other signals, a process we term "adaptive differentiation."


Comparative immunogenic properties of N-substituted phosphatidylethanolamine derivatives and liposomal model membranes. This study describes some of the parameters that quantitatively or qualitatively influence the immunogenicity in guinea pigs of synthetic lipid antigens: phosphatidylethanolamine (PE) derivatives in which the amino (N) group has been substituted with either dinitrophenyl (DNP), dinitrophenylaminocaproyl (DNP-Cap), fluoresceinthiocarbamyl (Fl), or mono (p-azobenzenearsonic acid) throsyl (ABA-Tyr) residues. Previous experiments have shown that the non-covalent insertion of DNP-Cap-PE and ABA-Tyr-PE into the same lipid bilayers of sphingomyelincholesterol-dicetylphosphate liposomes markedly enhanced anti-DNP-Cap antibody formation over that produced by liposomes sensitized with only DNP-Cap-PE. The humoral response to Fl-PE and CNP-PE-sensitized liposomes is also augmented by the simultaneous incorporation of ABA-Tyr-PE. Moreover, micelles containing both DNP-Cap-PE and ABA-Tyr-PE induce more antibodies to the DNP-Cap deteminant than do micelles of DNP-Cap-PE alone, or a mixture of DNP-Cap-PE and ABA-Tyr-PE micelles. Nevertheless, in regard to a humoral response, liposomes were more potent immunogens than were their micellar counterparts. Of all the N-substituted derivatives examined so far, ABA-Tyr-PE is unique in that it can elicit cell-mediated immunity in addition to antibodies. The cellular response to ABA-Tyr-PE is not, however, stimulated by incorporation into liposomal bilayers and requires administration of either micelles or liposomes in complete Freund's adjuvant. In contrast, the ability of ABA-Tyr-PE to enhance a  humoral response to another N-substituted PE derivative present in the same immunogen is also observed when the latter are given with incomplete Freund's adjuvant. The relationship of these findings to the immunogenicity of naturally occurring lipid antigens, as well as conventional immunogens having at least one determinant covalently attached to a protein carrier is discussed.


The L1210 leukemia cell bears a B lymphocyte specific, non-H-2 linked alloantigen. The L1210 murine lymphoblast cell line possessed a B lymphocyte-specific alloantigen which was detected with C57BL/Ks anti-L1210 serum. The antigen was found on splenic B lymphocytes but not on thymocytes, T lymphocytes, or erythrocytes. It was present on only 7% of bone marrow cells. The reactivity of C57BL/Ks anti-DBA/2-spleen serum was indistinguishable from that of the anti L1210 serum, confirming that the antigen was a normal component of B lymphocytes of the CBA/2 mouse. The strain distribution pattern of the antigen detected by the C57BL/Ks anti-L1210 serum indicated that this alloantigen was not an allele of the H-2K, H-2D, Mls, or Ig loci. Genetic analysis indicated that the antigen was inherited as a single, Mendelian dominant trait. Segregation analysis of (B10.BR X DBA/2)F1 X AKR/J offspring indicated that this B cell marker was not linked to geneslambdacoding for H-2.31 or Ly 4.2. The C57BL/Ks anti-L1210 serum identified a new polymorphic genetic locus, the product of which was B lymphocyte specific.


Studies on non-H-2-linked lymphocyte-activating determinants. II. Nonexpression of Mls determinants in a mouse strain with an X-linked B lymphocyte immune defect. Minor lymphocyte-stilulating (MIs) coded non-H-2-linked lymphocyte-activating determinants (LADs) are present on a late developing subpopulation of murine B lymphocytes. Spleen cells derived from CBA/N mice, a strain with an X-linked defect in B cell function, failed to stimulate a response in MIs-defined mixed lymphocyte reactions (MLRs) with H-2 identical CBA/J or C3H/N responder spleen cells. However, both CBA/J and C3H/N spleen cells induced significant responses in CBA/N responder spleen cells. The inability of CBA/N cells to induce an MIs-defined response was not due to the presence of the nonstimulatory MIsb genotype, since spleen cells of immunologically normal F1 mice derived from crosses of CBA/N and C3H/N mice were stimulatory in MLRs for C3H/N responder cells. Immunologically abnormal CBA/N female x C3H/N male) F1 male mice, which are hemizygous for the CBA/N X chromosome, were also unable to induce a response in MIs-defined MLRs. Although the MIs coded LADs are not X linked in immunologically normal mouse strains, these data indicate that the failure of CBA/N mice to functionally express the MIs-coded LADs is X linked. This characteristic was not secondary to the presence of suppressor cells and is most likely related to the absence of a late developing (mature) subpopulation of B lymphocytes in these mice.


