Observations on the ultrastructure of the choanoflagellate Codosiga botrytis (Ehr.) Saville-Kent with special reference to the flagellar apparatus. The ultrastructure of the choanoflagellate Codosiga botrytis is described with particular reference to the flagellar appendages, the flagellar rootlet system, the transition zone, the basal body and accessory centrioles, and the stalk. The controversial early reports of flagellar appendages in this species have been confirmed and they have been detected in 2 further species, Salpingoeca frequentissima and Monosiga sp. The appendages consist of a delicate bilateral vane 2 mum wide on either side of the axis, composed of extremely fine overlapping or interwoven fibrils. The flagellar root system consists of a large number of radiating microtubules associated with bands of electron-dense material near the basal body; striated roots are absent. The microtubules extend from several separate foci, those in any one group originating near a composite electron-dense band, and for a distance of 300 nm from the basal body they are separated by blocks of interstitial material. The flagellar basal body forms one of a diplosome pair of centrioles. The triplet microtubules of the accessory centriole are embedded in amorphous electron-dense material and the whole is enveloped in a sheath of similar appearance. The existence of a third centriole close to the diplosome pair is also reported. The relatively complex structure of the flagellar transitional zone is described. The stalk is composed of a core of circular lacunae, which may or may not contain finger-like protoplasmic extensions of the posterior end of the cell, surrounded by a continuation of the sheath material which encloses the remainder of the protoplast. In the stalk only there is a further closely sheathing layer about 15 nm thick which is regularly striated, the spacing of the striations in shadowcast material and sections being about 3 times that measured by negative staining. The structure of choanoflagellates differs widely from that of the algal class Chrysophyceae, the group in which they are included in some classifications, and from the remainder of the algae; they do not appear to have a place in either the algae or the plant kingdom. The structure of Codosiga botrytis is briefly compared with that of sponge choanocytes and collared cells in the Metazoa and some of the possible phylogenetic implications of this are indicated.


Hygiene in the prevention of udder infections. I. Comparative efficacy of four teat dips. Three trials of teat dips were conducted with experimental exposure to mastitis pathogens. A control group of animals and a group treated with a hypochlorite preparation were included in all trials while a third group was dipped with a different iodophor preparation in each trial. Four teat dips were highly effective in reducing the incidence of udder infection. Some teat irritation was transitory during each trial; most occurred in the hypochlorite group.


Hygiene in the prevention of udder infections. II. Evaluation of oil-based teat dips. Oil-based teat dips containing iodine at .5 and 1% were compared in a series of studies with two water-based iodophor products of the same concentration and also with undipped controls. When the two types of products were tested at .5% under conditions of natural infection, the water-based dip was four times more effective than the oil-based dip at preventing Staphylococcus aureus infections. In three subsequent trials teats were experimentally exposed to S. aureus and Streptococcus agalactiae. Five times more S. aureus infections developed in a group dipped with the oil-based dip at 1% iodine with an undipped control yielded no significant difference for S. aureus infections. The 1% iodine water-based product was nine times more effective in preventing S. aureus infections than the 1% oil-based product. Differences in the incidence of S. agalactiae infections were nonsignificant within the individual trials. Though ineffective in preventing udder infections oil-based products were effective in creating a supple condition of teat skin.


Hygiene in the prevention of udder infections. III. Effectiveness of 59 teat dips for reducing bacterial populations on teat skin. Fifty-nine teat dips were tested for effectiveness in reducing populations of experimentally applied Streptococcus agalactiae and Staphylococcus aureus on teat skin. The results varied widely among products. Populations of S. agalactiae recovered from dipped teats ranged from a 98.3% reduction to a 147.4% increase as compared to undipped controls. Reductions in recoverable S. aureus ranged from 98.5 to 43.6%. The products yielding the highest germicidal activity did not conform to a consistent pattern in type and concentration of active ingredients, presence or lack of specific emollients, or pH. Approximately 20% of the products yielded a low activity. Each of six oil-based products performed poorly and frequently increased numbers of organisms.


Estrus detection and estrus detection aids. There is no substitute for good management. Each animal should be identified carefully. The herds man should know all signs of estrus or impending estrus. In addition, for visual detection of estrus to be highly effective, sufficient time must be taken at least twice a day to catch animals with a short estrus period. Presently there are two visual aids to estrus detection which provide potential 24-h surveillance. One aid is a pressure sensitive device mounted on the back of each cow which can be triggered when the cow stands for mounting. The second one is a marking device worn by sexually aggressive animals which will stripe with colored ink the back of estrous animals as the marker animal mounts and dismounts. Both devices are effective when used properly. Other tests of changes in cervical mucus, vaginal characteristics, temperature, blood flow, and hormone changes in blood and milk are either not suffciently reliable or else simple enough yet to be practical aids for routine detection of estrus in dairy cattle. Milk progesterone can assist in characterizing problem cows.


Rehabilitation of the atrophic edentulous maxilla by bone grafting. Transoral bone grafting is a useful method for the reconstruction of the atrophic maxillary residual ridge in patients who cannot retain complete dentures. The indications, criteria for patient selection, and the technique used have been presented.


Further studies on gold alloys used in fabrication of porcelain-fused-to-metal restorations. Composition, microstructure, castability, mechanical properties, and heat treatment characteristics of two gold-palladium-silver-based alloys were studied. The materials exhibited compositional as well as microstructural differences. Clinically acceptable castings could not be obtained when manufacturers' recommended casting temperatures were used. Ultimate tensile strength, yield strength, modulus of elasticity, and Brinell hardness values for the alloys were comparable. The elastic limit of Cameo, however, was significantly higher than that of vivo-star. Maximum rehardening of annealed castings occurred on reheat treatment at temperatures between 1,200 and 1,300 F. As-cast specimens, however, were not heat hardenable. The sequence of heat treatments used in the application of porcelain reduced slightly the hardness of both alloys. Hardness of the metal substructures was not increased by return of porcelain-coated specimens to a 1,250 F oven for final heat treatment.


Humoral and cellular immunity in asthma. Parameters of humoral and cellular immunity have been measured in 91 asthmatic patients. Mean serum levels of IgG and IgE were raised. IgG levels were higher in those with a family history of asthma. IgE levels were higher in those with a past history of atopic eczema, but intrinsic and extrinsic asthma could not be differentiated on the basis of IgE levels. Thirteen of 74 patients failed to respond to tetanus immunization, while only 1 failed to respond to Salmonella typhi H antigen. Tetanus nonresponders had a raised mean serum IgA level, reduced spontaneous lymphocyte tritiated thymidine uptake, and reduced thymidine uptake in fetal calf serum. Eight of 87 patients failed to mount delayed hypersensitivity reactions to a battery of five intradermal antigens. The tritiated thymidine uptake of lymphocytes stimulated with phytohemagglutinin was normal in autologous serum, but reduced in fetal calf serum. The data support the hypothesis that asthma may be associated with immunodeficiency states.


The presence of IgE on the surface of lymphocytes in nasal polyps. The presence of IgE-bearing lymphocytes in nasal polyps was correlated with the patients' atopic status. Following surgical removal, the polyp tissue was treated with hyaluronidase and a single-cell suspension was obtained. Lymphocytes were isolated by gradient centrifugation, and the fluorescent antibody technique was used to study the presence of various immunoglobulin markers on the surface of lymphocytes. The presence of IgE-bearing cells was correlated with serum IgE levels, history of allergy, and skin test reactions. Patients with a positive atopic history had intermediate to high serum IgE levels. There was no correlation between IgE level and skin reactivity in these patients. Good correlation was obtained between the number of IgE-bearing cells in nasal polyps and serum IgE levels in atopic patients. The IgE-bearing cells represented 10 to 40 per cent of total immunoglobulin-bearing lymphocytes. No IgE-bearing cells were detected in five of six patients with a negative atopic history and negative skin tests. Thus IgE may be synthesized within nasal polyps of atopic patients, and the polyps in atopic patients may have a different etiology from those in nonatopic patients.


Clostridium botulinum in Scottish fish farms and farmed trout. Rainbow trout and specimens of pond mud were collected from three fish farms and examined for the presence of Clostridium botulinum. Two of the farms were constructed with concrete channels and one was mud-bottomed. Cl. botulinum was isolated only from the mud-bottomed farm (24% of muds), and the isolates were all non-proteolytic type B. The implications of the presence of Cl. botulinum spores in the mud of fish farms is discussed.


An "in-production" method for testing the sterility of infusion fluids. The addition of dehydrated broth powder to a random selection of bottles from each batch of infusion fluids before sterilization, followed by incubation of the bottles after sterilization, provides a method of sterilty testing which possesses many advantages over the traditional method of culturing small samples from bottles after sterilization.


Identification of two populations of immunoglobulin-bearing lymphocytes in man. Human peripheral blood lymphocytes bearing easily detectable surface immunoglobulin were quantitated with routine immunofluorescence procedures. The mean value in 17 healthy adults were 18% (range 9 to 30%). When the cells were preincubated at 37 degrees C and washed at the same temperature before staining, the mean value decreased to 9% (range 4.5 to 18%) (p less than 0.001). The mean value increased to 32% (range 15 to 49%) when the cells were prepared and washed at 4 degrees C, and this value was also significantly different from values at room temperature (p less than 0.01). These temperature-related effects were explained by IgG that remained bound to the cell membrane at 4 degrees C, but eluted at 37 degrees C in serum-free medium. The total number of Ig-bearing lymphocytes and T lymphocytes determined by rosette formation with sheep erythrocytes approached 100%. These studies indicate two populations of Ig-bearing lymphocytes in healthy subjects: one with surface-stable Ig determinants, and another that lacks these markers, but has receptors capable of binding IgG.


