The mechanism of acute renal failure after uranyl nitrate. Administration of 25 mg/kg uranyl nitrate (UN) to rats leads to a brief period of polyuria followed by progressive oliguria with death at 5 days. Factors that determine glomerular filtration rate (GFR) were examined in control Munich-Wistar rats (n equals 16) and 2 h after either 15 mg/kg (n equals 8) or 25 mg/kg (n equals 7) of UN (i.v.) utilizing direct measurements of hydrostatic and oncotic pressures and plasma flow. Total kidney GFR was reduced to 47% of control in the low dose group and to 21% in the high dose group. The simultaneous nephron filtration rate (sngfr) was 28.6 plus or minus 0.8 nl/min/g kidney wt in control, 29.1 plus or minus 1.0 in the low dose group, and 18.1 plus or minus 1.2 (P less than 0.001) in the higher dose group. This disparity in UN effect upon GFR and sngfr was due to tubular back-diffusion of solute through damaged epithelia beyond the early proximal tubule as demonstrated by microinjection of inulin and mannitol in the proximal tubule. Inulin "leak" persisted at 6 h after UN when tubular pressure had returned to normal. Comparison of sngfr measured in early vs. late proximal tubule revealed no difference after high dose UN, suggesting no significant leak of inulin from the early proximal tubule, and that the decreased sngfr was due to primary reductions in ultrafiltration. Nephron plasma flow was equal to control at both doses of UN. Also directly measured hydrostatic pressure gradient across the glomerular capillary was not changed. The effective filtration pressure achieved equilibrium in control of animals but became significantly positive at the efferent end of the capillary at both doses of UN and increased. Total glomerular permeability (LpA) was progressively reduced from control (0.089 plus or minus 0.005 nl/s/g kidney wt/mm Hg) at low dose UN (0.047 plus or minus 0.013) and high dose 0.024 plus or minus 0.003 nl/s/g kidney wt/mm Hg). Therefore UN decreases GFR by two mechanisms: (1) tubular damage leading to back-diffusion of solutes and (b) a primary reduction in sngfr due to reduced LpA.


Provisional tables on the zinc content of foods. A critical and exhaustive review of published and unpublished data on the zinc content of foods showed that, with few exceptions, the major dietary sources of zinc were foods of animal origin. Oysters, which contained more than 5 mg. zinc per 3-oz. cooked portion, were an outstanding source of zinc. Lean beef and beef liver were also among the foods highest in zinc and ground beef was a good source. Dark meat of poultry contained more zinc than light meat, and turkey was higher in zinc than chicken. Egg yolk, but not egg white, was relatively high in zinc, as were nonfat dry milk and Cheddar cheese. Foods of plant origin, like those of animal origin, varied widely in zinc content. Cocoa and the milling fractions of bran and germ of wheat were high in zinc. Seeds of plants, such as muture dry legumes and peanuts, were better sources of zinc than were leaves, stalks, fruits, or roots. Vegetables, fruits, oils, fats, tea, coffee, and carbonated beverages contained little zinc. Factors influencing the zinc content of foods were evaluated. Provisional tables, giving representative values for zinc in more than two hundred foods, both in 100-gm. portions and in common household units, based on this research, are presented.


Temperature ofthe pulp chamber during impression taking of full crown preparations with modelling compound. Temperature elevations were measured in the pulp chamber of full cast crown preparations during impression taking with modelling compound in copper bands. Uncontrolled flaming, controlled flaming, and water heating of the bands produced temperatures of 53, 44, and 41.5 C, respectively. The time involved in restoring the temperature in the pulp to 37 C was determined. The implications of delayed chilling are discussed.


Effect of ultrasonic ZnPO-4 cement removal on band adhesion and cement solubility under orthodontic bands. The use of ultrasonic energy to remove excess ZnPO-4 cement from orthodontic bands reduces the solubility of the cement but does not affect band adhesion unless excessive band contact is made. Excessive band contact weakens band-to-tooth adhesion. The solubility of the cement is increased by decreasing the pH and is increased when organic anions capable of binding metal ions are present.


Fluorescent antibody localization of the vitamin D-dependent calcium-binding protein in the oviduct of the laying hen. Fluorescein isothiocyanate-conjugated sheep antirabbit gamma-globulin was used in the indirect fluorescent antibody technique to localize the vitamin D-dependent calcium-binding protein (CaBP) in the shell gland of the laying hen. Intense specific fluorescence, indicative of the presence of CaBP, was found only in the tubular gland cells and not in the surface epithelium. CaBP was primarily associated with the luminal surface and other cell boundaries, and in lesser concentrations within the cells except for occasional intracellular regions of high concentration. The magnum, isthmus and vagina were essentially negative. Since the concentrations of intestinal CaBP is highly and directly correlated with the efficiency of calcium absorption, the present results suggest that the tubular gland cells might have a primary role in the transport of calcium associated with egg shell formation.


Niridazole. II. A potent long-acting suppressant of cellular hypersensitivity. Niridazole, a human anthelminthic drug, is shown to be a potent long-acting suppressor of delayed hypersensitivity. Much lower doses than used in the therapy of helminth infections suppressed granuloma formation around Schistosoma mansoni eggs for periods up to 32 days and inhibited delayed footpad swelling in mice previously sensitized with eggs. Marked prolongation of skin allograft survival in mice accross the H-2 area was produced. The drug is administered orally, has been used clinically on a massive scale, and appears to be relatively free of the cytotoxic effects of most immunosuppressive agents.


Increased risistance of immunoglobulin A dimers to proteolytic degradation after binding of secretory component. The contribution of secretory component to the stability of secretory IgA against proteolysis has been studied by a new approach, i.e., by comparing the proteolytic degradation of the complexes formed in vitro between these proteins and secretory component. The results show that attachment of secretory components to the dimeric backbone of the secretory IgA molecule is accompanied by a significantly increased resistance of this backbone against digestion by both trypsin and pepsin. This protective effect may be a physiologic function of secretory component or may be due merely to unspecific blocking by secretory component of one or more sensitive peptide bonds in the IgA backbone.


Increased immunologic reactivity between human glomerular basement membrane and group A type 12 streptococcal cell membrane after carbohydrase treatment. Anti-streptococcal cell membrane and anti-human glomerular antisera were evaluated on normal human kidney sections by an indirect immunofluorescent test both before and after absorptions with homologous and heterologous membrane preparations. The observed cross-reactions were potentiated by removal of carbohydrate units from both membrane-types by carbohydrase, a mixture of carbohydrate-cleaving enzymes.


Selection of a resin system for anterior fracture treatment. The tensile strength of the bond formed between etched bovine enamel and selected resin systems was evaluated; also, the need for a resin pulpal barrier was examined. Results indicate that the BIS-GMA unfilled primer is unnecessary for strength or as a pulpal protectant. The large scatter of results substantiates the clinical hypothesis that the effectiveness of the acid etch is highly variable.


Scanning electron microscopy of dentition: methodology and ultrastructural morphology of tooth wear. Scanning electron micrographs were taken of sets of human molars-those of paleo-Indians used in mastication of, ostensibly, a highly abrasive diet, and those of contemporary Americans. Different ultrastructural patterns of enamel wear were observed between the groups.


Blast transformation of rabbit B-derived lymphocytes by a mitogen extracted from Nocardia. Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.


Migration inhibitory factor and type II interferon in the circulation of mice sensitized with mycobacterial components. Migration inhibitory factor (MIF) and type II interferon were released into the circulation after mice had been sensitized i.v. with a bacillus of Calmette and Guerin (BCG) cell-wall-in-oil vaccine and then challenged four weeks later with 50 mg old tuberculin. At least two components of BCG, protein (PPD) and lipid (P-3), have been identified which are essential for the type of sensitization in which mediators are released after appropriate challenge. The route of sensitization had a marked effect on the development of delayed footpad reactions, release of lymphokines, and resistance to infection with virulent tubercle bacilli. Sensitization by the subcutaneous route tended to induce more pronounced delayed footpad reactions, whereas the i.v. route of sensitization was associated with maximum release of mediators. A close correlation existed between the conditions of sensitization that resulted in maximum production of lymphokines (MIF and Type II interferon) and those that caused protection against aerosol challenge with a virulent strain of Mycobacterium tuberculosis.


Stimulation of a T-independent primary anti-hapten response in vitro by TNP-lipopolysaccharide (TNP-LPS). The trinitrophenyl hapten (TNP) has been covalently conjugated to bacterial lipopolysaccharides (LPS) to give TNP-LPS. The site of attachment has been suggested to be in the core polysaccharide and lipid A region of the molecule and approximately 2.4 hapten molecules are bound per monomer LPS molecule. The TNP-LPS has been demonstrated to be immunogenic in vitro at very low concentration. This antigen has further been shown to initiate a T-independent TNP-PFC response. The immunogenicity of TNP-LPS is abrogated by mild alkaline hydrolysis, suggesting a requirement for intact lipid A in the initiation of an immune respose at the very low concentrations of antigen used.


Effects of concanavalin A on the in vitro responses of mouse spleen cells to T-dependent and T-independent antigens. ?The stimulatory and inhibitory effects of concanavalin A (Con A) on the in vitro primary immune responses to a T-dependent antigen, sheep erythrocytes (SRBC) and a T-independent antigen, TNP-lipopolysaccharide (TNP-LPS) have been studied. Inhibition of the response to both antigens was optimal when 2 mug Con A were added at the initiation of the culture period. The response to SRBC was considerably enhanced by the addition of Con A 24 hr later. In contrast, this late addition did not stimulate the TNP-LPS response and often inhibited it. Inhibition of the TNP-LPS response required the participation of T cells since it was not observed in cells from adult thymectomized irradiated bone marrow-reconstituted (ATXBM) mice. The response to TNP-LPS was somewhat enhanced in ATXBM cells, but the degree of enhancement was strikingly less than that observed for SRBC. LPS per se did not block the stimulatory effect of Con A on the SRBC response, and was observed to act synergistically with this lectin. None of the Con A effects observed required the participation of adherent cells. These observations are consistent with a model in which different subpopulations of T cells are responsible for the inhibitory and stimulatory effects. They further suggest that the Con A inhibitory activity acts via a T cell to inhibit directly the B cell response to antigen.