Antigen-specific and nonspecific mediators of T cell/B cell cooperation. IV. Development of a model system demonstrating responsiveness of two T cell functions to HGG in vitro. Previous work in this laboratory has defined two functionally distinct T cell subpopulations (viz., antigen-specific helper) and characerized some of the parameters of these subpopulations derived from the keyhole limpet hemocyanin (KLH)-primed mouse spleen. Characerization of further parameters such as relative susceptibility to tolerance induction and relative degree of specificity was not possible with the use of KLH as the antigen. In order to overcome this impediment, an experimental protocol was developed, as herewith described, to permit the study of these two T cell subpopulations in response to the antigen human gamma-globulin (HCG). This protocol qualitatively duplicates the parameters defined for the KLH system and provides an extremely useful model for the study of the response to serum proteins in the Mishell-Dutton culture system.


Antigen-specific and nonspecific mediators of T cell/B cell cooperation. V. Unresponsiveness to HGG in vitro of these two T cell subpopulations. In previously published experiments we have distinguished two subpopulations of helper T cells (specific helper and nonspecific helper) in the spleens of antigen-primed mice. These subpopulations can be differentiated by their modes of action, and their activities and dose-response characteristics in mouse spleens primed with human gamma-globulin (HGG). In experiments designed to test shether the two subpopulations were derived from the same pool of precursors, or different pools, mice were injected with limiting doses of a tolerogenic form of HGG. They were subsequently challenged with an immunogeneic dose of HGG and their spleens assayed for specific helper and nonspecific helper activities in response to HGG in vitro. Over the dose range of tolerogenic HGG studied, both the helper activities were found to be equally tolerized. The question of the possible involvement of suppressor T cells in the maintenance of the state of unresponsiveness was also addressed quantitatively by the use of this model system. No active suppression was demonstrable in the maintenance of tolerance in either T cell subpopulation. These results suggest that the two types of helper T cells are derived from a common precursor pool, or from different pools with identical susceptibilities to tolerogen. Suppressor cells do not appear to play a role in this form of tolerance.


Mutant lines of guinea pig L2C leukemia. III. The reaction of an alloantiserum detecting idiotypic determinants on a clonally derived guinea pig B cell leukemia with IgM and Ia molecules. An alloantiserum was prepared in a strain 13 guinea pig against the GH line of the strain 2 guinea pig L2C leukemia. This serum contained antibodies to both IgM and Ia molecules. After absorption with normal spleen cells from a strain 2 guinea pig, this antiserum no longer reacted with strain 2 cells, but detected idiotypes on the IgM molecules of the L2C leukemia. These idiotypes were on the same IgM molecules detected by a xenogeneic sheep anti-L2C Fab mu antiserum. As assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the idiotype-bearing IgM molecules were synthesized by the cell, composed of normal sized mu and light chains, appeared on the cell surface as monomeric IgM, and were the only immunoglobulin molecules present on the cell. Although the alloantiserum potentially contained antibodies to unique Ia idiotypic determinants, none were found. Furthermore, the anti-IgM idiotype antisera did not react with any Ia-like molecules.


Specific extraction of antigen in vivo by extracorporeal circulation over antibody immobilized in collodion-charcoal. A novel extracorporeal immunoadsorbent is described in which anti-bovine serum albumin (anti-BSA) was entrapped in collodion membranes adherent to activated charcoal particles. This immunoadsorbent was capable of specifically removing circulating BSA in vitro and in vivo in an extracorporeal system in dogs. In preparation of the immunoadsorbent, up to 81.2% of added anti-BSA was retained on collodion-charcoal. In vitro circulation studies demonstrated that anti-BSA collodion-charcoal removed 812% more 125I-BSA than control colloidon-charcoal. For in vivo studies, an extracorporeal circulation system was established and arterial blood was passaged through a continuous flow celltrifuge wherein plasma which was separated from formed elements of the blood was circulated over anti-BSA and control rabbit gamma-globulin collodion-charcoal. 125I-BSA was passively infused into mongrel dogs, and plasma was then circulated over extracorpeal immunoadsorbents for 2 hr. Results showed up to 896% greater uptake of circulating 125I-BSA on the charcoal containing immobilized anti-BSA compared to control charcoal. There was no evidence of release of anti-BSA from the immunoadsorbent since 125I-anti-BSA cpm on the charcoal before and after the experiments were unchanged. In addition, there were no significant alterations in hematocrit, leukocyte counts, serum sodium, potassium, calcium, magnesium, or creatinine levels before and after in vivo procedures. These data suggest that this immunoadsorbent consisting of anti-BSA immobilized in collodion membranes adherent to charcoal particles may specifically withdraw circulating antigens in vivo with minimal release of entrapped antibodies and no significant alteration in the host hematologic and biochemical status.