Subepidermal deposition of immunoglobulin in NZB/NZW F hybrid mice. The dermal-epidermal junction of NZB/NZW F1 hybrid mice is a site of immunoglobulin (Ig) deposition. This produces a fluorescent band which is very similar to that seen in the skin of patients with systemic lupus erythematosus. The deposition of Ig in the mouse skin is first observed at six months of age and increases in frequency thereafter. After an initial lag of 1 to 2 months, it parallels the age-related deposition of Ig in the kidney. The observed temporal relationship between glomerular and cutaneous Ig deposition supports the concept that the presence of immunoglobulin in the skin is highly indicative of the presence of immune complexes in the kidney.


E receptors on blasts from untreated acute lymphocytic leukemia (ALL):  comparison of temperature dependence of E rosettes formed by normal and leukemic lymphoid cells. The presence of receptors for sheep erythrocytes (E) and sufrace Ig on the bone marrow blasts was investigated in 29 children with untreated acute lymphocytic leukemia (ALL). In 6 of them more than 50% of the bone marrow blasts formed E rosettes, while in none of the 29 were surface Ig detected. The six children with rosette-forming blasts had WBS greater than 5 times 10-4/mm-3 at admission and 4 of them presented with thymic enlargement. E-positive ALL blasts and normal human thymocytes formed rosettes after incubation for 1 hr at 4 degrees C or 37 degrees C. In contrast, normal human peripheral T lymphocytes from blood, spleen, and pleural fluid also formed rosettes at 4 degrees C but these rosettes dissociated at 37 degrees C. In two patients with E-positive ALL, the disappearance from blood of cells forming rosettes at 37 degrees C during the 1st week of treatments paralleled the reduction in circulating blasts. Conversely, after 6 days of therapy almost one-half of the remaining cells formed rosettes at 4 degrees C and had the morphologic features of normal lymphocytes. We conclude that, in contrast to peripheral T cells, normal thymic cells and E-positive blasts share the property of forming E rosettes after 1-hr incubation at 37 degrees C. In patients with E-positive ALL this property may be used to evaluate drug effects upon leukemic and normal T lymphocytes.


Depression of humoral immunity to sheep erythrocytes in vitro by Friend virus leukemic spleen cells: induction of resistance by statolon. The in vitro antibody plaque-forming cell (PFC) response of normal spleen cells exposed to sheep erythrocytes in Marbrook chambers was depressed by the addition of Friend virus (FV) leukemic spleen cells. Fewer than 10-5 leukemic cells inhibited the PFC response of 10-7 normal cells. This immunodepression could not be produced with sonicated, irradiated, or mitomycin C-treated leukemic cells or with cellfree FV. It could be blocked by FV immune serum but was unaffected by high titers of purified interferon. Interferon did not inhibit the immune response of normal spleen cells to sheep erythrocytes. Spleen cells from mice treated with statolon or chlorite-oxidized oxyamylose statolon but not Newcastle disease virus or poly rI:poly rC were resistant to immunodepression in vitro by FV leukemic spleen cells. The unique ability of statolon to prevent immunodepression by leukemic spleen cells may be the basis of its FV leukemo-suppressive activity in vivo.


Immunosuppression by leukemia viruses. Effect of Friend leukemia virus on humoral immune competence of leukemia-resistant C57BL/6 mice. Infection of genetically resistant C57BL/6 mice with Friend leukemia virus resulted in a marked but transient immunodepression of the humoral immune response to sheep erythrocytes. The primary immune response to sheep erythrocytes was depressed in mice infected 1 or 3 days before challenge immunization, but no suppression was observed when the interval was greater. The suppression coincided with the time of transient viremia in the mice. The secondary immune response to sheep erythrocytes was inhibited when virus was injected within a few days before booster immunization. Induction of immunologic "memory" to sheep erythrocytes was also blocked in C57BL/6 mice given virus before priming with the SRBC. The immunosuppressive activity appeared due to a marked but transient effect of the virus on antibody precursor cells, as ascertained by cell transfer experiments. These observations are pertinent to the general question to the cellular site of action of immunosuppressive tumor viruses and the relationship between immunosuppression and the neoplastic properties of the RNA viruses.


Differential IgA repopulation after transfer of autologous and allogeneic rabbit Peyer's patch cells. The distribution and differentiation of rabbit Peyer's patch cells were studied in lethally x-irradiated autologou, and allogeneic recipients, 6 days after i.v. transfer. Immunoglobulin Ig-containing cells were detected in both frozen tissue sections and fixed smears of single cell suspensions of spleens using monospecific anti-alpha, -mu, and -iota antisera in direct immunofluorescence. The tissues of lethally x-irradiated rabbits which had not received any cells were almost devoid of Ig-containing cells whereas the intestinal lamina propria of both autologous and allogeneic Peyer's patch cell recipients had approximately equal numbers of predominantly IgA-containing cells. In the spleen of allogeneic recipients, many large colonies of IgA-containing cells were seen and these cells made up 4.6 to 16.9% (mean 8%) of all spleen cell suspension. The spleens of autologous recipients contained fewer and smaller colonies of IgA cells which made up only 0.5% of all cells in spleen cell suspensions. The numbers of IgM- and IgG-containing spleen cells were small in all animals, however, recipients of allogeneic Peyer's patch cells had four to five times as many as either non-reconstituted lethally irradiated rabbits or autologous recipients. These data confirm that rabbit Peyer's patches are relatively rich in precursors of IgA-producing cells and suggest that histocompatibility differences may either potentiate differentiation of IgA production or lead to trapping of allogeneic Peyer's patch cells in the spleen. Although these experiments did not elucidate the mechanisms of differentiation of IgA-producing cells, the fact that no differences were seen between numbers of IgA cells in gut lamina propria after either autologous or allogeneic cell transfer suggest that different mechanisms may be responsible for appearance of these cells in gut and spleen.


Studies on the composition of adjuvants which selectively enhance delayed-type hypersensitivity to lipid conjugated protein antigens. Hen egg albumin (HEA), heavily conjugated with dodecanoic acid (D-HEA), stimulated sustained delayed type hypersensitivity (DTH) specific for HEA without detectable antibody formation in guinea pigs. An oil-in-water emulsion containing purified BCG cell walls attached to the oil drops was found to be a very effective adjuvant for enhancing DTH to D-HEA, but not to HEA. Animals immunized with D-HEA in the BCG cell wall emulsion produced skin test reactions 2.4 cm in diameter when challenged with HEA 21 days after a single immunization. Control animals immunized with D-HEA in saline produced skin reactions 1 cm in diameter to similar challenge. Neither group of animals produced detectable antibody to HEA-OR D-HEAmThe emulsion had no adjuvant effect if the BCG cell walls were suspended in the aqueous phase and not attached to the oil droplets. Dodecanoic acid conjugated Salmonella typhi organisms could be used in place of the BCG cell walls to produce effective adjuvant preparations. Fruend's complete adjuvant and other water-in-oil emulsions, however, were found to be ineffective adjuvants for enhancing the degree of DTH produced by D-HEA. Experiments with autoradiography demonstrated that effective adjuvant preparations promote the localization and retention of both 125-I-labeled D-HEA and 125-I-labeled BCG cell walls in the paracortical area of lymph nodes where they are in close proximity to many T-type lymphocytes which proliferate in the induction of DTH.


Suppression and enhancement of the T cell-dependent production of antibody to SRBC in vitro by bacterial lipopolysaccharide. LPS induced the production of antibody to sheep red blood cells (SRBC) in cultures of spleen cells from normal and T cell-depleted mice, and addition of SRBC to the cultures enchanced this T cell-independent response very little. By contrast, the T cell-dependent production of antibody to SRBC in vitro was suppressed when lipopolysaccharide (LPS) was added at the time when the spleen cells were cultured. Later addition of LPS to spleen cell cultures caused enhancement of antibody production, but only when LPS had not been added before. Addition of T cells that had been primed with SRBC in vivo did not reverse the LPS-induced suppression of antibody production. The data are interpreted to mean that either B cells are rendered incapable of receiving T cell signals in the presence of LPS or that LPS interferes with the appropriate association of cellular components which cooperate in the immune response to SRBC.


Synergistic effects on DNA synthesis of phytohemagglutinin or concanavalin A and lipopolysaccharide in human peripheral blood lymphocytes. Mixtures of phytohemagglutinin (PHA) or Concanavalin A (Con A) and lipopolysaccharide (LPS) with human peripheral blood lymphocytes (PBL) resulted in a synergistic effect on DNA synthesis as measured by increased 3H-thymidine uptake. These data were compared to the mitogenic response of human PBL to PHA, Con A, or LPS alone. Various combinations of PHA and Con A usually suppressed DNA synthesis in human PBL. Titration experiments revealed that increasing amounts of LPS, in the presence of a constant amount of Con A, resulted in corresponding increases in the synergistic response of human PBL. Conversely, increasing amounts of Con A, in the presence of a constant amount of LPA did not lead to increases in the amount of synergism. The results are discussed in terms of the participation of additional cells in the synergistic response of human PBL.


Functional heterogeneity among the T-derived lymphocytes of the mouse. II. Sensitivity of subpopulations to anti-thymocyte serum. A number of T cell functions were examined in mice after treatment with rabbit anti-mouse thymocyte serum (ATS). Two populations of T cells were distinguished; the first, sensitive, and a second, resistant, to elimination by ATS. The helper cell involved in the primary humoral response to sheep red blood cells (SRBC) was found within the sensitive population, whereas the T-cells required for the priming of mice with SRBC for helper activity or delayed hypersensitivity were found within both populations. Previous work and experiments utilizing both adult thymectomy and ATS treatment indicated that the ATS-sensitive population is long-lived, whereas the ATS-resistant population is short-lived.