Induction of theta-positive lymphocytes and lymphoblasts in mouse bone marrow by mitogens. With a sensitive technique for the identification of lymphocytes and lymphoblasts bearing the surface antigens phi, TL, and immunoglobulin, the induction of phi-positive small lymphocytes in bone marrow could be observed. Dibutyryl cyclic adenosine 3',5'-monophosphate and thymosin were both effective in inducing precursor cells to express phi antigen. Concanavalin A and bacterial lipopolysaccharide were also shown to be inducers. After treatment with concanavalin A but not with lipopolysaccharide, lymphoblasts developed in marrow cultures which were positive for phi and TL antigens; when spleen was cultured with concanavalin A, lymphoblasts were phi-positive and TL negative. It is concluded that the cell responsive to concanavalin A (and phytohemagglutinin) in bone marrow is a phi-negative precursor (stem) cell, which is induced first to express phi and TL antigens, and then to differentiate to a lymphoblast.


Role of the clotting system in cell-mediated hypersensitivity. II. Kinetics of fibrinogen/fibrin accumulation and vascular permeability changes in tuberculin and cutaneous basophil hypersensitivity reactions. Radioactive tracers and immunofluorescence were employed to detect and quantitate fibrinogen/fibrin deposition in two types of cell-mediated hypersensitivity reactions in the guinea pig. Classic delayed hypersensitivity (DH) reactions to Old Tuberculin and to the azobenzenearsonate hapten were characterized by a progressive increase in the fibrinogen (125-I-HF) content which exceeded that of the albumin tracer (131-I-HSA) and paralleled the development of induration and erythema. Accumulation of 125-I-HF could be related both to increased vascular permeability to 125-I-HF and, more specifically, to retarded efflux of extra vascular 125-I-HF from tuberculin reaction sites. Warfarin inhibited 125-I-HF accumulation and the formation of urea-insoluble 125-I-HF (cross-linked fibrin) as well as induration in tuberculin reactions. Immunofluorescence studies revealed the site of Fib deposition to be extravascular, among the connective tissue fibers of the dermis, similar to that in DH reactions in man. In contrast, little 125-I-HF accumulated in cell-mediated reactions rich in basophils--cutaneous basophil hypersensitivity (CBH) reactions to keyhole limpet hemocyanin, ovalbumin, and dinitrochlorobenzene--due in part to less vascular leakage of macromolecules and to decreased formation of urea-insoluble fibrin. By immunofluorescence Fib deposits were found in CBH reactions in a pattern similar to that in DH reactions, but the intensity of staining was appreciably less. Thus, fibrin accumulation further distinguishes DH from CBH reactions and is very likely responsible for the induration characteristic of DH reactions.


Activation of guinea pig macrophages by bacterial lipopolysaccharide requires bone marrow-derived lymphocytes. The activation of guinea pig peritoneal macrophages by Salmonella typhimurium lipopolysaccharide (LPS) was studied by using 14C-glucosamine uptake. Peritoneal exudate cells incorporated significant amounts of 14C-glucosamine when stimulated with LPS but neither purified macrophages nor nonadherent lymphocytes by themselves incorporated glucosamine. The activation of macrophages could be restored by adding nonadherent peritoneal lymphocytes, spleen cells, and lymph node cells but not thymocytes. Removal of B lymphocytes abolished the restorative capacity from active lymphoid cell populations. In contrast, B lymphocytes would restore glucosamine incorporation by macrophages stimulated with LPS but T lymphocytes did not. In addition, cell-free supernatants from LPS stimulated B lymphocytes but not from T lymphocytes could restore glucosamine incorporation by macrophages. These experiments demonstrate that LPS does not directly activate macrophages as measured by glucosamine incorporation but stimulates B lymphocytes which in turn activate macrophages.


The role of antibody feedback inhibition in the regulation of the secondary antibody response after high and low dose priming. Spleen cells from mice immunized with a low dose of SRBC produce a greater secondary response to subsequent antigenic challenge in vivo than spleen cells from mice initially immunized with a larger dose. However, when the spleen cells are removed from the mice and given a secondary challange in tissue culture, a greater response is produced by cells from animals primed with the larger dose. Serum from the mice receiving the high dose of antigen contains low, but significant levels of specific antibody, and can inhibit further antibody production by lymphoid cells in vivo and in vitro. The characteristics of this suppression are similar to those known for passively administered specific antibody. Thus, antibody produced as a result of primary immunization may act through an inhibitory feedback mechanism in specifically limiting the magnitude of the secondary response to subsequent antigenic challenge in the normal animal


Rosette formation by canine peripheral blood lymphocytes. The surface membrane characteristics of canine peripheral blood lymphocytes were investigated using erythrocyte (E) and erythrocyte antibody complement (EAC) rosette assays and immunofluorescent staining techniques. Canine thymus-derived lymphocytes formed nonimmune E rosettes with human and guinea pig erythrocytes at approximately the same percentages (mean percentage 36.3 and 32.4, respectively). Rosettes did not form with erythrocytes from seven other animal species. Cell surface immunoglobulins were demonstrable in a small percentage (4%) of the E rosette-forming lymphocyte population. Fragments of human erythrocytes inhibited E rosette formation by intact human red cells, but did not result in a significant decrease in rosette formation by intact guinea pig erythrocytes; likewise, guinea pig fragments had no inhibitory effect on rosette formation by human erythrocytes, demonstrating that separate receptors were required for the two red cell types. EAC rosette formation was not affected by addition of intact or fragmented human and guinea pig erythrocytes. Canine bone marrow-derived lymphocytes were characterized by immunoglobulin on the cell surface and EAC rosette formation. Serial tests of lymphocytes from one dog revealed a wide variation in percentage of cells forming E and EAC rosettes. A close correlation was observed between the immunoglobulin-bearing cells (mean percentage 46.6) and those forming EAC rosettes (mean percentage 49.3).


Inhibition of the primary in vitro antibody response by interferon preparations. The primary in vitro plaque forming cell (PFC) response of mouse (C57BL/6J) spleen cells to sheep red blood cells (SRBC) was inhibited by two sources of crude and two sources of partially purified mouse interferon. The substance(s) in the interferon preparations which induced PFC and antiviral inhibitory activity have the following common characteristics: a) The interferon preparations were of different potencies and specific activities, but they inhibited the PFC response in proportion to their activities; 20 to 60 units were the minimal amounts required for greater than 90% inhibition of the PFC response. b) Both the antiviral activity and the PFC inhibitory activity of the interferons were neutralized by antibody specific for mouse interferon. c) Both activities were partially or completely inactivated by heating at 60 degrees C for 1 h. d) Human interferons had neither antiviral activity nor PFC inhibitory activity in mouse cells. e) Limited exposure (4 h) of cells to interferon significantly inhibits both viral infection and the PFC response. f) Both the antiviral activity and the PFC inhibitory activity of the interferon preparations are acid stable. It is concluded, therefore, that the inhibition of the primary in vitro PFC response is due to interferon in the preparations. Maximum inhibition of the PFC response occurred when interferon was added to cultured at the same time as SRBC. Significant inhibition was also observed when interferon was added 1 day later, and slight enhancement was observed when added at days 2 and 3 of SRBC addition. Kinetic data showed that the greater the concentration of interferon added to the cultures, the earlier the effect on the PFC response. The presence of interferon in cultures for the first 4 h is sufficient to inhibit the PFC response. Interferon, then, appears to affect some early event(s), which leads to inhibition of the PFC response. Since mature T cells can be one of the sources of interferon, we have demonstrated a suppressor effect of a T cell lymphokine. The precise relationship of interferon to various expressions of suppressor T cell effects remains to be determined.


Effects of bacterial lipopolysaccharide on the induction and expression of cell-mediated immunity. I. Depression of the afferent arc. Bacterial lipopolysaccharide (LPS), being a B cell mitogen, enhances antibody production. When given prior to an injection of sheep red blood cells (SRBC) it caused a premature but not significantly increased production of antibody forming cells in regionally stimulated lymph nodes. There was a corresponding depression of T cell activity, as measured by the relative levels of delayed-type hypersensitivity (DTH) in normal and LPS-treated mice. Although LPS also induced hyperplasia of the reticuloendothelial system, and a consequent reduction in the effective antigenic dose of SRBC, this was not the reason for the reduced level of T cell activity. The opposing effects of LPS on T and B cell responses was due to premature inhibition of the T cell response by an accelerated production of antibody. This caused an earlier onset of the inhibitory mechanism which regulates the T cell response and is specific for the inducing antigen.


Thymic regeneration after lethal irradiation evidence for an intra-thymic radioresistant T cell precursor. The data presented indicate the existence of a significant pool of radioresistant stem cells which are capable of partially restoring the thymus of heavily irradiated mice. 3-H-TdR incorporation by the thymus of lethally irradiated mice begins 48 to 72 hr after irradiation and increases throughout the next 8 days. By the 9th day after 760 rads, typical corticomedullary architecture has been restored. 890 rads markedly suppressed, but did not totally eliminate this regeneration. Injection of large numbers of syngeneic bone marrow cells immediately after irradiation was without effect on the rate or extent of regeneration. Mice whose bone marrow and spleen were shielded from irradiation showed an identical amount of thymic regeneration as those receiving total body irradiation indicating that the precursor cell pool responsible for the early post irradiation phase of thymic regeneration is most likely an intrathymic population. The cells repopulating the thymus were morphologically indistinguishable from normal thymocytes and were susceptible to cytotoxic antisera against the thymic differentiation antigens Thy-1, TL, LyA2 and LyC2.


Immunoglobulin D-bearing lymphocytes in primary immunodeficiencies. Surface IgD on blood lymphocytes was studied in 10 normal adults and 24 patients with primary immunodeficiencies by direct immunofluorescence, together with surface immunoglobulins of the other classes and with spontaneous rosette formation with sheep erythrocytes. In the normal adults, 8% of the lymphocytes bore delta chains (the figures for mu chains being 11%) and, among the cells positive for mu and/or delta, 70% were mixed stained, 22% and 8% being single stained for mu and delta respectively. In 10 patients with sex-linked agammaglobulinemia or variable immuno-deficiency, practically no cells bearing surface immunoglobulins, including IgD, were detectable. A normal distribution of surface immunoglobulins, including the results of double labeling for mu and delta, was found in five other immunodeficiency patients in whom there was a block of the terminal differentiation of B lymphocytes into plasma cells. A new kind of block in the differentiation of the B cell line was observed in two patients affected with sex-linked severe combined immunodeficiency and variable immuno-deficiency respectively. They showed high figures for IgD-bearing lymphocytes, some of which carried simultaneously mu chains, contrasting with the absence of lymphocytes carrying IgM without IgD and of IgG- or IgA-bearing cells. The data obtained in several other patients with low figures for IgG- and IgA-bearing lymphocytes and a predominance of IgD-carrying cells with an excess of single producers for delta chains over single producers for mu chains suggest an analogous but incomplete maturation arrest.