Isolation of a human lymphocyte mitogen from wheat germ with N-acetyl-D-glucosamine specificity. A lectin, isolated from wheat germ by affinity chromatography on chitin, was mitogenic for purified human peripheral blood lymphocytes. Peak incorporation of 3H-thymidine was observed after incubation of lymphocyte cultures with wheat germ mitogen for 7 to 10 days. When lymphocytes were separated into two fractions based on their ability to form rosettes with unsensitized sheep erythrocytes, the mitogen induced a negligible proliferative response in either fraction. Mixing experiments demonstrated a strong response in the T lymphocyte fraction which required the collaboration, but not proliferation, of cells present in the nonrosetting fraction. Stimulation was specifically abolished by addition of N-acetyl-D-glucosamine at initiation of culture.


Cytomegalovirus specific IgM and IgG response in humans studied by radioimmunoassay. An indirect solid phase micro-radioimmunoassay (RIA) was adapted for the measurement of anti-cytomegalovirus class-specific immunoglobulin M (IgM) and immunoglobulin G (IgG). Cytomegalovirus (CMV) antigen (Ag) was added to the wells of microtiter plates and desiccated onto the bottom surface of the wells. Serial dilution of human CMV antisera were added and allowed to react with the Ag. The amount of viral antibody (Ab) was determined by measuring the specific binding of 125I-labeled goat anti-human IgG (125I-AHIgG) and/or IgM (125I-AHIgM). The multiple factors which influence the test were determined. Serial samples of sera from CMV-positive patients showed progressive increases in Ab titers on the basis of specific binding of 125I-AHIgG. The titers of IgM class anti-CMV Ab were also determined with the same sera, and enhancement of the titers was demonstrated when the incubation periods of the first Ag-Ab reaction were extended from 1 to 3 hr. The competition between the different Ig classes of Ab for CMV Ag and the involvement of rheumatoid factor (RF) were investigated. The anti-CMV IgG that may compete with the IgM determination was removed by adsorption of sera with Staphylococcus aureus (Cowan 1). The RF that would cause false anti-CMV IgM results was adsorbed with glutaraldehyde-insolubilized IgG. Our results indicate that this RIA is a practical, specific, and reproducible technique for detection of specific IgG and IgM Ab to CMV.


T and B lymphocytes in the regulation of delayed hypersensitivity. A correlation was demonstrated between the transient nature of a) delayed intradermal responses of guinea pigs sensitized to hen egg albumin in incomplete Freund's adjuvant and b) the proliferative response of sensitized lymphocytes to the specific antigen. Spleen cells from sensitized animals suppressed the proliferative response of lymph node cells to specific antigen. This suppression was dependent on the dose of spleen cells and the time of their removal after sensitization. Thymus cells were suppressive to a lesser extent, and their activity was not correlated with the time of removal after sensitization. Separation of spleen and thymus cells into T and B populations indicated that the B cell was the major suppressor cell in the spleen, while the T cell in the thymus had a similar but less pronounced action.


Induction of antibody to the "y" determinant of HBsAg in a chimpanzee carrier of HBsAg subtype "adw". Antibody to the y determinant of hepatitis B surface antigen (HBsAg) was induced in a chimpanzee chronically infected with hepatitis B virus and circulating HBsAg subtype adw. The chimpanzee was immunized with purified preparations of HBsAg subtypes adw and ayw. Six weeks after immunization, antibody to HBsAg (anti-HBs) specific for the y determinant, appeared. No change occurred in titers of HBsAg or antibody to hepatitis B core antigen (anti-HBc) and "e" antigen remained detectable. The circulating HBsAg subtype adw remained present despite the persistence of anti-y for greater than 8 months.