Effect of polycations and polyanions on behavior of sheep red blood cell rosettes of human lymphocytes. The role of cell surface charges in the behavior of sheep red blood  cells (SRBC) rosettes with human lymphocytes was investigated by using various polycations and polyanions. Polycations (DEAE-dextran, polybrene, poly-L-lysine, spermadine) at appropriate concentrations (1) invariably increased the proportions of early rosettes. This was so irrespective of whether it was the SRBC or the lymphocytes that were being treated with the reagents. (2) The rosettes so formed also consisted of a greater number of red blood cells per rosette (3). On incubating the rosettes at 37 degrees C, they dissociated by capping. The rates of dissociation and capping were retarded by polycation treatment. (4) The resistance to rosette disruption was increased. These properties were very similar to those observed on cells treated with neuraminidase. Treatment of cells with the polyanions heparin and dextran sulfate induced the opposite effects: reduced the number of rosettes and increased the rate of dissociation, and decreased the resistance of rosettes to disruption. It was concluded that alterations of cell surface charges could drastically influence SRBC-lymphocyte interactions. To investigate whether rosette inhibitors could do so by altering cell surface charges, cell were treated with anti-lymphocytic sera, 2,4-dinitrophenol and sodium azide. All three agents inhibited rosette formation. Whereas DEAE-dextran could partially reverse the inhibition by the first two agents, it failed to do so with the third. This suggested that the first two may have inhibited rosette formation by altering the surface charges.


The effect of plasmacytomas on serum immunoglobulin levels of BALB/c mice. The effect of tumor growth on serum immunoglobulin levels and on the immune response to sheep erythrocytes (SRBC) was studied in BALB/c mice bearing MOPC-315 (IgA), MOPC-460 (IgA), MOPC-173 (IgG2a) and MOPC-104E (IgM) to gain insight into the immunologic competence of the plasmacytoma-bearing host. The initial increase of myeloma protein coincided with the first appearance of the tumor and increased as the tumor progressed. However, at the time of death there was little correlation between spleen weights, tumor size, and myeloma-protein levels. The mean serum concentration of the myeloma proteins reached a higher level in the mice bearing tumors transplanted i.p. compared to those with tumors transferred subcutaneously (s.c.). Non-myeloma immunoglobulin levels in the serum were reduced: IgM was significantly lowered in the presence of MOPC-315 injected i.p. and MOPC-460 injected s.c. and the IgG2 levels were depressed in mice injected i.p. with MOPC-315 and MOPC-104E. The only significant reduction of IgA levels was seen when MOPC-173 was transplanted i.p. The decreases observed in immunoglobulin levels correlated with plasmacytoma growth. They were specific for myeloma and were not due to tumor growth per se since the levels of all immunoglobulins tested increased in the presence of Sarcoma 37, a pleomorphic neoplasm. The primary plaque-forming cell (PFC) response of tumor-bearing animals after the injection of 0.5 ml of 10% SRBC was either similar or enhanced compared to the controls. However, with a lower SRBC dosage (0.5 ml of 2% SRBC) the indirect PFC were reduced with mice bearing MOPC-104E and MOPC-173. Tumor sizes did not seem to correlate with reduction of the PFC response. MOPC-460-bearing mice had a comparable number of PFC per spleen to those of the controls, but reduced numbers when calculated per 10 spleen cells. Consistently, hemagglutination titers were reduced in all tumor-bearing animals. The number of direct and indirect PFC per spleen was increased in mice bearing Sarcoma 37, compared to the controls. The possible implications of these findings are discussed.


Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. II. Functional studies. The alloantigenic specificity Ly-4.2 can be detected on a proportion of lymphocytes by the antiserum (BALB/c times SWR)F1 anti-B10.D2. In the preceding study it was shown that these lymphocytes were not thymus-derived (T) cells, as they were Thy-1 (theta)(-), and were therefore presumably B (bone marrow-derived) cells. Evidence is now presented for the reaction of the Ly-4.2 antiserum with functional B cells. Thus, the LY-4.2 and Thy-1.2 specificities were detected on antigen-binding rosette-forming cells (RFC) in mice both immune and non-immune to sheep red cells (SRC). RFC formed to endotoxin lipopolysaccharide (LPS) were also Ly-4.2(+). Memory cells to both SRC andLPS could be detected with anti-Ly-4.2 and anti-Thy-1.2 antisera, thereby indicating that both T and B cells are involved in memory to these antigens. Both direct and indirect antibody-forming cells (the PFC) could be inhibited, in vitro, by anti-Ly-4.2 antiserum, although it is likely that not all PFC are Ly-4.2(+). Neither of the specificities Ly-4.2 nor Thy-1.2 were detected on the bone marrow precursor of the splenic colony forming unit (the CFU). In an assay for B cells, the treatment of lymph node or spleen cells with anti-Ly-4.2 before transfer to irradiated recipients could inhibit the ability of these cells to make PFC to SRC, and this capacity could only be restored by bone marrow cells and not by thymus cells. These studies provide clear evidence for the presence of the Ly-4.2 specificity on antibody-forming cells and their precursor (B cells).


Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity. A transplantable, murine reticulum cell sarcoma is described which exhibits the cytologic, adherence, and phagocytic properties of macrophages. It forms specific rosettes with erythrocytes in the presence of the corresponding anti-serum. The ascites cells mediate antibody-dependent cellular immunity as assayed by release of radioactivity from 51Cr-labeled erythrocytes. The contribution of contaminating host cells in the cytotoxic reaction was ruled out by growing the tumor in F1 mice and removing the host cells by anti-H2 serum and complement. The tumor cells have receptors for IgG2a and IgG2b immunoglobulins. The availability of a pure population of effector cells in the immune system allows study of the biochemical processes pursuant to lysis of foreign cells.


The plaque cytogram assay. II. Correlation between morphology and density of linear bovine serum albumin buoyant density gradient-separated immunocompetent cells. The plaque cytogram assay has been combined with pH 6.8, linear bovine serum albumin (BSA) gradients to demonstrate the correlation between the morphology and buoyant density of immunocompetent cells. Ten types of plaque-forming cells (PFC) and four types of plaques have been identified and enriched in the density gradients. Reproducible density profiles of mouse erythrocytes, lymphoid cells, and PFC have been obtained with BSA gradients. The density peak for mouse erythrocytes was about 1.080 g/ml, while the lymphoid cell density profile had several peaks with a maximum concentration at 1.064 g/ml. Three distinct peaks or shoulders were found in the density profile of hemolytic PFC. Most of the density distributions of the individual morphologic classes of PFC were considerably narrower than that of the entire PFC distribution and were discrete from each other. The combined result that the PFC could be subdivided by morphology and density, and that the two phenomena were correlated establishes that the PFC are truly a heterogeneous population. Whether this heterogeneity is due to different stages in development or due to the existence of differing classes of cells will have to await future experimentation.


Demonstration of in vitro lymphocyte-mediated cytotoxicity against measles virus in SSPE. Patients with subacute sclerosing panencephalitis (SSPE) are presumed to lack specific cellular immunity against measles virus. In order to test this hypothesis in vitro, the interaction between peripheral lymphocytes and measles virus-infected tissue culture cells was investigated in 10 SSPE patients. Human fibroblasts, either uninfected or carrying a persistent measles virus infection, were labeled with 51Cr and incubated with lymphocytes for 18 to 20 hr in the absence of antibody and complement. Peripheral lymphocytes from measles sero-positive and sero-negative individuals were tested, and the system was found to be virus specific. The lymphocytes from the 10 SSPE patients caused specific cytotoxicity of target cells. A correlation was not found between antibody titer and specific 51Cr release. It could also be domonstrated that target cell destruction was not mediated by monocytes or B lymphocytes. These in vitro studies suggest that SSPE patients do not have a specific defect of cellular immunity against measles virus.


Characterization of measles virus-specific cytotoxic antibodies by use of a chronically infected cell line. The complement-dependent cytotoxic effects of measles virus specific antibodies on a chronically infected cell line were studied by a microcytotoxicity assay. Cell damage was determined by trypan blue staining. Monospecific antisera against the hemagglutinin (HA) and the hemolysin (HL) of the virus were both cytotoxic. Rabbits, immunized with measles virus, developed cytotoxic antibodies in parallel with the appearance of antibodies against the measles virus HA and HL. Human sera with high antibody titers against HA and HL also gave a specific cytotoxic reaction. The main part of the activity was carried by IgG in rabbit sera tested within 2 weeks after primary immunization and only IgM. The serum reaction was characterized by a prozone. The maximal degree of cytotoxicity varied between tests performed on different occasions. Complement, at higher concentrations than were used for analysis of antibody-mediated cytolysis, caused a significant lysis of infected cells. Cerebrospinal fluid (CSF) samples containing measles virus-specific antibodies were investigated. CSF from patients with subacute sclerosing panencephalitis displayed a pronounced cytotoxicity without a prozone. CSF samples from four patients with multiple sclerosis were identified by a specific cytotoxic reaction despite low titers of antibodies against HA and HL.


Studies on the mechanism of stimulation of the humoral response of murine spleen cultures by culture fluids from human monocytes. It has been demonstrated that media derived from cultures of human monocytes can stimulate the IgM and IgG anti-sheep erythrocyte response of normal or T cell-deficient murine splenocytes in vitro. This culture fluid also causes a slight stimulation in the rate of B cell division and in the number of antibody-secreting cells occurring in the absence of added antigen. The active factors, which are released from the monocytes during the first 2 days of culture, appear to affect the murine B cells throughout the culture period.