Enhancement of nitroblue tetrazolium dye reduction by leukocytes exposed to a component of complement in the absence of phagocytosis. Enhancement of in vitro nitroblue tetrazolium dye reduction by human polymorphonuclear leukocytes may be mediated by a low molecular weight fluid phase component of the complement system. This occurs in the absence of phagocytosis and is associated with an increase in leukocyte hexose monophosphate shunt activity. The stimulatory component may be generated by activating the alternate complement pathway in serum, and shares many of the properties of human C5a. Thus, the enhanced spontaneous reduction of nitroblue tetrazolium by leukocytes from patients with bacterial sepsis may not necessarily be due to the phagocytic activity of these cells, but rather may occur as a consequence of in vivo complement activation by either intact microorganisms or their products.


Effect of cortisone on the disappearance kinetics and tissue localization of soluble immune complexes. The effect of cortisone treatment on the disappearance kinetics, the hepatic uptake, and the glomerular deposition of i.v. administered, soluble immune complexes (HSAantiHSA) was examined in mice. An initial rapid disappearance of complexes from the circulation occurred after the injection of complexes into control mice, which was caused by increased vascular permeability. This phase was absent in the cortisone-treated group. The half life of complexes composed of more than two antigen and two antibody molecules (greater than Ag2Ab2)was prolonged from 1.93 hr in control mice to 4.71 hr in cortisone-treated mice while the half life of Ag2Ab2 complexes was unchanged (11.40 hr vs 12.04 hr). Although the clearance velocity of greater than Ag2Ab2 complexes was suppressed in cortisone-treated mice, the quantity of complexes specifically located in the liver at 1, 2, and 4 hr was not significantly different in the two groups. Persistence of circulating greater than Ag1Ab2 complexes was associated with enhanced and prolonged glomerular depostion of complexes in the cortisone-treated mice.


Partial characterization of leukocyte inhibitory factor by concanavalin A-stimulated human lymphocytes (LIF Con A). Leukocyte inhibitory factor (LIF) obtained from normal human lymphocytes stimulated by Concanavalin A was characterized by Sephadex gel filtration, disc electrophoresis, isopycnic centrifugation, enzymatic treatment and by immunoabsorbent studies. LIF activity was found after Sephadex G-100 gel filtration in a fraction containing molecules having the size of albumin (68,000 daltons). This is the same region were LIF activity derived from antigen-stimulated lymphocytes has previously been found to elute. On disc gel electrophoresis at pH 9.1, peak LIF activity was eluted from the acrylamide gel fraction containing molecules that migrate with albumin. In isopycnic centrifugation studies, human LIF was shown to have a buoyant density similar to that of pure protein. Human LIF was inactivated by treatment with chymotrypsin but not by neuraminidase treatment. LIF activity could not be removed by incubating the material on columns containing Sepharose 6b coupled with anti-human serum albumin or anti-Fab. The characteristics of human LIF and migration inhibitory factor were compared.


Non-random distribution of surface immunoglobulins on murine B lymphocytes. An indirect labeling technique was used to map the surface immunoglobulin (Ig) on murine splenic B lymphocytes by freeze-etching. Cells were labeled first with fluorescein conjugates of monovalent (papain-digested) anti-Ig antibody followed by monovalent anti-fluorescein antibody coupled to ferritin. The technique avoids cross-linking and aggregation of surface Ig. Freeze-etched replicas of cells labeled at 4 degrees C, as well as of cells prefixed with paraformaldehyde showed that surface Ig was distributed in small clusters with interconnecting networks. The observed pattern was analyzed statistically by comparing it with the expected random (Poisson) distribution and shown to be non-random to a high degree of statistical significance. The deviation from randomness could be explained by the presence of clusters and relative excesses of bare membrane. Such an observed distribution of surface Ig suggests that this membrane macromolecule may be organized in a specific manner. The distribution may also play a role in the function of surface Ig as the antigen receptor on B lymphocytes.


Capping of the lymphocyte C3 receptor and temperature-dependent loss of C3 rosettes. The percentage of mouse spleen cells rosetted by bacteria-antibody-complement complexes (BAC) was found to be temperature dependent. Maximal rosetting was observed when BAC and spleen cells were incubated in ice (0.5 degrees C), whereas less rosetting resulted from 37 degrees C incubations. When rosetted cells were incubated at 37 degrees C, cap formation was found to occur, and indirect evidence suggested that the capped bacteria were subsequently shed from the lymphocyte surface. Neither capping nor shedding occurred at 0.5 degrees C, suggesting that these phenomena were responsible for the decreased percentage of rosetted cells found after incubation at 37 degrees C. An incidental observation indicated that a second factor potentially contributing to the loss of rosetted cells at 37 degrees C was the removal of BAC from the surface of cells by phagocytes.


Electrophoretic fractionation of guniea pig lymphocytes: evidence for different subsets of T and B cells in spleen and lymph node. Guinea pig lymph node and blood lymphocytes have been physically fractionated in preparative cell electrophoresis into two functionally viable populations, the high mobility cell population (HMC) and the low mobility cell population (LMC). By using cell surface markers and functional tests known to be specific for T and B lymphocytes, respectively, it is shown that the T lymphocytes localize in the HMC population and the B lymphocytes in the LMC population. The spleen lymphocytes do not separate into the two populations. They move into one single broad peak containing both T and B cells. This finding indicates the presence of electrokinetically different subsets of T and B lymphocytes in the spleen on one hand and in the lymph node and blood on the other hand.


Humoral immunity in rodent malaria. III: Studies on the site of antibody action. Serum which protects rats against Plasmodium berghei infections fails to sensitize parasitized erythrocytes in vitro for in vivo destruction. Further, the efflux of 86Rb from parasitized erythrocytes in the presence of complement is not accelerated. On administration to animals with preexisting malaria, it does, however, produce a relatively slow decline in parasitemia, a phenomenon interpreted in terms of the gradual production and/or release of reactive antigenic determinants associated with maturation of the parasites. Protective activity is elaborated in the serum of animals during the development of parasitemia, thus creating a situation in which there is apparently the coincident presence of antibody and its putative antigen in the circulation. Little absorption of protective activity by parasitized cells or parasites was observed in vitro. However,  in vivo removal of the protective activity during an ongoing infection could be demonstrated. These observations favor the notion that protective antibody exerts its influence not on the bulk of the parasitized erythrocytes, but rather on some subpopulation thereof. The cumulative evidence of this and other studies suggests that the stage(s) involved are the schizonts and/or merozoites.


Activation of T and B lymphocytes in vitro. IV. Regulatory influence on specific T cell functions by a thymus extract factor. In the presence of a preparation of purified thymus extract factors (TEF), spleen cells from athymic nude mice develop in vitro immune responses to soluble DNP-protein conjugates, to particulate sheep red blood cells, and to allogeneic cells. TEF has similar effects also on the in vitro antibody responses of normal mouse spleen cells, enhancing antigen-dependent plaque-forming cells responses to dinitrophenyl carrier complexes of unprimed and primed mice. TEF fails to reconstitute helper cell function in cultures of T cell-deprived spleen cells. The thymic factor increases the reactivity or normal thymus cells in the in vitro mixed lymphocyte reaction and has at high doses mitogenic effects. The role of humoral thymus factors on the differentiation process of T cells is discussed.


The role of macrophages in the production of lymphokines by T and B lymphocytes. The role of macrophages in the production of two lymphokines, monocyte chemotactic factor and macrophage activating factor, was investigated. Lymphokine production by guinea pig lymph node and spleen cells required macrophages for thymus-dependent antigens and mitogens. In contrast, B cell stimulants which also induce the synthesis of lymphokines were macrophage independent. When populations of relatively pure B or T lymphocytes were isolated, it was found that T cells required viable macrophage cooperation to produce these two lymphokines and to undergo propliferation in response to specific antigens, whereas B cells could be directly activated in the absence of macrophages. These findings suggest that T and B cells have different requirements for activation and for macrophage cooperation. Futhermore, since lymphokine synthesis is evident within the first 4 hr of stimulant presentation, these observations demonstrate that macrophages play an essential role in the earliest events of lymphocyte activation.


Regulation by antibody of phytolectin induced lymphocyte proliferation. I. Evidence for two mechanisms of suppression. Studies were conducted to determine the mechanisms of antibody suppression of phytohemagglutinin (PHA)- and concanavalin A (Con A)-induced lymphocyte proliferation. Initial experiments incubated lymphocytes with either PHA or Con A, and at various times the respective anti-phytolectin was added to the cultures. DNA synthesis was less than 20% of the total response if anti-PHA was added within 2 hr and if anti-Con A was added within 10 hr of addition of mitogens to the culture. At the concentrations used, anti-PHA reduced PHA binding to the lymphocytes, while anti-Con A did not reduce Con A binding to the cells. Antibody was non-toxic and specific for its respective mitogen. Additional experiments incubated lymphocytes with either PHA or Con A and then the cells were washed and placed in mitogen-free media. The addition of antibody to cultures which had been washed markedly suppressed proliferation. Using radiolabeled PHA and Con A in one group of experiments and fluorescent labeled anti-mitogen antibodies in others, it was determined that antibody did not remove Con A or PHA from the cell, but instead antibody slowed the release of mitogen from the lymphocyte. Anti-mitogen antibody remained attached to mitogen on the surface of lymphocytes for at least 24 hr. The experiments suggest that antibody can suppress the lymphocyte reaction by blocking the necessary phytolectinlymphocyte interaction and interrupting stimulation by cell-bound mitogen.