Blocking of HLA and B lymphocyte alloantigens with F (ab')2 fragments of rabbit antibodies. F(ab')2 fragments of rabbit antibodies specific for human B lymphocytes will block human anti-B lymphocyte alloantibodies. F(ab')2 of anti-beta2m will block HLA alloantibodies. The F(ab')2 fragments can be used to improve the typing of B cell lines and certain leukemia cells which express both HLA and B lymphocyte alloantigens. Pretreatment of these cells with anti-beta2m F(ab')2 preparations allows the observation of B cell alloantigen reactions independently of HLA reactions. Similar use of rabbit anti-B cell F (ab')2 allows HLA reactions to be observed free from the "extra reactions" caused by B alloantibodies that are present in many HLA typing sera.


The genetics of cell-mediated lympholysis. The role of HLA antigens in the generation of cytotoxic cells in CML has been investigated. Cytotoxic effector cells were generated in MLC among HLA-A or HLA-A and HLA-B  disparate, HLA-D identical siblings, and among HLA-A and HLA-B disparate, MLC identical (%RR less than or equal to 2 3.6) unrelated individual. The data indicate that HLA-D differences and poliferative MLC responses as measured by 3H-thymidine incorporation are not requisite for the in vitro generation of cytotoxic cells and suggest the existence of a CML-S locus (loci) distinct from HLA-A, HLA-B and HLA-D. The degree of cytotoxicity generated in a proliferative versus a "nonproliferative" MLC was comparable. In addition, these studies demonstrate that antigens other than the currently definable HLA-A, HLA-B, HLA-C, and HLA-D can serve as target determinants in cell-mediated lympholysis.


Analysis of mononuclear cell surfaces with fluoresceinated staphylococcal protein A complexed with IgG antibody of heat-aggregated gamma-globulin. Fluorescein-conjugated staphylococcal protein A (SPA) was complexed with either: 1) heat-aggregated IgG, 2) B cell specific antibody, or 3) T cell specific antibody and then used for an immunofluorescent analysis of mononuclear cell surfaces. Cellular Fc receptors failed to recognize the Fc region of aggregated IgG that had been blocked by SPA. Moreover, fluoresceinated SPA that had been complexed either with anti-Fab (B-cell specific) or T cell-specific antisera prevented the nonspecific binding of these reagents to the IgG-Fc receptors on mononuclear cells, thereby permitting the latter to be properly identified as B or T lymphocytes. In addition, when unconjugated SPA was added to presensitized target cells in a test for antibody-dependent cell-mediated cytotoxicity, cytolysis was abrogated.


The role of hydroxylation of proline in the antigenicity of basement membrane collagen. Rabbit antibodies to bovine basement membrane collagen were used to compare the antigenic determinants of rat parietal yolk sac basement membrane [14C]procollagen with [14C]protocollagen. Basement membrane [14C]protocollagen was found to be less antigenic than basement membrane [14C]procollagen. Hydroxylation of basement membrane [14C]protocollagen, either intracellularly or in vitro with protocollagen prolyl hydroxylase, resulted in restoration of antigenicity. The difference in antigenicity observed between basement membrane [14C]procollagen and basement membrane [14C]protocollagen appeared to depend primarily upon the presence of hydroxyproline in the collagen molecule. Glucosylgalactosylhydroxylysine was found to be unimportant for antigenicity.


Maintenance of hyporesponsiveness to antigen by a distinct subclass of T lymphocytes. Immunization with increasing doses of SRBC, in excess of 10(8), results in a progressive decline in the anti-SRBC PFC response. This hyporesponsive state is antigen specific and is reflected in a decrease of both T helper and B antibody-forming activity. We asked whether the apparent defect of T helper activity reflected a) an absence of alphaSRBC helper T cell activity, or b) the presence of SRBC-specific suppressor T cells within the hyporesponsive population. Our results indicate that at least a portion of hyporesponsiveness noted after antigen exposure to large doses of antigen can be ascribed to specific suppressor T cell activation. Fractionation of the suppressive T cell population using Ly antiserum showed that specific suppressive activity was mediated by a subclass of T cells (Ly2+), distinct from that committed to express helper function (Ly1).