Antigen-specific and nonspecific mediators of T cell/B cell cooperation. I. Evidence for their production by different T cells. Evidence is presented for two types of helper T cells in the mouse specific for a protein antigen, keyhole limpet hemocyanin (KLH). The first cell is able to help B cells respond to the hapten, trinitrophenyl (TNP), when coupled to KLH presumably through the participation of KLH-specific T cell factors. The second helper cell when stimulated with KLH is able to help B cells respond to red blood cell antigens, but not TNP-KLH, through the production of a non-antigen-specific factor. Both types of helper cells were shown to be present in the spleens of KLH-primed mice, although they occurred with different frequencies. They were shown to be different T cells by their segregation from each other in vitro at limiting dilution.


Acute destruction of rat skin grafts by alloantisera. Rat skin transplanted to mice or to allogeneic rats can be acutely and severely damaged by injecting the graft recipients with alloimmune serum of appropriate specificity. This susceptibility of rat skin to alloantisera has been obscured in previous studies by the fact that the extent of serum-mediated damage that occurs is decisively influenced by the interval of time between placement of the grafts and the injection of antiserum. Grafts are resistant to damage during the 1st week after transplantation; they rapidly develop sensitivity during the 2nd week, reaching a peak at 14 to 16 days; and during the next few weeks the sensitivity is lost. Grafts surviving beyond 35 to 40 days become totally resistant to antisera. LBNF1 GRAFTS Are more resistant than Le or BN grafts to BN anti-Le serum or Le anti-BN serum, respectively. However, combinations of the two sera can act synergistically to destroy LBN skin that has been grafted to immunosuppressed mice. This form of serum-mediated tissue damage is thought not to play a significant role in skin graft rejection in the usual circumstances of transplantation since cell mediated immunity is well developed before the acquisition by the grafts of sensitivity to humoral agents. Furthermore, antisera from animals that have rejected a single graft have little or no destructive potential, and hyperimmune animals reject skin very shortly after grafting, often before the grafts have become vascularized and hence before they are sensitive to antiserum.


Receptor sites for antigen-antibody complexes on cells derived from solid tumors: detection by means of antibody sensitized sheep erythrocytes labeled with technetium-99m. Surface receptor sites for the Fc portion of antigen-antibody complexes were demonstrated on cells derived from three methylcholanthrene-induced fibrosarcomas, one of strain C3H and two of strain BALB/c origin, two spontaneously occurring malignant melanomas (B16 in strain C57BL/6 and Harding-Passey in strain BALB/c mice), a Moloney sarcoma virus-induced tumor of strain BALB/c origin and the Walker 256 carcinosarcoma of Holtzman rats. Primary cell cultures derived from these tumors adsorbed technetium-99m labeled, antibody-sensitized sheep erythrocytes (99mTc EA) as determined either by visual scoring of adherence or radioisotopic quantitation. Depending upon the tumor tested, from 20% to greater than 95% of the target cells absorbed 99mTc EA. All cells lost their reactivity after 1 or 2 passages in vitro, but this was regained after a single passage in vivo. Indicator erythrocytes coated with F(ab')2 fragments of the sensitizing sheep erythrocytes (SRBC) antiserum did not adhere thereby demonstrating that the hemadsorption required an intact Fc portion of the antibody molecule. Adherence of 99mTc EA was blocked by soluble immune complexes prepared with ovalbumin and rabbit antibody directed against it and Escherichia coli 055:B5 lipopolysaccharide and mouse antibody directed against it. Normal rabbit or mouse serum, immune serum, or antigen alone did not block adherence of 99mTc EA thereby demonstrating that the receptors had greater affinity for immune complexes than for either antigen or antibody alone. The existence of membrane receptors on tumor-derived cells which react with the Fc portion of antigen-antibody complexes may provide an explanation for the mechanism by which immune complexes are capable of blocking cell-mediated tumor cell destruction irrespective of whether the receptors are on the tumor cells themselves or on admixed lymphocytes and macrophages.


Immune response to liposomal model membranes: restricted IgM and IgG anti-dinitrophenyl antibodies produced in guinea pigs. Guinea pigs were immunized with liposomal model membranes actively sensitized with 2,4-dinitrophenyl-aminocaproylphosphatidyl-ethanolamine. The immune response was characterized by the formation of both IgM and IgG anti-DNP plaque-forming cells (PFC) which paralleled the appearance of anti-DNP antibodies in the serum. Plaque inhibition by DNP-lysine indicated that the PFC produced after liposomal immunization fell in a much narrower range of avidity groups than the PFC obtained after immunization with DNP-albumin and, in this regard, resembled MOPC 315. The restricted nature of the serum IgG anti-DNP antibodies was confirmed by isoelectric focusing and revealed the expression of a limited number of clones in all cases.


Biological defense mechanisms. Evidence for the participation of superoxide in bacterial killing by xanthine oxidase. Incubation of either Staphylococcus epidermidis or Escherichia coli with a sufficiently high concentration of xanthine oxidase, an enzyme capable of reducing oxygen to superoxide (O2-), resulted in the death of the microorganisms. Protection against the killing os S. epidermidis by xanthine oxidase was afforded by superoxide dismutase, an enzyme which converts O2- to O2 and H2O2, and also by catalase, which destroys H2O2. These findings indicate that neither O2- nor H2O2 were able to kill S. epidermidis under the experimental conditions, but that the bactericidal agent was the product of a reaction between O2- and H2O2. By contrast, E. coli was protected by catalase but not by superoxide dismutase. With this organism, therefore, H2O2 appears to have been the bactericidal agent.


The management of rhinoscleroma.

The management of rhinoscleroma. This investigation showed that the organism klebsiella rhinoscleromatis was present consistently in our cases. It was found to be difficult to eradicate completely although seemingly sensitive to common antibiotics in vitro. A prolonged treatment with bactericidal antibiotics is therefore necessary to eradicate it. Nasal dilatation rather than excision of granulations is required to restore nearly-normal physiological nasal function. Excision or cautery of granulations inside the nose is discouraged so as to avoid atropic changes.


The presence of IgA on the surface of rat thoractic duct lymphocytes which contain internal IgA. The presence of lymphocytes with internal IgA among cells from rat thoracic duct lymph wdy. The number of cells detected was greater in animals kept in a convential animal house compared with those maintained under specific pathogen-free conditions. Thoracic duct lymph from B rats and adult thymectomized rats also contained cell with internal IgA. The surface Ig of the IgA-containing cells was studied using a double-labeling technique with (126I) anti-Ig to detect surface Ig, and fluorescein-conjugated anti-IgA  in large amounts, but very little IgM and no surface IgG2. The surface IgA was not acquired passively.


Cross-reactivity between H-2K and H-2D products. I. Evidence for extensive and reciprocal serological cross-reactivity. Serological cross-reactivity between the products of the H-2K and H-2D genes has been demonstrated by a design in which antibody was produced against determinants controlled by one locus (e .g . H-2K(k)), and then tested against the product of the opposite locus (e .g . H-2D(d)). A total of 13 out of 18 such test combinations exhibited H-2K-H-2D cross-reactivity. The presence or absence of cross-reactivity was reciprocal in most cases (i.e. antibody directed against the H-2K(k) gene product reacted with H-2(d) determinants, and antibody directed against the H-2D(d) gene product reacted with H-2K(k) determinants). An Ia-like reaction was detected with one antiserum which implied possible cross-reactivity between the products of two discrete la genes.


Demonstration of an antibody-mediated tolerance state and its effect on antibody affinity. We have described a model of immunological tolerance induced, in adult mice, by a single injection of a moderate dose of a hapten-protein conjugate. The data suggest that the mechanism of this tolerance state is the production of small amounts of high affinity antibody in response to the tolerance-inducing antigen injection. This antibody acts to inhibit the response to a subsequent challenge with antigen in complete Freund's adjuvant by a mechanism comparable to that of passive antibody-medicated immune suppression. It was shown that a small but high affinity. Tolerance was not terminated by transfer of normal syngeneic spleen or peritoneal cells into tolerant animals. Spleen cells from tolerant mice, when transferred into lethally irradiated, syngeneic animals, produced  a PFC response which is greater in magnitude and tolerance state had a significant degree of carrier specificity which was shown to be comparable to the carrier specificity of antibody-mediated immune suppression. hus, evidence was presented to show that one mechanism of tolerance in adult animals in the suppressive effect of small amounts of high affinity antibody formed in response to the tolerizing injection of antigen.


Inhibition of multiplication of Toxoplasma gondii by human monocytes exposed to T-lymphocyte products. The multiplication of Toxoplasma gondii was quantitated in human monocytes in vitro by phase-contrast microscopy. Toxoplasma multiplication was identical in monocytes from subjects byt was significantly inhibited in cells from both sources if the monocytes were preincubated with immune lymphocytes and toxoplasma monocytes were preincubated with immune lymphocytes and toxoplasma antigen. Supernates prepared from toxoplasma-immune lymphocytes incubated with toxoplasma antigen were also effective in inducing in monocytes the capacity to inhibit toxoplasma multiplication. Supernative acitivty was evident after lymphocytes and antigen were incubated for as little as 15 min. The instruction of monocytes was also repid and reversible. Monocytes were fully induced to inhibit toxoplasma multiplication after a 2 h exposure to an active supernate, but they lost their inhibitory capacity on culture in vitro for 48 h in the absece of immune cells or their products. The lymphocytes particupating in the monocyte induction were identified as t cells. The in vitro stimulation of monocytes appeared to exhibit some specificity, since no inhibition of toxopreotein derivative and lymphocytes from tuberculin-positive subjects, concanavalin a-stimulated lymphocytes, or their supermates. Supernates which induced monocytes to inhibit toxoplasma multiplication did not influence parasite growth in HeLa cells.