Normal function of immunologic stem cells from aged mice. Marrow or spleen grafts from aged donor mice produced antibody-forming cells as effectively as did grafts from younger controls in recipients tested 3 to 10 months after the transplantation. All recipients were lethally irradiated, and the T6 chromosome marker was used to demonstrate that they were populated by donor cell lines. Recipients of aged or younger control grafts gave similar responses when stimulated with varying doses of antigen and when tested at different times after the transplantation except in two cases: 1) Recipients of aged spleen grafts gave significantly lower responses than younger controls for the first few weeks after the transplantation. 2) If recipients had been thymectomized before lethal irradiation, aged cell lines (pooled marrow and spleen cells) gave only 37% of the responses of younger controls. Given sufficient time and intact young recipients, immunologic stem cell lines from old donors populated recipients with cells having normal immune responses. These results suggest that age-related immunologic defects are not intrinisically timed in the precursor cell lines that populate the immune system.


Antibody production and interaction with lymphoid cells in relation to tumor immunity in the Moloney sarcoma virus system. The temporal development of antibodies to Moloney sarcoma virus (MSV)-induced antigens in relation to tumor progression was followed with the membrane immunofluorescence (MF) and antibody-dependent normal lymphocyte cytotoxicity (ADNLC) assays. Antibody was detected at 14 days following virus infection by MF. In mice that developed primary tumors which regressed, MF titers developed to high levels following a period between 2 and 4 weeks post-innoculation during which the titers remained at low and constant levels. The increase in MF titers corresponded closely on a temporal basis to the initiation of the regression process. In contrast, antibody remained at low levels in those mice that developed progressively growing tumors. ADNLC was  detected in the sera of regressor mice approximately 6 weeks post-inoculation but was not detectable at any of the time interavals in sera from mice with progressively growing tumors. Attempts to enhance the cytotoxic activity of normal spleen cells directly with immune serum were unsuccessful. The synergistic effect of serum on the cytotoxic activity of immune lymphocytes was less pronounced than with normal lymphocytes in this system. Temporal studies  indicated that there was an inverse relationship between the development of cellular immunity and the capablility of these cells to be activated in the ADNLC assay.


Antibody affinity. VIII. Measurement of affinity of anti-lactose antibody by fluorescence quenching with a DNP-containing ligand. The method of fluorescence quenching by bound ligand for the measurement of antibody affinity has been extended to anti-lactose antibodies. The basis for this extension is the use of a lactosyl ligand covalently linked to the 2,4-dinitrophenyl (DNP) group. This group serves as a sensor of the bound state by causing fluorescence quenching similar to that observed with anti-DNP antibody. The synthesis and characterization of this new ligand, N-(Nalpha-acetyl,Nepsilon-DNP-L-lysyl)-rho-aminophenyl-beta-lactoside, are described. The affinity of its interaction with three rabbit IgG and one equine IgM antilactose antibody preparations has been measured. The Qmax values range from 24 to 47% and the calculated association constants from 2.5 times 10-5 M-1 to 7.2 times 10-5 M-1. For one IgG preparation, measurement of the association constant by equilibrium dialysis with the tritiated ligand gave a value of 6.75 times 10-5 M-1 compared to the value of 7.2 times 10-5 M-1 obtained by fluorescence quenching. It is evident that with monoclonal anti-lactose antibody the accurate determination of Qmax, which is possible in this system, provides another structurally dependent clonal characterization of the variable region of the antibody. This optical probe along with others can be used for the identification of individual clones of antibody-producing cells through time and inheritance.


Interference of anti-allotype antisera with antigen-antibody binding. Rabbit anti-b4 antisera are capable of inhibiting the antigen-binding ability of antibodies carrying b4 allotype. The data support the hypothesis of the localization of at least one b group allotypic determinant in the V region.


Demonstration and assaying of IgG antibodies in tissues and on cells by labeled staphylococcal protein A. Staphylococcal protein  A (SpA) labeled with FITC was used to detect IgG antibodies to tissue and cell surface antigens. Radioiodinated SpA was employed in a radioimmuno technique to assay IgG antibodies which had reacted with cell membrane antigens. Red blood cells coated with SpA were used as an indicator system in the mixed haemadsorption technique to detect IgG antibodies on tissue culture cells. The results showed that SpA was highly specific in the various systems tested, although it was not as sensitive in the immunofluorescence test as FITC anti-IgG. It was confirmed that SpA reacts with IgG antibodies of human, monkey, rabbit, sheep and mice origin. The reactivity with human and monkey IgG was stronger than with IgG of the other species. We consider labeled SpA as an alternative to monospecific anti-IgG for detection and assaying of IgG antibodies to tissue and cell surface antigens.


Combined activity of amphotericin B and 5-fluorocytosine against Cryptococcus neoformans in vitro and in vivo in mice. The in vitro and in vivo activities of amphotericin B and 5-fluorocytosine (5-FC) alone and in combination were studied to determine possible drug interactions against two strains of Cryptococcus neoformans, one sensitive to 5-FC and one resistant to 5-FC. In vitro tube dilution studies demonstrated only additive effects with the 5-FC-sensitive organism but antagonism with eth 5-FC-resistant organism. A mouse model of cryptococcal meningitis allowed comparative drug trails in a new model for the detection of drug interactions. Drug combinations were no more effective against meningitis caused by the 5-FC-sensitive organism than the additive effects of the individual drugs. However, meningitis caused by the 5-FC-resistant Cryptococcus responded less to drug combinations than to either drug alone. Serum levels of amphotericin B and 5-FC were comparable in all groups. No evidence of toxicity from the drug combinations was found. No inhibition of development of resistance to 5-FC by the combination with amphotericin B was detected.


Use of erythrocytes sensitized with purified enterotoxin from Vibrio cholerae for the assay of antibody and antibody-forming cells. A method was developed for the sensitization of ovine erythrocytes with a purified enterotoxin from Vibrio cholerae. Sensitized cells were used for the titration of serum antibody by passive hemagglutination and in a hemolytic plaque assay for both IgM and IgG antibody-secreting cells. Inhibition experiments with various antigens of V. cholerae indicated that the toxin, whether unheated or heat-inactivated, significantly reduced the expected antitoxic plaque-forming cell response, whereas a lipopolysaccharide-rich extract from homologous vibiros was not inhibitory.


Microplaque reduction: new assay for neutralizing antibody to lymphocytic choriomeningitis virus. A plaque-reduction neutralization test for lymphocytic choriomeningitis virus was developed; microtiter dilution techniques and assay plates were used for the new test. Plaques develop by day 4 after inoculation of serum and lymphocytic chorio-meningitis virus into 5-mm cups containing agarose suspension of baby hamster kidney cells. The method lends itself well to the titration of neutralizing antibody and gives rapid results with great economy of reagents. When tested with one batch of 55 lymphocytic choriomeningitis virus-positive sera, the plaque reduction test was more senstive for detection of specific antibody than either the immunofluorescence or the complement fixation method.


Rheumatoid behavior in normal human synovial fibroblasts induced by extracts of Gram-negative bacteria. We have previously shown that exposure of normal human synovial cells to whole extracts of Gram-negative bacteria or purified endotoxins results in increased synthesis of hyaluronic acid, and increased glucose uptake and lactate output. Derivatives of Gram-negative bacteria, therefore, duplicate the major known actions of connective tissue activating peptide (CTAP). The studies presented here demonstrate that, in addition to the hypermetabolic changes, extract-treated synovial cells become hyposensitive to CTAP and acquire accelerated growth rates. Furthermore, the cellular alterations which occur following a short bacterial product-synovial fibroblast interaction period (72 hours) persist for varying lengths of time, in some cases for as long as the cells are followed in culture (up to 38 days). Additional data presented suggest that bacterial products induce CTAP formation or activation within synovial fibroblasts. Pre-incubation of bacterial endotoxin in fresh normal human serum does not alter its ability to activate synovial cells. Many of the cellular alterations induced by bacterial products and endotoxin (derivatives of infectious agents) resemble the abnormal metabolism of rheumatoid synovium in vivo and are characteristic of the behavior of rheumatoid connective tissue fibroblasts grown in vitro. Furthermore, the persistence of the bacterial extract-induced behavior resembles the semi-autonomy of the cultured rheumatoid synovial cell, at least for a limited period of observation.


Effects of castration, and testosterone in vitro, on the hypothalamic synthesis of different peptide fractions. The incorporation of [3H]tyrosine ([3H]tyr) into different hypothalamic peptide fractions isolated from normal and castrated rats on a Sephadex G-25 column has been studied in vitro. Luteinizing hormone releasing factor (LH-RF) activity was determined in the different fractions by measuring their ability to elicit release of radioimmunoassayable luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in ovariectomized rats treated with oestrogen and progesterone. For a further purification, the fraction with LH-RF activity was applied to a CM-Sephadex G-25 column eluted with a gradient of ammonium acetate. The large radioactive peptides emerged from the Sephadex G-25 column in fraction S-1, while the small peptides with LH-RF activity were eluted in fraction S-2. Gonadectomy significantly increased the incorporation of [3H]tyr into the peptides of fractions S-2. Only in the purified fraction with LH-RF activity was the radioactivity incorporated higher in gonadectomized than in normal rats. The enhanced incorporation in fraction CM-3 observed after castration implies an increase in the hypothalamic synthesis of peptides with LH-RF activity. The addition of testosterone (2 mug/ml) to the incubation medium of hypothalamus from gonadectomized rats, corrected these modifications. Gonadectomy decreased the incorporation of tyrosine into the large peptides, and incubation with testosterone corrected this change. The modifications in the incorporation of [3H]tyr into the large and small peptides produced by castration appear to indicate that gonadectomy, as well as stimulating the production of LH-RF, enhances the synthesis of other hypothalamic peptides while inhibiting the synthesis of proteins. On the other hand an increase in the breakdown of large peptides into small peptides cannot be excluded.


The temporal relationship between the secretion of luteinizing hormone and testosterone in man. It has been suggested recently that testosterone secretion by the human testis may be controlled by factors other than luteinizing hormone (LH). In order to re-examine this hypothesis, plasma LH and testosterone concentrations were determined throughout the day in eight studies. A new method of data analysis revealed that the levels of the two hormones were closely related, but that the testicular response to LH was sluggish. These results explain some inconsistencies in the literature. It was demonstrated that average values for LH varied throughout the day, with a morning maximum and an evening minimum. It was also shown that injections of LH releasing hormone in man resulted in an increase in plasma testosterone above control levels. These results are consistent with the concept that LH controls the major changes in testosterone secretion in men. They do not exclude, however, the possible existence of other factors which might affect the peripheral concentration of testosterone, such as changes in testicular blood flow.