Cell-mediated immune response in vitro. II. The mechanism(s) involved in the suppression of the development of cytotoxic lymphocytes. Spleen cells harvested from mixed leukocyte cultures (MLC) on day 2 or 3 suppress the development of CL from a fresh MLC across a cell-impermeable membrane, but day 4 MLC cells which have the maximum level of CL showed only a limited effect. Inhibition was observed only when suppressor cells were restimulated with the same H-2 type cells used during induction. However, the suppressive effect was not strain specific; that is, CBA-induced C57BL/6 spleen cells effectively inhibited the development of CL from DBA/2-induced C57BL/6 cells. In addition, DBA/2-induced C57BL/6 spleen cells effectively inhibited the development of CL from CBA cells. B10 spleen cells stimulated by B10.D2 cells gave rise to a suppressor cell population, indicating that H-2 differences alone can induce the response. The suppressive effect seemed to be exerted on an early phase of the response since no detectable inhibition was observed when suppressor cells were added 48 hr after culture initiation. The suppressive effect is not exerted on the accessory cell function but seems to inhibit DNA synthesis of the reacting cells in the MLC.


The covalent coupling of proteins to periodate-oxidized sephadex: a new approach to immunoadsorbent preparation. Proteins were covalently coupled in good yield to periodate-oxidized Sephadex. The reaction was studied in detail with respect to Sephadex type, sodium m-periodate concentration, protein (albumin or immunoglobulin) concentration, time course, temperature dependence, and pH optimum. The complexes were stable and were used successfully for the immunoadsorption of sheep anti-rabbit immunoglobulin antibodies.


Role of ionic strength in salt antagonism of aminoglycoside action on Escherichia coli and Pseudomonas aeruginosa. Studies were designed to determine whether ionic strength (mu) is a significant factor in salt inhibition of aminoglycoside action against Escherichia coli and Pseudomonas aeruginosa. In both nutrient broth (a low mu medium) and Mueller-Hinton broth (a relatively high mu medium), protection of E. coli from dihydrostreptomycin or gentamicin action by MgCl2, NaCl, or Na2SO4 was attributed to ionic strength alone. The percentage of protection increased with ionic strength and was independent of the particular salt used. Antagonism of aminoglycoside action against P. aeruginosa appeared to involve both a specific, divalent caption-dependent mechanism, revealed in Mueller-Hinton broth, and a nonspecific, ionic strength effect, elicited by sodium salts in nutrient broth. With media of relatively low salt content, variation in ionic strength itself over a range of mu of 0.02-0.14 significantly influences the effectiveness of aminoglycoside antibiotics against E. coli and P. aeruginosa.


Amikacin, an aminoglycoside with marked activity against antibiotic-resistant clinical isolates. A total of 319 clinical isolates known to be resistant to one or more aminoglycoside antibiotics were tested for their susceptibility to 10 aminoglycosides. The percentages of isolates found by an agar dilution method to be susceptible were: amikacin, 83.7%; tobramycin, 41.4%; butirosin A, 33.2%; dideoxykanamycin B, 32.6%; gentamicin C, 27.3%; lividomycin A, 17.6%; neomycin B, 10.7%; paromomycin, 10.3%; kanamycin A, 10.0%; and ribostamycin, 7.2%. The effectiveness of the antibiotics was related to their degree of resistance to bacterial enzymes; e.g., of the nine enzymes known to inactivate antibiotics containing 2-deoxystreptamine, amikacin was affected by one enzyme, tobramycin by five, and gentamicin and kanamycin by six. Examination of cell-free extracts from the 52 strains resistant to amikacin revealed that only four contained the amikacin-inactivating enzyme aminoglycoside-6'-acetyltransferase, a finding indicating that this mechanism of resistance is rare. Other experiments suggest that most amikacin-resistant strains, which are almost invariably resistant to all aminoglycosides, lack the ability to accumulate effectively either amikacin or presumably the other antibiotics intracellularly.