Scanning electron microscopy of tobacco mosaic virus-labeled lymphocyte surface antigens. The study of surface antigen by immunoelectron microscopy has been hampered by the fact that thin sections of cells provide only a view of the cell perimeter in an essentially two- dimensional fashion. Although the reconstruction of the entire cell from serial sections has been accomplished (1), it remains too exacting a technique and will find only exceptional application. Carbon-platinum replicas (2) allow the inspection of larger surface areas and therefore are better suited for studying the distribution of antigens (3). But since only relatively smooth surfaces will yield stable replicas, cells with large numbers of microvilli are not amenable to this technique. Despire its limited resolution, scanning electron microscopy (SEM) seems to be the method of choice because it can provide a view of almost half of the surface of a cell close to its natural configuration, particularly after critical point or freeze drying (4, 5). Immunological-labeling methods have not yet been routinely applied to SEM although both latex spheres (6) and hemocyanin (7) have been used with some success. The optimal visual marker should possess the following properties: be of a distinctive shape, chemically stable, and have per se a low binding affinity for cell surfaces. Tobacco mosaic virus (TMV), a marker with which we are familiar in transmission electron microscopy (8), seems to meet these demands; it has rod-like shape and defined dimensions (15 x 300 nm) and in addition it can easily be distinguished from surface microvilli. As the hybrid antibody technique (9) is also applicable to TMV, we have attempted to combine such immunological labeling with SEM. We present evidence that surface antigens can indeed be visualized by SEM, using the TMV marker in conjunction with the hybrid antibody technique.


Torulopsis bacarum, Torulopsis pustula and Torulopsis multis-gemmis sp. nov., three new yeasts from soft fruit. Three new species of yeasts isolated from fresh soft fruit are described. Torulopsis pustula was isolated from blackcurrants, Torulopsis bacarum from blackcurrants, strawberries and raspberries, and Torulopsis multis-gemmis from raspberries. All differ from known species in the test substrates they utilize. In addition, cells of Torulopsis pustula often form a bulbous projection near their apices from which budding takes place. These buds are frequently pear- or heart-shaped.


Thermosensitive production of their transfer systems by group S plasmids. Transfer of plasmids of group S is much more efficient at low temperatures (e.g. 22 degrees C) than at 37 degrees C. This is due to failure of the donor strain to produce the transfer system during growth at the higher temperature.


Isolation and chemical characterization of plasma membranes from the yeast and mycelial forms of Candida albicans. It has been possible to induce the yeast-mycelium transformation in Candida albicans by growth of the organism under completely defined conditions in batch culture. Protoplasts have been obtained from the two forms by using a lytic enzyme preparation from Streptomyces violaceus. A plasma membrane fraction was prepared by osmotic lysis of these protoplasts and fractionated by using a combination of differential and discontinuous sucrose density-gradient flotation centrifugation. The purity of this fraction was determined by radioactive dansylation and iodination of plasma membranes of intact protoplasts followed by localization of the radioactivity upon fractionation. This procedure demonstrated less than 4% contamination of the plasma membrane fraction with other cell membranes. Chemical analysis of this fraction revealed that the major components were protein and lipid. Membranes from the yeast form contained (w/w): 50% protein, 45% lipid, 9% carbohydrate and 0.3% nucleic acid. Plasma membranes from the mycelial form contained significantly more carbohydrate and were found to be composed of (w/w): 43% protein, 31% lipid, 25% carbohydrate and 0.5% nucleic acid. Marked differences were also observed between the phospholipid, free and esterified sterols, and total fatty acids of membranes from the two forms of the organism.


Chromosome transfer in Proteus mirabilis mediated by hybrid plasmid. A previously-described fused plasmid, P-lacRIdrd19, was found to mediate chromosomal transfer between cells of Proteus mirabilis strain PM5006; PM5006-(P-lacRIdrd19) was usually the donor and various auxotrophs of PM5006 resistant to nalidixic acid and/or streptomycin were recipients. The donor was usually counterselected with nalidixic acid and/or high concentrations of streptomycin. Recombination experiments with single markers indicated a 40-fold variation in recombination frequencies for different markers. Mapping double-auxotrophic markers by their gradient of transmission confirmed this variation and placed each of two independent isolates of eight markers in a linkage group his-ser-ura-pyrB-trp-cys-ade-ilv. Some donor markers did not register. Despite low recombination frequencies, interrupted mating experiments showed a polarity of early marker transfer. The segregation of unselected markers confirmed the order of some markers and showed that genetic material passed from the presumptive donor to the recipient. Recipients with two auxotrophic markers which could not be cotransduced by phage 5006M were converted to prototrophy by conjugation. The plasmid transferred to recipients at high frequency and all recombinants carried it. Recombinants could act as donors in further matings. Recombinants were fully susceptible to phage 5006M, unlike transductants of PM5006 by this phage. Direct involvement of the plasmid was indicated by drastically diminished recombination frequencies in crosses with recipients carrying P-lac as resident. P-lac had previously been shown to reduce the frequency of transfer of the hybrid plasmid to cells harbouring it. The histidine region was the first to register in recipients and recombined at the highest frequency of 5 times 10-6/donor cell. Some temporary association of plasmid and perhaps only the histidine region of the chromosome is favoured as the mechanism of chromosomal transfer. This could explain why not all donor markers could be mapped. Transduction and transformation were excluded as the cause of results.


Mutations which affect amino acid transport in Aspergillus nidulans. Mutants deficient in amino acid transport (aau), and unable to utilize L-glutamate as a sole carbon and nitrogen source, have been isolated. There are four unlinked genes involved: aauA, aauB, aauC and aauD. The transport levels of certain amino acids, and the growth characteristics on certain nitrogen and carbon sources and toxic amino acid analogues, indicate that: aauAI has defective transport of acidic amino acids; aauBI has defective transport of acidic and neutral amino acids; aauCI and aauDI have defective transport of acid, neutral and aromatic amino acids. aauAI and aauBI are recessive for all three characteristics in the heterozygous diploid; aauCI and aauDI are dominant.


Sub-cellular location of mercury in yeast grown in the presence of mercuric chloride. The distribution of 203Hg in Saccharomyces cerevisiae grown in the presence of mercuric chloride has been examined by physical and chemical fractionation procedures and autoradiography. The major fraction of the bound mercury is tightly bound to the wall. A significant quantity of mercury penetrates to the cytoplasm but only a minor fraction is present as low molecular weight components. The wall associated mercury is not readily released by extraction with sodium hydroxide or ethylenediamine but a major fraction is solubilized by pronase and Helicase treatment. Isolated walls are capable of binding their own weight of mercury to high-affinity adsorption sites. The major role of the cell envelope in the in vivo binding of mercury and the penetration to the cytoplasm of mercury was confirmed by autoradiography.


R factors from Serratia marcescens.

R factors from Serratia marcescens. In recent years, Serratia marcescens has become established in certain localities as an agent of hospital infection and cross-infection (Clayton & von Graevenitz, 1966; Wilfert, Barrett & Kass, 1968; Davis, Foltz & Blakemore, 1970; Wilkowske, Washington, Martin & Ritts, 1970). In general, strains of S. marcescens isolated from infective lesions differ from those from other sources in being non-pigmented and antibiotic resistant (Ewing, Johnson & David, 1962; Clayton & von Graevenitz, 1966). Medeiros & O'Brien (1969) and Schaefler et al. (1971) described strains of S. marcescens, isolated from hospital patients, which were able to transfer R factors to Escherichia coli. Transfer of resistance to E. coli has also been reported from strains of S. marcescens isolated in France (Grimont & Dulong de Rosnay, 1972; Scavizzi, 1972; Lemosquet-Villemon, Morel & Freymuth, 1973)9 We have collected strains of S. marcescens, most, but not all, clinical isolates, from widely separate geographical areas; each strain was tested for antibiotic resistance and for R factors transmissible to E. coli K129 The R factors were classified by compatibility in K12 (Datta, 1974). Our purpose was to find out how much of the antibiotic resistance observed in S. marcescens is characteristic of that genus and to what extent it is shared with other bacterial genera (Coetzee, Datta & Hedges, 1972; Datta & Hedges, 1972a; Hedges, 1974). The R factors described by Medeiros & O'Brien (1969) and Lemosquet-Villemon et al. (1973) were included in this study.


Sphere-rod morphogenesis of Escherichia coli. The morphogenetic capacity of E. coli was studied by converting the rod-shaped cells into spheres and then determining whether these spheres could revert to rods. The morphogenesis of cells was followed by immobilizing them in a viscous Methocel-containing medium. Two different types of spheres were prepared: cells which retained a mechanically intact sacculus, and osmotically sensitive sphaeroplasts lacking a sacculus. The sphaeroplasts were not able to revert to rods although they were able to synthesize a new sacculus. In contrast, spheres which had retained an intact sacculus were able to reshape themselves into rods. The were also able to form new ends at (or near) the sites of the ends on the original rods.


Characterization of the growth of Pseudomonas putida LP on lipoate and its analogues: transport, oxidation, sulphur source, and enzyme induction. Pseudomonas putida LP, which grows on lipoate, NH4NO3 and mineral salts, converts most of the organic substrate to bisnor-lipoate (1,2-dithiolane-3-propanoic acid) and acetyl-CoA. D-, L-, or DL-lipoate serve equally well as carbon and sulphur sources. There was no growth on or bacterial oxidation of the chemically synthesized bisnor- or tetranor-(1,2-dithiolane-3-carboxylic acid) chain-shortened analogues, but these, as well as lipoate, could supply the sulphur needed for growth when acetate was provided as the sole source of carbon. The uptake of lipoate by the bacterium is very slow and non-inducible, while the uptake of acetate is faster than octanoate. The oxidation of octanoate is more rapid and extensive than that of lipoate. Levels of acyl-CoA synthetase are not affected by the source of carbon, but activities of isocitrate lyase and malate synthase are higher when the cells are grown in acetate, octanoate or lipoate and lower when glucose is the carbon source. The glyoxylate cycle is induced to facilitate utilization of acetyl-CoA derived from lipoate, which is also degraded to water-soluble catabolites that yield the much smaller amount of sulphur required for growth.