Luteinizing hormone release in entire and castrated rams following injection of synthetic luteinizing hormone releasing hormone, and effect of testosterone propionate pre-treatment. Plasma luteinizing hormone (LH) levels and LH responses to intravenous administration of 100 mug luteinizing hormone releasing hormone (LH-RH) were studied in entire rams, long-term castrated animals (operation performed six months previously), long-term castrated animals treated with testosterone for the two prededing weeks and short-term castrated animals (castrated 3 h before LH-RH injection). LH was measured by radioimmunoassay in samples taken at 5 or 15 min intervals. Basal LH levels were lower in entire rams (0-9 ng/ml) than in long-term castrated animals (6-0 ng/nl). After LH-RH treatment the LH response was much smaller (peak level 9-6 ng/ml), total response 13-3 ng/ml/1 h) and slower (120 min to peak) in entire than in long-term castrated animals (peak level 61-8 ng/ml, total response 141-2 ng/ml/1 h, 29 min to peak). Testosterone treatment after long-term castration depressed the basal LH level and delayed the peak LH response after LH-RH to values similar to those for entire rams. After short-term castration the response to LH-RH was already as great (peak level 70-1 ng/ml, total response 133-6 ng/ml/1 h) as after long-term castration. The latency to peak LH level (82 min) was intermediate between that for untreated and testosterone-treated long-term castrated animals (130 min). Testosterone treatment was considered to have acted on the hypothalamus to depress basal levels. The results provided evidence for the presence of two inhibitory actions of the testis at the pituitary level in the ram: a qualitative delaying action of testosterone and a quantitative inhibitory action of the testis on LH release after LH-RH injection. The latter may also be related to plasma testosterone levels.


The secretion and metabolic clearance rates of oestradiol in the rat. The secretion rate (SR) of oestradiol in rats at different stages of the oestrous cycle was determined by measuring the concentration of this hormone in ovarian vein plasma by a competitive protein binding method utilizing rabbit uterine cytosol. The plasma metabolic clearance rate (MCRP) for oestradiol was determined in groups of rats of the same strain and housed under the same conditions by giving a single intravenous injection of tritiated oestradiol-17beta and measuring the rate of disappearance of labelled hormone from the plasma. From the relationship SR equals MCRP times plasma concentration, predicted values for the plasma oestradiol concentration were obtained and shown to be close to those obtained by direct analysis of peripheral plasma. The MCRP for oestradiol was not greatly influenced by the stage of the oestrous cycle, the dose of oestradiol-17beta injected, ovariectomy 18 h previously or by a sham-operation immediately before the injection of labelled hormone. The anaesthetic used did affect the MCRP ; the value for adult female rats injected under ether anaesthesia was about 970 ml/h but under Avertin anaesthesia clearance rates of about 1500 ml/h were obtained. Ligation of the bile duct acutely reduced the MCRP but functional nephrectomy had no immediate effect. Sub-total hepatectomy 18 h previously reduced the MCRP considerably. A small decrease in MCRP was observed when the labelled hormone was administered in third trimester human pregnancy plasma and the MCRP was lower in rats on Day 14 of pregnancy than in rats examined during the oestrous cycle.


Development of pathways of insulin secretion in the rabbit. Insulin release was studied in vitro using pieces of pancreas from rabbits of between 24 days gestational age and 6 weeks postnatal age. When allowance was made for the fraction of pancreas which was endocrine, 16-5mM-glucose caused increasing stimulation of insulin release as development advanced and 3-3 mM-glucose caused a similar rate of secretion at all ages. Secretion was not significantly influenced by insulin destruction in the incubation medium. Glucagon (5 mug/ml) did not stimulate insulin secretion from 24-day foetal pancreas but did so postnatally. Theophylline (1 mmol/1) stimulated insulin release at all ages and was equipotent on 24-day foetal pancreas in 3-3 or 16-5 mM-glucose. The stimulation of insulin release from 24-day foetal pancreas by 1 mM-theophylline occurred in the absence of extracellular glucose, pyruvate, fumarate and glutamate and in the presence of mannoheptulose and 2-deoxyglucose (each 3 mg/ml). Adrenaline (1 mumol/1) and diazoxide (250 mug/ml) abolished or attenuated the stimulation of insulin release by glucose, leucine plus arginine or theophylline from 24-day foetal, 1 day and 6 weeks postnatal pancreas. The stimulation of insulin release from 6-week-old pancreas by 1mM-barium was blocked by adrenaline and diazoxide but the effect became less with increasing immaturity. The experimental results illustrate some of the ways in which insulin secretion by the rabbit beta cell changes as a function of development and draw attention to the importance of glucose and cyclic adenosine monophosphate in this process.


Mass isolation and culture of rat kupffer cells. Collagenase perfusion of the liver followed by pronase treatment of the cell suspension thus obtained gave a quantitative recovery of viable nonparenchymal liver cells (NPC). From these NPC, Kupffer (K) cells can be purified by attachment to tissue culture dishes. Tail vein injection of carbon 1-2 h before liver perfusion permitted stepwise calculation as well as visualization of carbon-containing K cells. When these K cells have been put into tissue culture medium with serum and incubated overnight, they exhibit typical macrophage characteristics. Phase-contrast and transmission electron microscopy showed typical macrophage morphology and scanning electron microscopy revealed well-spread cells with cytoplasmic projections and ruffled membranes. Endocytosis studies using radioactive colloidal gold and inert latex particles also indicated that these cells are highly active in pinocytosis and phagocytosis. Further characterization of K cells is the identification of Fc receptor on their membranes. Studies on lysosomal enzymes showed that purified K cells possess higher specific activities in beta-glucuronidase, acid DNase, and cathepsin D than in purified parenchymal cells.


Cell surface immunoglobulin. XI. The appearance of an IgD-like molecule on murine lymphoid cells during ontogeny. An Ig molecule containing L chains and H chains similar to human delta-chains has been detected on the surface of radioiodinated murine lymphoid cells. Newborn mice have only IgM on their splenocytes. Between 10 and 15 days, the IgD-like molecule appears and increases in amount until 3 mo of age, when it is the predominant cell surface Ig in terms of radioactivity. IgD is found only in peripheral lymphoid tissues and is present in larger amounts on peripheral lymph node cells (approximately 85% of surface Ig) than on splenocytes (approximately 50%). IgD is also present in comparable amounts on cells from both nu/nu and germfree mice, indicating that its expression may be independent of both thymic influence and antigenic stimulation. These studies suggest that there is a switch from cell surface IgM to IgD that occurs during differentiation of virgin B lymphocytes in the spleen.


Cell interactions between histoincompatible T and B lymphocytes. VII. Cooperative responses between lymphocytes are controlled by genes in the I region of the H-2 complex. The results of this study provide compelling evidence for the existence of the gene or genes controlling optimal T-B-cell cooperative interactions in the designated I region of the H-2 gene complex. Previously, we have speculated that the relevant gene(s) involved may well be located in this region based on several observations from our earlier work in this area (3, 5, 6). Thus, in the preceding paper, we showed that T and B cells from B10.BR and A strain mice developed effective cooperative interactions in vitro to DNP-KLH in a system identical to the one reported herein. Since these mice differ for genes in the S and D regions of H-2 but are identical for K and I region genes, we were able to localize the critical genes to the K-end of H-2.


Guinea pig immune response-related histocompatibility antigens. Partial characterization and distribution. We have previously demonstrated that guinea pig alloantisera directed at strain 2 and strain 13 membrane antigens block specific lymphocyte activation in immune response gene-controlled systems. In this communication we describe the partial characterization of the antigens against which these antisera are directed (the 2 and 13 antigens) and, in addition, that of the B antigen which by distribution resembles the human HL-A and mouse H-2 major histocompatibility antigens. Lymphoid cells from strain 2 and strain 13 guinea pigs were surface labeled with 125I by the lactoperoxidase technique. Nonidet P-40 extracts of these labeled cells were precipitated by sandwiches of strain 2 antistrain 13, strain 13 antistrain 2, or outbred anti-B antisera, followed by rabbit antiguinea pig immunoglobulin antisera. Precipitates were dissolved in sodium dodecyl sulfate (SDS) and electrophoresed on SDS polyacrylamide gels. Radioactive peaks representing the 2 and B-cell membrane antigens were obtained from strain 2 lymph node cells, as well as from a B-lymphoid cell population (L2C leukemia cells) and a T-lymphocyte population (STRAIN 2 PERITONEAL EXUDATE LYMPHOCYTES [PELs]). Radioactive peaks representing the 13 and B-cell membrane antigens were obtained from strain 13 lymph node cells and strain 13 PELs. All anti-B precipitates produced two peaks when electrophoresed on SDS polyacrylamide gels; one representing an antigen with a mol wt of approximately 45,000, and one representing an antigen with a mol wt of about 12,000. Both may be components of a single protein. All anti-2 and anti-13 precipitates produced a single peak when electrophoresed on SDS polyacrylamide gels. Both the 2 and 13 antigens were found by this technique to have mol wt of approximately 25,000. By molecular weight criteria, as well as by previously investigated distributional criteria, the B antigen is similar to the human LA and Four antigens, and to the mouse D and K antigens, and the 2 and 13 antigens are similar to the mouse Ia antigens.