In vitro activity of amikacin and ten other aminoglycoside antibiotics against gentamicin-resistant bacterial strains. Sixty-nine strains of gentamicin-resistant gram-negative bacilli obtained from different geographical sources were tested for susceptibility to 11 aminoglycoside antibiotics. From the results of determinations of minimal inhibitory concentrations, patterns of resistance were established for 45 strains of Enterobacteriaceae and 24 strains of Pseudomonas aeruginosa. Overall, 81% of the strains were sensitive to amikacin and 33% of the strains were sensitive to butirosin, the next most active compound. Results indicated that 54% of the P. aeruginosa strains were sensitive to amikacin and 33% were sensitive to tobramycin. From resistance patterns, enzymes responsible for inactivation of the antibiotics were deduced. The most common enzyme was aminoglycoside nucleotidyltransferase(2''), either alone or combined with either aminoglycoside phosphotransferase(3')-I or aminoglycoside phosphotransferase(3')-II. Aminoglycoside acetyltransferase(2) was identified exclusively in strains of Providencia stuartii. Specific enzymes could not be identified for 30 strains, 21 of which were P. aeruginosa.


Modulation of the alternative complement pathways by beta 1 H globulin. C3b inactivator accelerator (A-C3bINA) was isolated from human plasma. An antiserum produced against the purified protein gave a reaction of identity with beta 1 H, a well-documented contaminant of C3 preparations. Beta 1 H appears to be composed of a single polypeptide chain containing a significant quantity of carbohydrate, and having a sedimentation coefficient of 5.6 on analytical, and 6.4 on sucrose density gradient ultracentrifugation. Its mol wt based on SDS polyacrylamide gel electrophoresis and equilibrium sedimentation is approximately 150,000, whereas it elutes from Sephadex G200 with an apparent mol wt of 300,000, suggesting that beta 1 H is an asymmetric molecule. Beta 1 H potentiates the inactivation of C3b by C3b inactivator, binds to EAC43 to limit the formation of EAC43bB and EAC43bBP, and in contrast to  C3b inactivator, it increases the rate of loss of hemolytic sites from  EAC43bB and EAC43bBP. For the C3b inactivator-potentiating effect, beta 1 H and C3b inactivator must necessarily be simultaneously present. The kinetics of inactivation of C3b by C3b inactivator and beta 1 H are first order, suggesting that potentiation is not a multistep process. The mechanisms of binding to C3b and inhibition of the alternative pathway convertases C3bB and C3bBP are currently unknown.


Nature of the antigenic complex recognized by T lymphocytes. I. Analysis with an in vitro primary response to soluble protein antigens. In order to analyze the genetic factors involved in the regulation of macrophage-T-cells interaction we have developed an in vitro primary response to soluble protein antigens in which nonimmune guinea pig T cells can be sensitized and subsequently challenged in tissue culture with antigen-pulsed macrophages. Antigen-specific T-cell activation, as measured by increased DNA synthesis, occurred when syngeneic antigen-pulsed macrophages were used for both initial sensitization and secondary challenge. No T-cell activation occurred when allogeneic antigen-pulsed macrophages were used for secondary challenge of cells primed when syngeneic macrophages. When allogeneic antigen-pulsed macrophages were used in both primary and secondary cultures it was difficult to assess antigen-specific stimulation due to the substantial mixed leukocyte reaction. However, when T cells from F1 animals were primed with parental antigen-pulsed macrophages they responded only to the parental macrophages used for initial sensitization but not to those of the other parent. These results are discussed with respect to T-cell recognition of a complex antigenic determinant which may include I-region gene products.


Expression of equivalent clonotypes in BALB/c and A/J mice after immunization with phosphorylcholine. Analysis of A/J antibody to phosphorylcholine (PC) revealed a striking degree of similarity to PC-binding myeloma proteins of BALB/c origin. By quantitative idiotypic analysis A/J anti-PC antibody was composed to antibodies bearing binding site idiotypic determinants indistinguishable from two different BALB/c myeloma proteins, T15 and M511. Idiotypic determinants of three other PC-binding proteins, W3207, M167, and M603 were not detected. Isoelectric focusing of the light chains verified the presence of antibodies similar to T15 and M511 and indicated the presence of a third antibody whose light chains had a pI identical to that of M603. When the sequence of A/J heavy chains were compared to the heavy chains of T15, M511, and M603, both the framework and first complementarity regions were identical in all cases. Sequences analysis of the light chains through part of the first complementarity region revealed three chains, one similar to each of the myeloma proteins T15, M603, and M167-M511. The latter two sequences differ by only a single amino acid (a single base substitution) in the first 23 residues, suggesting that the two light chains may be very similar if not identical. Thus, BALB/c and A/J mice which differ genetically at multiple loci including the heavy chain allotype complex locus show a remarkable preservation of their anti-PC antibodies. These results indicate that the genes encoding these antibodies are contained in the germ line.