Polarity of the cysJIH operon of Salmonella typhimurium. Certain point and deletion mutants with lesions in the cysJ gene of Salmonella typhimurium have low levels of enzymes coded by the cysI and cysH genes. These results support the hypothesis that an operon exists comprising genes cysJ, I and H which is transcribed in the direction from cysJ to H. The nearby cysC and cysD genes do not form part of this cysJIH operon.


Some morphometric methods for the central nervous system. It is the quantitative study of the central nervous system to enumerate neurone populations. Since neurones are generally arranged in non-random patterns the conventional method of counting with a square lattice graticule in the ocular of the microscope is severely restricted. Appropriately tailored methods of (a) counting, or (b) estimating the density of a neurone population must therefore be used. Four types of neuronal distribution pattern are discussed and morphometric methods adapted to them are presented, as follows: (1) Where the whole population is circumscribed, as in the anterior horn motor neurones of the cord, direct counting is feasible. Only those cells whose nucleoli are visible should be included; this diminishes the bias in favour of large cells, and if one corrects for the fact that larger neurones have larger nucleoli the bias can be eliminated completely. (2) Where neurones are situated on an "interface", as Purkinje cells are, the length of its profile (in this instance the boundary between the molecular and granular zones) is estimated by superimposing parallel lines on the microscopical image and counting intersections as these lines cross the interface. The number of Purkinje cells in each particular field is counted at the same time. A "linear dinsity" (PL) is then calculated from the formula pl = 2n/pid where n is the quotient of (a) the number of cells counted along (b) an estimated length of profile, and d is the distance between the parallel lines of the graticule. For comparative studies between cerebella of different sizes a correction factor is easily introduced. Those who prefer to work on photomicrographs can use a mapping wheel to measure a length of profile and then proceed as before. (3) Where neurones are organised in a curving band, as in the dentate nucleus, another form of cell density can be established. The microscope is focused on a neurone at random, and the distance from this cell to its nearest neighbour is measured, preferably by means of a screw-micrometer eyepiece. The mean value r of a number of such measurements is substituted in a formula which gives the "areal density" of the neurone population: pa = 1/4r2. This method can be applied to photomicrographs by using a pair of dividers and/or an accurate rule to measure the set of r values. (4) In large collections of neurones, e.g., in the thalamus, three methods are available: (a) the nearest neighbour method; (b) a conventional squared graticule count, and (c) a count of cells intersected by a line probe as in Haug's (1972) technique (fig. 5), or a modified form of Strong's (1966) transect method.


Immunohistochemical demonstration of antigen associated with the post-synaptic lattice. The binding of amtosera against synaptosomes from rat cerebral cortex to dissociated cells from both cerebral cortex and liver was assessed by immunofluorescent labelling. This showed that none of the liver cells bound antisynaptosome antibodies but that some of the cerebral cortex cells bound antibody on their surfaces. Immunofluorescent labelling showed that all the particles in the crude mitochondrial fraction from rat cortex bound antibodies present in the unadsorbed antisera. However, when the antisera were absorbed with purified mitochondria and myelin, only a proportion of the mitochondrial fraction particles then bound antibody. Isolated IgG from the adsorbed antisera was labelled with ferritin and incubated with the crude mitochondrial fraction. Examination in the electron microscope showed that the ferritin and hence the antisynaptosome antibody was bound to the post-synaptic thickenings of about 20% of synaptosomes having their junctions in the plane of section.


Review of basic concepts of cell kinetics as applied to brain tumors. The authors review and discuss the basic concepts of cell kinetics as applied to brain tumors. Uncontrolled growth of a neoplasm represents an expanding tumor cell population. Four growth parameters characterize the behavior of a neoplastic population: cell cycle time, growth fraction, tumor doubling time, and cell loss. The concept of provisionally nondividing cells explains the disparity between cell cycle time and tumor doubling time. Human gliomas, like many non-neural solid tumors, contain variable proportions of actively proliferating and nonproliferating tumor cells; this ratio is expressed by the growth fraction. The major kinetic difference between glioblastomas and differentiated astrocytomas resides in their respective growth fractions, in all likelihood an inherent biological characteristic of each tumor. Glioblastoma proliferates at a rapid rate, and only a high rate of cell loss prevents this tumor from doubling its volume in less than 1 week. The selection of drugs and design of drug schedules for treatment of glioblastomas should be made with the knowledge that 60% to 70% of the cells in this tumor are resting (nonproliferating). If experience with other solid tumors is any guide, judicious selection and combined use of drugs according to kinetically sound schedules will produce more effective chemotherapy of brain tumors.


Gallium-67-citrate scanning for the localization and staging of lymphomas. One-hundred-eight 67Ga-citrate scans were evaluated in Hodgkin's and non-Hodgkin's lymphomas for staging, diagnosis of recurrence, and for determing the results of treatment. Accuracy as confirmed by comparing the scans with pathologic material or roentgenologic and clinical findings was found to be 83%. Known lymph node involvement was diagnosed correctly in 87% but accuracy was only 48% for extranodal areas. Bone lesions were diagnosed correctly in 83% compared with only 48% of lesions of the lungs and liver. Scans were 75% accurate in 28 patients scanned for initial staging. False negatives were present in 12%; recurrent tumor in an area of prior radiation therapy appeared to be the most common cause. There were 5% false positives. Gallium scanning is a useful adjunct to other methods of detecting lymphoma.


A rapid method for evaluation of regional cerebral blood flow after intra-arterial injection of 133Xe. A technique based on the stochastic method is described for the computation of regional blood flow (RBF) after intra-arterial infection of 133Xe. Results are presented on scintigrams, defined by 600-800 channels in a 64 times 64 matrix. The content of each channel is equal to the computed RBF value. The standard deviation is also computed for each channel. The total computation cycle is achieved within 10 min. The method has been used to investigate the influence of anesthesia or drugs on regional blood flow. For this purpose, a differential printout program was written in which variations of RBF values between the two measurements are expressed in units of a fixed percentage; provision is made for calculation of RBF variations in units of differential standard deviations. Data are in good accordance with other clinical findings. This method is suitable for clinical use when blood flow values in gray and white substances are not requested.


Evaluation of portable radionuclide method for measurement of left ventricular ejection fraction and cardiac output. Seventeen patients with coronary artery, valvular, or myopathic heart disease were studied to determine correlations of the cardiac output and ejection fraction when comparing the results obtained with a portable probe technique using 113mIn with those obtained with standard methods (cineangiographic, Fick, and dye dilution). With ejection fractions ranging from o.10 to 0.85, the coefficient of correlation was 0.90 when comparing cineangiographic and radionuclide techniques. Cardiac output determinations by the radionuclide technique also correlated well with standard methods (r equals 0.88). The radionuclide method shows promise as an accurate, safe, and simple method in the evaluation of cardiac function at the bedside.


Reconstruction of the Fresnel-coded gamma camera images by digital computer. An algorithm for the digital reconstruction of the Fresnel-coded gamma camera image consists of the spatial Fourier transform of the hologram after applying a quadratic phase shift. Background subtraction is implemented by use of positive and negative zone plates. Tomographic planes are reconstructed in 27 sec from a single zone-plate shadow image by successively varying a parameter in the reconstruction algorithm on a small computer system.


An experimental study of autoclaved autogenous mandible in combination with cancellous bone and marrow. Results of a study in eight dogs indicate that a combination of cancellous bone and marrow contained within autoclaved bone can produce a successful graft.  Four implants failed because of breakdown of the oral mucosa and exposure of the graft; rejection may have been the cause of the breakdown in one instance.  Although most of the factors that caused exposure of the grafts probably could be controlled in a clinical situation, the technique cannot be recommended without reservation.


Hepatitis B in a pediatric hemodialysis unit. Serial HB Ag determinations were obtained on 62 children undergoing hemodialysis during a 31/2-year period. Thirty six (58%) of the patients had atleast one positive HB Ag determination (titer larger than 1:8) either during the period of dialysis or within 2 months after transplantation. Of the children who became HB Ag positive during the period of dialysis, 89% demonstrated hepatitis B antigenemia during the initial 6 months of dialysis. Becuase of the temporal relationship between the development of HB Ag positivity and the duration of dialysis, contamination of equipment was proposed as a contributing source for transmission of HB Ag. Although 67% of the patients who were HB Ag positive during dialysis developed biochemical evidence of hepatic dysfunction, only two patients had clinical manifestations. The incidence of hepatic involvement appeared to be related to age; only one of nine children less than 12 years of age had evidence of hepatic disease. Intermittent persistence of HB Ag for periods larger than 3 years was observed. Both the incidence and persistence of HB Ag indicate the need to prevent its acquisition by pediatric patients undergoing hemodialysis.


Hypertension after renal transplantation in children. Hypertension persisted for longer than 6 mo or developed de novo after the first month following transplantation in seven of 77 pediatric recipients of renal allografts; concomitantly there were an elevation of PRA and renal angiographic abnormalities. In two of the four patients who developed RAS there was evidence of diminished allograft function. Successful correction of the stenotic lesion in these two recipients resulted in a return of the blood pressure, PRA, and biochemical function of the allograft to normal. Unsuccessful attempts at surgical repair led to loss of the allograft in the other two patients with RAS. Intrarenal vascular and/or parenchymal lesions were evident in the other three recipients with hypertension. Although an explanation was not apparent, subclinical rejection was hypothesized. Treatment effected reduction of the hypertension in these three patients and no deterioration of allograft function was observed for periods of 5, 34, and 38 mo, respectively. Renal angiographic studies and determinations of PRA are recommended in any pediatric recipient of an allograft who develops hypertension after the first month following transplantation or has hypertension which persists for longer than 6 mo after transplantation.