A stable form of delayed-type hypersensitivity. An antigen dose below the level needed to provoke an antibody response produces in mice a persistent, but minor degree of delayed-type hypersensitivity (dth) to sheep red blood cells. The DTH is unstable. It is erased by larger doses of antigen and cannot be built upon by further antigenic stimulation. The much higher levels of DTH resulting from immunization under the modulating influence of cyclophosphamide (CY) or BCG persist under strong secondary antigenic stimulation, though the former is subject to partial suppression unless CY is used to prevent the secondary humoral response. The DTH produced by a BCG-modulated primary response is not subject to this suppressive effect of a secondary antibody response. In this case the anamnestic T-cell response is very brisk and cannont be potentiated by giving CY at the time of the secondary antigenic stimulus. This effect is not due to the modulating influence of a residual BCG infection. It results from a permanent change induced during the primary response. The mediator cells formed under the influence of BCG are apparently resistant to inhibition by blocking serum containing immune complexes. Even the actively dividing T cells which are susceptible to vinblastine, and most readily blocked in the absence of BCG, are highly resistant to blocking by immune complexes. It is not clear whether these cells are intrinsically different or whether their insensitivity to blocking results from features peculiar to the humoral response that accompanies a BCG-modulated primary response. The mediator cells produced by both BCG- and CY-modulated responses become vinblastine resistant, relatively insensitive to humoral blocking factors, and capable of surviving in a functionally active form in syngeneic recipients with an apparent half-life of about 50 days. There were indications, however, that their effective life-span may be greatly extended in some circumstances by persisting antigenic stimulation; and in the case of BCG-modulated immunity the prevailing level of T-cell activity can be greatly augmented by a further antigenic stimulus without the necessity for renewed exposure to BCG.


Rosette-forming ability of thymus-derived lymphocytes in humoral and cell-mediated immunity. II. Helper cell activity. The technique of velocity sedimentation at unit gravity was used to determine the size and rosette-forming ability of helper cells in nonimmune and immune C57BL/6 mice. Helper T cells were assayed by the ability to cooperate with bone barrow (B) cells in the antibody response to sheep erythrocytes (SRBC) in vivo. 19S helper cells in nonimmune animals were nonrosette-forming small lymphocytes; after immunization with SRBC in complete Freund's adjuvant, 19S helper cells were nonrosette-forming medium lymphocytes. 7S helper cells in immune animals were small lymphocytes which did not form rosettes. No SRBC binding by helper cells was observed under the conditions of rosette formation used.


Role of C'3 and Fc receptors in B-lymphocyte activation. Attempts were made to identify the non-Ig lymphocyte receptor responsible for B-cell induction by antigen and polyclonal B-cell activators (PBA). As a first step, the role of C'3 and Fc receptors was analyzed. It was shown that complement could be fixed onto B cells to such an extent that the lymphocytes could not bind complement-coated red cells, but this did not result in induction of polyclonal antibody synthesis, nor did it inhibit the lymphocytes response to PBA. However, the C'3 receptros possessed a passive focussing role in the induction of polyclonal antibody responses. Thus, PBA that had fixed complement activated polyclonal responses at lower concentrations than the same substances that had not fixed complement. Most likely the dual binding of PBA molecules to B cells by the PBA and the C'3 receptors caused more PBA molecules to be bound to each cell. However, the focussing function of the C'3 receptors was several orders of magnitude smaller than that of the Ig receptors. Analogous studies were carried out with Fc receptors. Binding of different types of antigen-antibody complexes did not cause activation of polyclonal or specific antibody synthesis, nor did it significantly interfere with induction of antibody synthesis by PBA substances.


The Clifford Irving hoax of the Howard Hughes autobiography. The problem was to determine whether Howard R. Hughes, Edith Irving, or Clifford Irving wrote the questioned material Exhibits Q-1 through Q-35. The findings were as follows. 1. Howard R. Hughes was eliminated as the possible writer of the questioned handwriting on Exhibits Q-1 through Q-35. Although there were pictorial resemblances between Hughes' handwriting and the questioned material enumerated above, significant characteristic differences were also present which unequivocally eliminated him as the writer. 2. Edith Irving was identified as the writer of the "Edith Sommer for deposit only" endorsement on Exhibit Q-11. 3. Edith Irving also very probably wrote the "Hanne Rosenkrantz" on Exhibit Q-27 through Q-34, but the questioned writings were restricted to the examination of poor quality photocopies which did not provide proper basis for the handwriting comparison. 4. There were indications that Edith Irving also may have written the questioned endorsement "H. R. Hughes" on Exhibit Q-3 and the questioned signatures "H. R. Hughes" on Exhibits Q-4, Q-5, Q-6, Q-7, and Q-35, by simulating the handwriting of Howard R. Hughes on those questioned exhibits. She could not be identified, however, based on the available handwriting specimens obtained from the federal grand jury on 8 Feb. 1972. 5. Clifford Irving was identified as the writer of the questioned material on Exhibits Q-1, Q-2, Q-8, Q-9, Q-12a and Q-13a and Q-13b, Q-14, Q-15a through Q-15c, Q-16a and Q-16b, Q-22a through Q-22i, Q-23, Q-24, Q-25, and Q-26. A study of the questioned handwriting with the known handwriting of Howard R. Hughes revealed a definite attempt on the part of Irving to simulate the handwriting of Hughes, having seen Hughes' writing in earlier issues of Time-Life.


Lithium in the treatment of aggression.

Lithium in the treatment of aggression. Lithium has become a widely accepted treatment for manic-depressive psychosis. It is dramatically effective for many cases of mania and is useful in the prevention of manic and depressive episodes. Hyperaggressiveness and hypersexuality are frequent components of manic-depressive illness and abate under the influence of lithium. A brief review is presented of the behavioral and biochemical pharmacology of lithium. This documents the inhibitory role which lithium can play in several examples of animal aggressive behavior including pain-elicited aggression, mouse killing in rats, isolation-induced aggression in mice, p-chlorophenylalanine-induced aggression in rats, and hypothalamically induced aggression in cats. The use of lithium to control human aggressive behavior has resulted in controversial findings. In epileptic conditions, improvement has been reported in interseizure aggressivity, but other reports indicate the possibility of increased seizures. Improvement in aggressive behavior in childhood has occasionally been reported as well as in emotionally unstable character disorders in young female patients. Te was a single blind study and the other a large but uncontrolled study. Both studies reported an improvement in aggressiveness as indicated by fewer recorded reports (tickets) for fighting. The final study reported is a study of 12 male delinquents age 16 to 23. They received lithium or placebo for 4 months inside an institution and then a trial of lithium for 1 to 12 months on an outpatient basis. Analysis of results in terms of the number of aggressive antisocial acts showed fewer serious aggressive episodes when the lithium level was between 0.6 and 1 meq/liter than when it was between 0.0 and 0.6 meq/liter. These results must be viewed with caution and are only suggestive since the study was not double blind.


Computerized EEG: predictor of outcome in schizophrenia. Based on a double blind cross-over study, it was determined that schizophrenic patient who have more high frequency fast activity and a lesser degree of alpha and slow waves in computerized EEG before the treatment have a better therapeutic outcome to the major tranquilizer (neuroleptic) treatment. The correlation between pretreatment high frequency computer EEG measurements and better therapeutic outcome reached the level of statistical significance. "Therapy resistant" schizophrenic patients were characterized by a lesser degree of very fast beta activity, more alpha waves and slow waves, higher amplitudes in computer EEG, and a lesser degree of acute (florid) psychotic symptomatology but more "negative" symptoms such as motor retardation and blunted affect. One of the most striking results of the study is the finding that schizophrenic patients with certain psychopathological profiles also have similar computer EEG profiles.


Brain function and behavior. I. Emotion and sensory phenomena in psychotic patients and in experimental animals. For the past 25 years, the research program of the Tulane University Department of Psychiatry and Neurology has been directed primarily to the development of treatment for patients with certain psychiatric and neurological disorders that have been resistant to commonly used therapy. In the course of investigations, using a variety of approaches, new techniques have evolved which have permitted simultaneous exploration of brain activity and behavior. The data reported substantiate an anatomical localization in the brain for the syndrome of psychotic behavior. Further, observations in patients, coupled with animal investigations, have led to the demonstration of brain pathways and previously undisclosed anatomical connections which provide a physical substrate for the clinically observed relation between perception and emotionality. These findings provide a basis for the development of specific biological methods for the treatment of behavioral disorders.


Sexual functioning in patients with chronic renal failure. There is little information on the sexual activity of patients with end-stage renal failure in spite of its being mentioned as a common side effect of kidney disease and chronic hemodialysis. In this report 32 married, male dialysis patients, 19 of whom had also received renal homografts, were interviewed and a detailed sexual history obtained. Twenty per cent of this population had no decrease in sexual functioning after the onset of kidney disease or the institution of dialysis, 45 per cent had reduced sexual potency after the onset of kidney disease, and another 35 per cent after beginning dialysis. Forty per cent of those patients receiving a kidney transplant had a subsequent increase in sexual potency.


Fat embolism: a comprehensive review.

Fat embolism: a comprehensive review. Fat embolism as a cause or as a contributing factor to the death of injured patients is probably not diagnosed in many instances. Treatment is beneficial in moderate cases of fat at embolism; severe cases result in death and mild cases usually resolve without treatment. Awarness of the symptoms is important. Treatment should be directed to the symptoms rather than to the disease. Respiratory support seems to be the most effective treatment.


Development of infectivity by the Plasmodium berghei sporozoite. Studies were done on the development of infectivity during ontogeny of the sporozoite of the rodent malaria parasite, Plasmodium berghei. Populations of sporozoites were separated from the oocysts, the hemocoel, and the salivary glands, with special precautions being taken to avoid cross-contamination between the different populations. The results indicated that populations of salivary gland sporozoites were more than 10,000 times as infective as populations of oocyst sporozoites from the same mosquitoes. The development of this infectivity appears to be asynchronous, in some cases taking place in the hemocoel, while in other cases not occurring until after the sporozoites have invaded the salivary glands. Thus, the development of infectivity seems to be time-dependent rather than site-dependent. There is also a continued increase in sporozoite infectivity during their residence in the salivary gland. The development of infectivity may be associated with other aspects of sporozoite maturation, including changes in their antigenicity and motility.


A controlled trial of intraperitoneal cephaloridine administration in peritonitis. From a controlled therapeutic trial extending for more than 2 yr and involving 69 patients with appendicitis and peritonitis a clear-cut statistically significant result emerged. There was a major reduction in the incidence of intraperitoneal abscesses using cephaloridine by the intraperitoneal as opposed to the systemic route. After randomized selection into treatment and control groups, cephaloridine, 25 mg/kg was given by injection every 6 hr for 48 hr to the treatment group by i.p. installation and to the control group by systemic injection. Both groups received initial intraoperative peritoneal lavage with normal saline and also continued systemic injections of cephaloridine on postoperative days 3, 4, and 5. Only one out of 36 patients in the treatment group developed a residual intraperitoneal abscess, as opposed to six abscesses developing in 33 patients in the control group. Technical problems and complications of the method were trivial and have not prevented us from continuing and extending the applications of the method.