Production of gramicidin S synthetases by Bacillus brevis in continuous culture. The effects of different nutrient limitations on the production of the two enzymes of gramicidin S biosynthesis were studied during continuous culture of Bacillus brevis. Gramicidin S synthetases I and II were produced in the chemostat under carbon, nitrogen, phosphorus or sulphur limitation. The growth rate, rather than the nature of the limitation, was the major controlling factor in regulating the level of the gramicidin S synthetases. Synthetase production was low at high dilution rates (0.45 to 0.50 h-1) but increased as the dilution rate was lowered. The highest specific activities occurred at dilution rates that were different for each type of limitation: 0.40 h-1 for nitrogen, 0.32 h-1 for carbon, 0.24 h-1 for sulphur and 0.20 h-1 for phosphorus. Phosphorus limitation gave the highest specific activities. At low dilution rates (0.10 to 0.15 h-1), enzyme activities were again low. Sporulation occurred under carbon limitation, but at a lower dilution rate than that which supported optimal gramicidin S synthetase formation. The specific productivity of the synthetases in the chemostat was higher than the highest productivity obtained in batch growth.


Morphological, biological and antigenic properties of Neisseria gonorrhoeae adapted to growth in guinea-pig subcutaneous chambers. Gonococci (strain BS3) passaged three times and harvested directly from plastic chambers implanted subcutaneously in guinea pigs were compared with the parent strain (BS) grown in vitro. The strain grown in vivo produced smaller colonies than that grown in vitro and when examined directly in chamber fluid was sometimes not pilated. It was more resistant to the bactericidal action of human serum and more infective for guinea-pig chambers. In gel diffusion, extracts of the organisms adapted in vivo and cultured once on agar appeared to contain one or two antigens that were different from those in extracts of the in vitro grown organisms; and on polyacrylamide gels, electrophoresis of similar extracts showed one or more protein components for strain BS3 which were not seen for strain BS. Gonococci grown in guinea-pig subcutaneous chambers appear to be suitable for studies on the determinants of gonoccal pathogenicity.


Expression of virus structural proteins on murine cell surfaces in association with the production of murine leukaemia virus. We have used a quantitative radiolabelled antibody procedure to measure the amount of certain virus structural antigens on the surface of BALB/c RAG cells producing endogenous B-tropic type C virus. RAG cells expressed group specificities of MuLV p30 on their cell surface but did not express gp70 group specificities. However, type specificities of gp70 were expressed on BALB/c cell lines infected with Moloney leukaemia virus. The majority of p30 antigens detected on the RAG cell surface were removed by trypsin and their reappearance was prevented by cycloheximide, even in the presence of 'conditioned medium' containing MuLV. Passive adsorption of exogenous MuLV p30 to the surface of virus negative BALB/c fibroblasts reached a maximum of 20% of the protein detectable on virus producing RAG cells. These data support the hypothesis that much, but not all, of the surface p30 is expressed de novo on the cell membrane and not derived from passive adsorption of p30 released from shed virus or as a by-product of virus infection of a cell.


Lipid storage myopathies. A review of metabolic defect and of treatment. Various cases of lipid storage myopathies have been described. The biochemical defect could be determined in only some of these cases. The syndromes identified to date are as follows: carnitine deficiency (type I lipid storage myopathy), carnitine-palmityltransferase (CPT) deficiency and pyruvate-decarboxylase deficiency. In the last two diseases the vacuolization in muscle is not marked. The case of a 10 year old carnitine deficient patient with a history of insidious muscle weakness in the proximal limb and neck muscles is presented. The patient was treated with oral carnitine and a medium chain triglyceride diet for 18 months and her clinical status has remained improved. In other lipid storage patients prednisone treatment resulted in improvement. In cases of suspected lipid storage myopathy the following studies are indicated: 1) examination of ketone bodies in serum and urine during fasting, long chain and medium chain triglyceride diets; 2) serum triglyceride and serum carnitine; 3) study on fresh muscle and fibroblasts with labeled substrates, biochemical determination of carnitine and CPT in muscle.