Comparison of House-Tree-Person drawings of young deaf and hearing children. The House-Tree-Person (H-T-P) drawings of a matched sample of 80 deaf and 80 hearing school children, ages 7 through 10 years, were compared to assess differences related to the handicap, and the capacity of the H-T-P to distinguish between children rated by their teachers as poorly adjusted and those rated well adjusted. No differences were found between deaf and hearing in the drawing of the ear or mouth of the human figure. There was a very significant difference in the drawing of the branch structure of the tree. There were no differences in numbers of indicators of disturbance in the drawings of the children rated as more or as less well adjusted.


A scaled-projective measure of interpersonal values. A projective instrument entitled the "Kilmann Insight Test" (KIT) was devised to measure the Interpersonal Value Constructs (IVCs) of individuals, defined as; the mental categories through which an individual perceives and interprets the desirable and undesirable features of interpersonal behavior. The KIT requires an individual to differentiate on a seven-point scal, 18 IVCs according to how relevant they are to a series of six ambiguous pictures of interpersonal situations. A factor analysis of the KIT's values resulted in two personal Restraint versus Boldness. Comparing the KIT with a self-report instrument containing the same value items as the KIT, seems to be tapping a different concept of values than the self-report assessment.


Hostility measurement and its relationship to fantasy capacity. Two-hundred and three male high school freshmen and sophomore students were administered a daydreaming questionnaire, Buss-Durkee Hostility Inventory, and Rotter ISB. The latter was scored for hostility by the Renner, Maher, and Campbell method. The Subjects also rated one another on a sociometric scale measuring behavioral hostility. Subjects were divided into high and low fantasy groups. Analyses of variance were computed on the hostility measure data for 108 Subjects. The independent variables were obtained for the fantasy capacity measure and not for the peer rating measure. There was no positive correlation between the sociometric ratings and the hostility measures. In view of the results, the hostility measurement instruments are interpreted as measures of an individual's capacity to regard himself as hostile, rather than as measures of the actual hostility of his behavoir.


Studies of biologic parameters for denture design. Part I. Comparison of masseter muscle activity during chewing of crisp and soggy wafers in denture and dentition groups. The motor response to two textures was studied by comparing the EMG activities of the ipsilateral muscle during 5 minute periods of eating crisp and soggy wafers in each group of subjects. In both groups, the activity of the ipsilateral muscle was significantly greater for eating crisp wafers than for eating soggy wafers. The ratio of EMG activities for crisp and soggy textures was 1.48 in the dentition group and 1.72 for the denture wearers. These differences between the activities of ipsilateral muscles for the two textures were significant in both groups (P smaller than 0.05). Thus, in terms of muscle effort, denture wearers display discrimination of textural characteristics of food during chewing similar to that shown by subjects with natural teeth.


Selectively retained vital roots for partial support of overdentures: a patient report. Vital mandibular canine roots were submerged submucosally ina 52-year-old man. The events that occurred during 27 months of observation, while the patient wore dentures over the retained roots, were described.


A ceramic restoration bonded by etched enamel and resin for fractured incisors. This report describes a technique for making ceramic restorations for fractured incisors without operative interference. The ceramic block is baked in the laboratory on a 24 karat gold matrix cast. A resin is bonded to a silane-treated porcelain block and etched enamel.


Malignant lymphomas of follicular center cell origin in man. I. Immunologic studies. Lymphomas with histologic features indicating a follicular center cell (FCC) origin were analyzed from 26 patients of a group of 45 consecutive non-Hodgkin's lymphoma patinets whose tumors were studied for B- and T-cell characteristics. They were compared with benign, reactive lymphoid tissue from 14 patients. Cell suspensions from biopsy material, blood, or bone marrow were examined for surface Ig and for rosette formation with sheep erythrocytes (E rosettes). Of the 26 patients with FCC lymphomas, 22 had 40% or more Ig-bearing cells; all patients with FCC lymphoma tissues had 25% or less E rosette-forming cells. Cells from most FCC lymphomas of the cleaved type had surfac IgM; those from several FCC lymphomas had both IgM and IgD. Cells from lymphomas of noncleaved cell type had surface IgG or IgA. Light-chain analysis showed that cells from FCC lymphomas bore a predominant light-chain type, which indicated their monoclonal nature. Neoplastic cells from several FCC lymphomas synthesized the surface Ig which they bore. Reactive tissues usually contained fewer Ig-bearing and more E rosette-forming cells than FCC lymphomas; the Ig-bearing cells, with one exception, had a polyclonal distribution. Correlation of histologic and immunologic observations indicates that most lymphomas identified as FCC in origin by light micorscopic criteria mark as B cells with the use of immunologic techniques and that FCC lymphomas are the most common type of non-Hodgkin's lymphoma.


Immune complexes in cancer: demonstration of complexes in mice bearing neuroblastomas. Immune complexes occurred in a large proportion of mice with syngeneic neuroblastomas. Circulating immune complexes formed in all mice, whereas almost 50% had deposits of host IgG and complement in a granular pattern along the mesangia and basement membranes of the renal glomeruli. The bound IgG was eluted and recovered from the glomeruli and demonstrated immunologic specificity to neuroblastoma cell-surface antigens.


Malignant lymphomas of follicular center cell origin in man. II. Ultrastructural and cytochemical studies. Tissues from malignant lymphomas with both nodular and diffuse growth patterns, thought by light microscopy to be composed of cells of follicular center cell (FCC) origin, Were examined by electron microscopy; the tumor cells were similar to lymphoid cells found in reactive follicular centers. Tumor cells from neoplasms thought to be composed of cleaved FCC often had more pronounced nuclear folding than did cleaved FCC of reactive follicles, whereas cells in tumors of noncleaved FCC type were indistinguishable from their presumed counterparts in reactive follicles. Large cell noeplasms, previously classified as "histiocytic" lymphomas were composed of cells with ultrastructural characteristics of transformed lymphocytes; they showed neither ultrastructural nor cytochemical features of mononuclear phagocytes. These findings support the concept that a major group of lymphomas arises from lymphocytes of follicular centers.


Detection of T-cell surface antigens in a Marek's disease lymphoblastoid cell line. Indirect membrane immunofluorescence was applied in the study of a lymphoblastoid cell line derived from Marek's disease lymphoma. The cell line tacked surface immunoglobulins and reacted specifically with antisera prepared against chicken thymus cells. Cells transformed by Marek's disease virus were of thymus origin.


Enhancement and retardation of spontaneous reticulum cell neoplasm development in SJL/J mice. The enhancement or delay in the appearance of spontaneous reticulum cell neoplasms (RCN-B) in SJL/J mice was tested. Immunosuppressive treatment with antithymocyte serum and prolonged antigenic stimulation by repeated injections of sheep erythrocytes, leukemogenic cell-free centrifugates, and multichain synthetic polypeptides had no effect on the incidence or latency of spontaneous tumor appearance. Treatment with mineral oil or incomplete Freund adjuvant markedly enhanced tumor appearance (though the abundance of plasma cells induced by these treatments did not evoke the development of plasmacytomas, nor did it affect the incidence of serum paraproteinemia among mice with spontaneous tumors). Treatment with cortisone acetate, however, markedly retarded spontaneous tumor development. Tumors occurring early due to mineral oil and adjuvant treatment all had the characteristics of "RCN-B plasma cell type," and tumors occurring late due to cortisone treatment were all of the "RCN-B reticulum cell type." The possible involvement of the stem cell pool size in the enhancement or delay of spontaneous tumor development was discussed.


Process of infection with bacteriophage phiX174. XXXVII. RNA metabolism in phiX174-infected cells. The RNA produced in vivo from bacteriophage phiX174 DNA has been analyzed by polyacrylamide-agarose gel electrophoresis and sedimentation in dimethyl sulfoxide gradients, and the results of Hayashi and Hayashi (1970) have been confirmed and extended. An efficient procedure for recovery of RNA from gels, followed by a hybridization assay, has indicated the presence in infected cells of 18 distinct RNA species with sizes up to and greater than the unit (viral) length. The sizes of phiX mRNA's were similar irrespective of whether material was analyzed on gels or in dimethyl sulfoxide gradients. When virus-induced RNA was detected by a double-label method, seven additional low-molecular weight species were observed on gels and the resolution of dimethyl sulfoxide gradients was enhanced. The present results lend support to aspects of the model of Hayashi and Hayashi (1970) for the generation of these discrete mRNA species; an alternative model is also discussed.


Mutations in the new gene stIII of bacteriophage T4B suppressing the lysis defect of gene stII and a gene e mutant. Mutations of bacteriophage T4B were found which suppress the lysis defect of both gene stII mutants and gene e mutants. The suppressor mutations belong to a new gene, stIII, of phage T4B. Gene stIII is located on the genetic map of T4B between genes stI and e. stIII mutants sometimes form star plaques on Escherichia coli B. The latent period on E. coli 594, but not E. coli B, is shorter with stIII mutants than that with wild-type phage. The premature lysis of E. coli 594 infected with stIII phage does not depend on the expression of both stII+ and e+ function. StIII allele is dominant over the stIII+ with respect to both the ability to suppress the stII defect and the early lysis of infected E. coli 594 cultures.


Host DNA degradation after infection of Escherichia coli with bacteriophage T4: dependence of the alternate pathway of degradation which occurs in the absence of both T4 endonuclease II and nuclear disruption on T4 endonuclease IV. Escherichia coli cells infected with T4 phage which are deficient in both nuclear disruption and endonuclease II exhibit a pathway of host DNA degradation which does not occur in cells infected with phage deficient only in endonuclease II. This alternate pathway of host DNA degradation requires T4 endonuclease IV.