Hemodialysis and renal transplantation in childhood: description of a program. Over a 2-yr period eight kidneys have been transplanted in seven patients at the University Children's Hospital of Berne. Six of these patients are alive 4-24 mo after transplantation. The results seem encouraging and the procedure justified, especially in view of the chance of full rehabilitation, which is obviously less complete with chronic hemodialysis.


The effects of two ultrasonic instruments on root surfaces. The Orbison ultrasonic instrument and the Cavitron ultrasonic dental unit were compared for the smoothness of the root surfaces they produced. Twenty teeth were analyzed with the scanning electron microscope and classified as to rough or smooth. Both instruments produced similar degrees of smoothness on the root surfaces with minimal scarring. Application of these instruments to the root surface apical to the epithelial attachment produced residual smearing of some of the collagenous fibrils.


The subcutaneous implantation of xenogeneic decalcified teeth. Decalcified matrices obtained from bone and dentin have been used in xenogeneic implantation experiments. It is observed that such implants generally do not induce new bone but in many cases result in recalcification of old matrices. Treatment of the matrices with the enzyme inhibitor, iodoacetic acid, prior to implantation also did not enhance or improve the osteoinductive potentiality of these matrices. It is therefore suggested that the bone morphogenetic protein (BMP) to which the osteoinductive potentiality has been attributed by Urist et al. is a highly species-specific moiety and that the xenogeneic implant is hence a poor choice for its determination.


A diagnostic wax-up technique.

A diagnostic wax-up technique. A procedural outline has been presented for the accomplishment of a diagnostic wax-up as a preliminary step to actual preparation of the teeth as suggested by the proponents of gnathologic techniques. 8.9) The use of this technique will decrease the possibility of error in the construction of any extensive fixed partial denture prosthesis. The success of any procedure required the willingness of general dentists and specialists to, 'Spend more time planning than doing.'


Factors affecting airborne beryllium concentrations in dental spaces. Air sampling for beryllium concentrations produced during finishing procedures for a beryllium-containing alloy was conducted in two rooms with capacities of 700 and  10,000 cubic feet. The clearance rate of beryllium in the air and the effect of ventilation and room size on these  concentrations were investigated. With local lathe ventilation, no beryllium was found. Without local lathe ventilation, mean  10 minutes concentrations of about 23 mug per cubic meter were found at the breathing zone of the lathe operator in both rooms. At 4 and 8 feet from the breathing zone, sizable concentrations of beryllium above the maximum acceptable standard were found only in the small room. These levels decreased to zero 10 minutes after completion of the finishing and polishing procedure. It was concluded that there was little hazard to dental personnel when local lathe ventilation was used; however, our finding of high concentrations of beryllium in the air when lathe ventilation was not used indicates that continued vigilance must be maintained.


Modified hygroscopic gold casting technique. A  modified hygrosopic gold casting technique has been described. It utilizes a dual-wax pattern with controlled amounts of water added to the setting investment to better influence both the location and amount of investment expansion. This technique has been used since 1970 with excellent clinical results.


An impression technique for cast restorations. A technique has been described for making impressions for cast restorations. It can be used for a single preparation or for multiple preparations in a single arch. The most unique feature of the teachnique is that the tray is formed over the prepared teeth, either directly or indirectly. The impression material is confined to a relatively small area and is forced by positive pressure into the gingival sulci around the prepared teeth. The result is an impression that is dense and void free and which records marginal and coronal surfaces with accurate detail. A dentist who is trained and experienced in the use of tray techniques can easily make the transition to this method.


A macromolecule-free partially defined medium for Trypanosoma cruzi. A macromolecule-free medium, containing in its defined part 3 salts, glucose, hemin, 21 amino acids, 3 lipids, and some undefined components obtained by dialysis of liver infusion, was developed for serial cultivation of Trypanosoma cruzi at 28 C.  The medium allows prolonged cultivation of T. cruzi by serial transfers and growth comparable to that obtained in more complex media, including those containing blood serum.


Three centuries of protozoology: a brief tribute to its founding father, A. van Leeuwenhoek of Delft. It was exactly 300 years ago this month (August 1974) that the 17th century modest draper from Delft, Holland--Antony van Leeuwenhoek--discovered protozoa.  Describing them, often with amazing accuracy considering the optical equipment he was using (simply a home-made "glorified" hand lens), in letters to the Royal Society of London, he established himself, certainly, as the founding father of protozoology.  It is particularly appropriate for an assemblage of protozoologists to pay homage to this intrepid "philosopher in little things," a man with an insatiable curiosity about his wee animalcules, on the tricentenary of his discovery of them, since it was an event of such long-lasting significance.


Antibodies and sperm survival in the female tract of the mouse and rabbit. Rabbit and mouse spermatozoa from male and female tracts have been examined for their species-antigenic surface character, and for adherent antibodies, by double immunofluorescence techniques. Mouse spermatozoa from the ductus deferens showed an area over the acrosome which was positive to anti-mouse serum that had been absorbed with some male mouse somatic tissues including blood, but those from the uterus and oviduct were not stained. Spermatozoa from the uterus were shown to have an antibody coat on the acrosome, with anti-mouse IgG, but those from the ductus deferens and oviduct did not. Rabbit spermatozoa were more variable but their activity was similar: ejaculated spermatozoa sometimes already had antibody of male origin; the majority of the spermatozoa arriving early in the uterus were coated, but in general those that attained the oviducts were not coated. The results are interpreted as evidence for selection by the female tract of a small antigenically different population; the majority of spermatozoa are rejected and/or destroyed.


Cervicectomy with uterovaginal anastomosis in the rhesus monkey: a new surgical model for reproductive studies. Cervicectomy with uterovaginal anastomosis in the rhesus monkey allows easy and non-traumatic access to the uterine cavity through the vagina for such purposes as biopsy, flushing, aspiration of fluid, and placement of IUDs. Endometrial and tissue samples cannot be contaminated by cervical mucus. The surgical procedure and results on eleven animals are described. Neither uterine nor ovarian function appeared to be significantly altered. The surgical model seems well suited to long-term studies on IUDs.


Progesterone: sites of action in inhibiting ovulation in hamsters. A single subcutaneous injection of progesterone (0.5 mg/animal at 17.00 hours) on Day 3 of the cycle (Day 1 = day of vaginal discharge) interfered with ovulation and external vaginal discharge in all the animals. Lower doses were only partly active. Intravenous administration of LH, FSH or their combination at 14.00 hours on Day 4 failed to restore ovulation in such animals, implying a direct action of the administered progesterone on the ovary. A large dose of oestradiol benzoate (400 mug/animal) also proved inactive suggesting that progesterone did not interfere with ovulation solely through antioestrogenic action. Depletion of the pituitary LH stores normally found in association with ovulation and ovulatory peak of LH in plasma seen in control animals were not observed in the progesterone-treated hamsters suggesting a further action of progesterone at the pituitary-hypothalamic level in inhibiting ovulation. Administration of synthetic LRF (100 ng/animal, i.v.) on Day 4 to hamsters treated with progesterone caused an increase in plasma LH 15 min later which was comparable to the rise observed in control animals given a similar injection of LH-RF, implying lack of action of progesterone at the pituitary level. It is concluded that progesterone acts centrally, principally on the hypothalamus, and peripherally on the ovary to interfere with ovulation in hamsters. It is possible that the anovulatory state accompanying pregnancy results from these actions.


The burn wound from the surgical point of view. The surgical approach cannot, and should not, be different for a mechanical wound and for a burn wound. Thin autografts applied over an excised burn wound act as the best antibiotic and the best analgetic, and at the same time provide a definite cover. The removal of thin skin grafts does not represent any additional danger to the burned patient. Homografts applied on excised dermal surfaces act as the best protection for potentially damaged layers of dermis, assuring spontaneous epithelization after their rejection. The clinical results obtained by primary necrectomies in extensive deep burns justify the conclusion that more of our effects should be concentrated towards an earlier and more adequate treatment in the early postburn phase. I am sure that the only chance of survival of critically burned patients depends on efficient action. Like Evans, I do believe in the value of whole blood transfusion in badly burned patients. We have to try to save their lives by changing their blood, as we do without hesitation in cases of badly matched blood transfusions.


Purification and properties of a bacteriophage T5-modified form of Escherichia coli RNA polymerase. A modified form of Escherichia coli RNA polymerase that contains one of the three T5-specific polypeptides known to interact with the host enzyme was purified from bacteriophage T5-infected cells. The properties of this T5-modified enzyme appeared identical to those of the RNA polymerase derived from uninfected non-colicinogenic cells and to a fully active enzyme isolated from T5-infected ColIb+ cells after the limited in vivo transcription of T5 genes allowed by the plasmid had ceased.


Occurrence of the bacteriophage lambda receptor in some enterobacteriaceae. In Escherichia coli K-12, the receptor for phage lambda is an outer membrane protein which inactivates the phage in vitro. Lambda receptor activity was found in extracts from all wild strains of E. coli tested, although most of them fail to support growth of the phage. In some cases this failure is due to a masking of the receptor in vivo, the bacteria being unable to adsorb the phage or to react with antireceptor antibodies. In other cases, adsorption does occur, and the nature of the block in phage growth was not investigated. Most Mal+ strains of Shigella have lambda receptor, whereas most Mal- strains do not have it. Synthesis of the lambda receptor in Shigella is thus presumably controlled by the positive regulator gene of the maltose regulon as is the case in E. coli K-12. Phage lambda adsorbs on many Mal+ strains of Shigella and even yields plaques on some of them, although at a low frequency. No lambda receptor activity could be found in extracts of several strains of Salmonella and Levinea.


Growth studies of three phi chi 174 mutants in tsDNA mutants Escherichia coli. Three mutants of phi chi 174 were examined for their abilities to grow in temperature-sensitive dna,A, dnaC, dnaE, or dnaG mutants of Escherichia coli. The results indicate that the phage mutants have acquired the ability to grow in some tsDNA mutants that normally block the replication of wild-type phi chi 174. Evidence is presented indicating that the phage mutants contain one or more altered structural proteins. Several models are presented to explain how altered phage structural proteins could affect phi chi 174 replication.


Bacteriophage-host interaction and restriction of nonglucosylated T6. Nonglucosylated T6 phage (T6gtam 16am30, hereafter called T6alpha gt-) were found to have two structural anomalies when compared with wild-type T6. The DNA of T6alpha gt- phage contains single-strand interruptions. These can be seen both during infection, in the pool of replicating DNA, and in DNA extracted from purified phage. In addition, the sodium dodecyl sulfate-polyacrylamide gel pattern of T6alpha gt- phage structural proteins reveals a protein band not found in T6. The altered protein has a mobility slightly faster than that of the major head protein, and it is not removed by osmotic shock. The restriction activity of Escherichia coli B directed against T6alpha gt- phage is abolished by preinfection of the cells for 4 min with T4 im m2. The shut-off of restriction is observed either by the rescue of superinfecting T6alpha gt- or by the failure to detect degradation of incoming T6alpha gt- DNA. This effect is resistant to rifampin and chloramphenicol.


Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase. Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.


Inhibition of group B arbovirus antigen production and replication in cells enucleated with cytochalasin B. A comparative study of the growth of Sindbis (SIN) virus, a group A arbovirus (togavirus), and Japanese encephalitis (JE) virus, a representative group B arbovirus (togavirus), was conducted in enucleate and nucleate cells. Immunofluorescent tests and yield measurements demonstrated that chicken embryo cells which had been enucleated and subsequently infected with SIN virus produced virus-specific antigens and infectious virus. By contrast, JE failed to replicate or produce virus-specific antigen in cells which had been enucleated before or even 2 h post infection. Studies of the effect of enulceation at various times after infection demonstrated that a nucleus must be present at least 2 and possibly as long as 4 h after infection to produce either JE-specific antigen or infectious JE virus. These studies demonstrate that the replication of SIN, a group A arbovirus (togavirus), which has no nuclear requirement, contrasts sharply with that of a group B arbovirus (togavirus), JE, which may have an initial dependence on a nucleus-associated process.


Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants. New mutants of bacteriophage T4 that overproduce the enzyme dihydrofolate reductase were investigated. Unlike previously described overproducers of this enzyme (Johnson and Hall, 1974), these mutants did not overproduce deoxycytidylate deaminase. Overproduction of dihydrofolate reductase by the new mutants occurred because enzymatic activity continued to increase for a longer period of time in cells infected by the mutants than in cells infected by wild-type phage. This continued increase occurred even in the presence of rifampin, indicating that the overproduction is probably due to a post-transcriptional event. Both these new overproducers and the previously  described overproducers were studied by using polyacrylamide gel electrophoresis. The two types of overproducers appeared to be very different. The previously described overproducers showed a delay and/or reduction in the synthesis of several proteins that normally started to be made 4 to 6 min after infection. Several proteins could be seen to be overproduced on the gels. The new overproducers did not show the delay in the synthesis of some proteins and only overproduced a few proteins. The new gene defined by the new overproducers is between the gene coding for thymidine kinase and the gene coding for lysozyme.


Escherichia coli capsule bacteriophages. III. Fragments of bacteriophage 29. A glycanase activity, catalyzing the depolymerization of host capsular polysaccharide, is associated with Escherichia coli capsule bacteriophage no. 29, a small virus with an isometric head, carrying a base plate with a set of spikes. The bacteriophage particles were disrupted by mild acid treatment (5 to 8 min at pH 3.5 and 37 C), and the enzymatically active fragments were isolated and subjected to sodium dodecyl sulfate-gel electrophoresis as well as to electron microscopy. Of the at least nine different polypeptide chains found in the complete virion, three (of 57,000 plus or minus 3,000, 29,500 plus or minus 2,000 and 13,500 plus or minus 1,000 daltons) were detected in detached base plates. They had the appearance of six-pointed stars of about 14 nm in outer diameter, with a central hole or prop, carrying six (or, possibly, a multiple thereof) spikes. Two sizes of polypeptide chains (57,000 and 29,500) were found in pure spikes, cylindrical particles of about 14.5 to 15 nm in length and 5 nm in diameter, and one (57,000) in -- still capsule depolymerizing -- spike subunits of roughly 5 nm in diameter. Phage 29 spike preparations, homogeneous in analytical ultracentrifugation and immunoelectrophoresis, were found to have a molecular weight of 245,000, as determined from the sedimentation equilibrium, and to contain equimolar amounts of the two polypeptides, probably three copies of each per organelle. The amino acid analysis of the isolated spikes revealed that aspartic acid, alanine, serine, and glycine are their dominant constituents; no amino sugars or other carbohydrates were detected in the preparations.


Escherichia coli capsule bacteriophages. IV. Free capsule depolymerase 29. The free host capsule depolymerase, induced by Escherichia coli capsule bacteriophage no. 29, and causing the formation of haloes around its plaques, has been purified to homogeneity. As judged from the following facts, this "enzyme" consists of free phage 29 spikes. (i) Detached phage organelles and depolymerase 29 particles exhibit the same molecular weight (about 245,000, as determined from the sedimentation equilibrium), contain polypeptide chains of the same two sizes (57,000 plus or minus 3,000 and 29,500 plus or minus 2,000, as determined by SDS-PAA gel electrophoresis), and have (within experimental error) the same sedimentation coefficient, isoelectric point, and amino acid composition. (ii) Isolated depolymerase and phage spikes in situ both catalyze the hydrolysis of glucosidic bonds in host capsular polysaccharide, leading ultimately to the formation of oligosaccharide fragments of one, two, and three hexasaccharide repeating units. (iii) Depolymerase 29 and phage 29 spikes have roughly the same electron optical dimensions. As tentatively estimated from the total and the virus-associated capsule depolymerase activity in the lysates, phage 29 infection seems to produce eight to seventeen times more free than incorporated spikes.


Escherichia coli capsule bacteriophages. V. Lysozyme 29. In addition to the spike-associated host capsule depolymerase, infection by Escherichia coli capsule bacteriophage no. 29 also induces the synthesis of a large bacteriolytic enzyme which has been purified to homogeneity. On incubation of isolated host murein sacculi with this enzyme, no amino groups but reducing sugar groups were liberated, and muraminitol, but no glucosaminitol, was found in the degraded sacculi after subsequent reduction with NaBH4. The bacteriolytic enzyme is thus another lysozyme (mucopeptide N-acetylmuramylhydrolase; EC 3.2.1.17). Electron optical visualization of negatively stained lysozyme specimens showed oblong particles of roughly 4.5 to 5.5 nm in diameter and 15 to 19 nm in length. Although the material tended to dissociate, a crude estimate of its molecular weight (270,000 plus or minus 30,000) could be obtained from these dimensions, from its sedimentation equilibrium, and from its behavior in gel chromatography. After disintegration of homogeneous lysozyme 29 by heating in solution with sodium dodecyl sulfate and dithiothreitol, polypeptides of one size only (about 46,000 dalton, probably six copies per molecule) were found in sodium dodecyl sulfate-polyacrylamide electrophoresis. The amino acid analysis of the enzyme accounted for more than 90% of its dry weight. One percent or less of the bacteriolytic activity in phage 29 lysates was found to be associated with the intact or disrupted virus particles, and a polypeptide of 46,000 daltons was not detected in the virions. These results strongly suggest that, in contrast to the host capsule depolymerase also induced by the same phage, and in spite of its comparatively large size, "lysozyme 29" does not constitute an integral part also of the homologous bacteriophage particles.


Extravesical ureteroneocystostomy in 170 renal transplant patients. Urinary tract reconstitution was done in 170 consecutive renal transplant patients, using an extravesical ureteronecystostomy. The urologic complication rate was 8.5 per cent but complications associated directly with the anastomosis occurred in only 5 per cent of the cases. There have been no anastomotic complications in the last 104 transplants. Vesicoureteral reflux occurred in 20 per cent of 50 patients surveyed. Death in 2 cases and loss of the kidney in 1 were associated with urologic complications.


Transverse colon conduit: a preferred method of urinary diversion for radiation-treated pelvic malignancies. In the management of pelvic malignancies treated by radiation the standard ileal conduit is subject to many hazards related to the use of damaged tissues. The transverse colon affords the use of a short isolated segment of non-irradiated bowel as a urinary conduit. The ureters can be dissected well above the field of pelvic irradiation. Eight patients with bladder or cervical carcinoma treated with high doses of external radiotherapy are presented to demonstrate the usefulness of the transverse colon in supravesical urinary diversions.


The ileocecal bladder: a new method for urinary diversion after radical cystectomy (a preliminary report). Ten patients underwent radical cystectomy and ileocecal bladder reconstruction for carcinoma of the bladder. The ileocecal region was used as an artificial bladder, its valve protecting the kidneys from back pressure exerted by the external urethral sphincter. The ureters were anastomosed to the ileal part and the cecum was anastomosed to the prostatic urethra in 7 men and to the bladder neck in 2 women and 1 man. Two patients died postoperatively. All patients had perfect control of micturition during the day. Those in whom the bladder neck was preserved were also fully continent at night. The remaining patients had a minor degree of nocturnal enuresis with only a few drops of urine voided involuntarily during sleep. All patients feel the desire to void when the bladder is full. Voiding cystograms in all patients did not reveal reflux in the ureters. IVP's done up to 11 months postoperatively showed preservation and improvement of kidney function and configuration. Followup cystometric studies and serum electrolyte determinations are under investigation.


Ileocecal conduit for temporary and permanent urinary diversion. The ileocecal intestinal segment has been used as a diverting conduit with a satisfactory colonic stoma in 6 patients with potentially reversible bladder disorders. Followup has been from 1 to 5 years. The ileocecal valve has been modified successfully by a fundoplication procedure similar to the Nissen esophagogastric junction operation to prevent ileocecal and ureteral reflux. IVP and renal function studies revealed resolution of pre-existing hydronephrosis and preservation of previously normal upper urinary tracts. One patient has undergone reversal of the diversion by cecocystoplasty and simultaneous bladder augmentation, and has been followed for 5 years with sterile urine and normal IVPs. The anatomic and functional advantages of a conduit with an antireflux mechanism that is applicable to the hydronephrotic collecting system are discussed.