Canavanine-mediated depletion of polyamine pools in Escherichia coli: effect of head morphogenesis and DNA synthesis. We have found that L-canavanine inhibited the synthesis of polyamines in T4-infected Escherichia coli. These polyamines are known to be required for T4 DNA synthesis and may be involved in phage morphogenesis. The new data indicate that the inhibition of polyamine synthesis is not primarily responsible for the L-conavanine-mediated inhibition of DNA synthesis nor does it seem to be involved in the induction of lollipops. L-Canavanine does influence the relative amounts of putrescine and spermidine found in the phage particle, but it does not influence the amount of DNA phosphate neutralized by polyamines.


Growth of a capsid mutant of bacteriophage phi X174 in a temperature-sensitive strain of Escherichia coli. A capsid mutant of phiX174 is capable of forming replicative form and synthesizing single strands at the restrictive temperature in a dnaB mutant of Escherichia coli. Under similar conditions, the wild-type bacteriophage is incapable of either step in viral synthesis.


Biological activity of T4 DNA synthesized in toluene-treated Escherichia coli cells. T4 DNA synthesized in a toluene-treated cell system can act as the genetic donor in a DNA transformation assay. This material transforms a variety of markers at high efficiency. We present evidence that the genetic activity is due to newly synthesized, double-stranded DNA.


In Vitro system for the study of bacteriophage phi chi 174 adsorption and eclipse. An in vitro system was developed for the study of the initial stages of bacteriophage phi chi 174 infection. Escherichia coli C cells were incubated with 20% sucrose and then subjected to cold osmotic shock in 5 mM MgSO4. The concentrated supernatant shock fluid inactivated phi chi 174 with the same kinetics and requirements as for normal infection. Shock fluids prepared from phi chi 174-resistant strains of E. coli did not show this effect. The 114S phage were initially converted into 70S particles, the process termed "eclipse". These structurally altered phages then attached to a component of the shock fluid, producing fast-sedimenting complexes, and eventually released at least a part of their DNA into the medium. The fast-sedimenting complex could be radioactively labeled with oleic acid. Radioactivity was found to co-chromatograph with both biological activity and the majority of the high-molecular-weight carbohydrates present in the shock fluid. It is concluded that E. coli C osmotic shock fluid contains isolated phi chi 174-specific receptor sites composed of lipopolysaccharides. This system conveniently separates the early stages of phage phi chi 174 infection from the intracellular events.


Technique of reductive ureteroplasty in the management of megaureter. Management of megaureter may be conservative or surgical. Most operative procedures are multistaged and directed toward segmental ureteral repair. We have treated succesfully a number of patients with the total linear ureteral reduction technique.


Identification and characterization of the in vitro synthesized gene products of bacteriophage M13. Bacteriophage M13 replicative form (RF) DNA was used to direct coupled transcription and translation in cell-free extracts prepared from Escherichia coli. By using RF DNA, isolated from cells infected with appropriate amber mutants of this phage, it has been possible to identify the products of genes I through IV. By using the same methods no gene-product relationship could be demonstrated for genes VI and VII. Coupled in vitro protein synthesis studies on RF-III DNA, a linear double-stranded DNA molecule, obtained after cleavage of either RF-I or RF-II DNA with the restriction endonuclease R.Hin11 from Haemophilus influenzae, indicated that the cleavage site for this enzyme is located in gene II. The in vitro products of both gene III and gene VIII are about 30 and six amino acids longer, respectively, than their native counterparts present within the virion. These results suggest that the latter proteins arise in vivo by cleavage of precursor molecules. Coupled transcription and translation studies on a DNA fragment which only contained the genetic information coding for gene IV protein, obtained after cleavage of RF DNA with the restriction endonuclease R.Hap11 from Haemophilus aphirophilus, indicated that a large number of the in vitro synthesized polypeptides are the result of premature chain termination.


Bench surgery for tumor in a solitary kidney. Bench surgery which included ex vivo partial nephrectomy with subsequent autotransplantation was successfully performed on a solitary kidney containing renal cell carcinoma. Helpful adjuncts included perfusion with a hyperosmolar intracellular-like solution, perfusion with a preservation machine and upside-down ipsilateral transplantation.


Dorsal rectotomy and full thickness skin graft for repair of prostatic urethrorectal fistula. Management of a case of postoperative prostatic urethrorectal fistula is presented wherein the standard perineal repair failed. Successful repair was achieved by using a full thickness penile skin patch graft placed through a dorsal rectotomy incision.


A safe, simple and reliable method for percutaneous (closed) renal biopsies in children: results in 100 consecutive patients. A safe, simple and reliable technique for percutaneous renal biopsy in children has been described. Specimens adequate for pathologic interpretation were obtained in 97 patients. Significant complications (gross hematuria lasting more than 36 hours, severe pain, perirenal mass or unexplained fever) occurred in 8 patients. No secondary operations or transfusions were required. No mortailty or late complications occurred.


Renal transplantation in pediatric patients. Thirty-five transplants have been performed in 29 children, 1 week to 16 years old. Of these patients 79 per cent are surviving from 9 months to 14 years post-transplantation. Eighteen of these patients have required different surgical procedures for transplantation than adult patients. Immunosuppressive therapy has been essentially the same as in adult patients. A striking difference between the living related donor and the cadaver donor transplant functional survival as seen in this series is unexplained at the present time. Linear growth has been good in those children who have required minimal doses of corticosteroids to maintain adequate renal function.


Cryptococcus neoformans as a cause of lytic bone lesions. Cryptococcosis is a disseminated infection of man and animals that involves many organs, especially the central nervous system. Isolated bone involvement may cause lytic bone lesions. Diagnosis may be made by pathologic sections and culture of specimens obtained through surgical biopsy. When Cryptococcus is recovered from bone, care must be taken to study other organ systems, especially the central nervous system. Isolated bone disease without meningitis is very uncommom. Intense medical therapy is necessary for treatment of meningitis; isolated osteomyelitis may respond to surgical drainage alone.


Ascorbic acid and the common cold. Evaluation of its efficacy and toxicity. We reviewed the clinical data relating to the efficacy and safety of pharmacologic doses of ascorbic acid in the prevention and treatment of the common cold. Although one study tentatively supports the hypothesis that such doses of ascorbic acid may be efficacious, a second study by the same group did not confirm the significant findings, and no clear, reproducible pattern of efficacy has emerged from the review of all the evidence. Similarly, there is currently little adequate evidence on either the presence or the absence of serious adverse reactions to such doses of ascorbic acid, although many such reactions have been hypothesized. The unrestricted use of ascorbic acid for these purposes cannot be advocated on the basis of the evidence currently available.


Antimicrobial resistance of Shigellae isolated in Michigan. Patterns of antimicrobial resistance were studied for 213 strains of shigellae isolated in rural and urban areas of Michigan by quantitative techniques: 66% of these organisms were resistant to ampicillin, tetracycline, cephalexin, or chloramphenicol or had multiple resistance to various combinations of these antibiotics. The incidence of resistant organisms was higher in the Detroit-Wayne County area, which is the most urban part of the state. Nalidixic acid and oxolinic acid were noted to be quite effective in vitro against shigellae and perhaps warrant further clinical study as potential agents for therapy of shigellosis, especially in view of the appearance of isolates with multiple resistance. Sulfamethoxazole alone was effective against only 62% of the Shigella isolates tested, but mixture with trimethoprim in a 20:1 ratio yielded a highly active in vitro combination.


A method for quantitative detection of tricuspid regurgitation with double injection--single sampling dye-dilution technique. A method for quantitative detection of tricuspid regurgitation is proposed. In cases without tricuspid insufficiency or intracardiac shunt, 1/slope (ts-fv) of the earpiece dye-dilution curve after injection of dye into the femoreal vein (fv curve) was significantly correlated with that (ts-pa) of the curve after injection into the pulmonary artery (PA curve) within each range of right heart blood volume (rbv) calculated as a product of cardiac output and mean transit time difference between FV curve and PA curve. The relationship among the 3 parameters was expressed as a statistically significant regression equation; log (tsfv)  equals 0.9858 log (ts-pa) plus 0.2980 log (RBV) minus 0.6418 (p small than 0.005, sd of predicted ts-fv equals plus or minus 0.591). The assumption was made that tricuspid regurgitation caused prolongation ot ts-fv without any changes in ts-pa or RBV, being based on the concepts of the indicator dispersion and of the path-length distribution. The ratio of tricuspid regurgitation to cardiac output was calculated from (observed ts-fv-predicted ts-fv)/predicted ts-fv, in which predicted ts-fv is the value calculated from ts-pa and RBV using the equation shown above. In 2 patients who manifested clinical tricuspid insufficiency, large amount of tricuspid regurgitant fraction was determined by this method. It is suggested that the present method utilizing double injections--single sampling dye-dilution technique is useful for clinical quantitation of tricuspid regurgitation.


Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. A series of 67 liver biopsies (20 kidney transplant recipients and 47 outpatients with hepatitis) was investigated for the presence of hepatitis B antigen core (HBc) and surface (HBs) components by immunofluorescence and electron microscopy. The variable appearance of the core in liver cell nuclei and of the surface in the cytoplasm allowed the recognition of expression patterns which, together with histologic parameters, could be integrated into four reaction types of diagnostic and prognostic implications: Type I (Elimination Type). No components or only occasional expression of HBc; histologically, classic lobular hepatitis; clinically, acute, self-limited viral hepatitis. Type II (HBc Predominance, or Immunosuppression Type). Abundant core expression in each liver cell nucleus and moderate appearance of HBs; histologically, nonaggressive inflammation (nonspecific reactive or portal hepatitis); clinically, mild, chronic, persistent hepatitis in transplant patients. Type III (HBs Predominance, or Nonaggressive Type). Prominent HBs expression largely in the absence of HBc; histologically, nonaggressive inflammation (nonspecific reactive and portal hepatitis) or normal liver tissue, together with ground-glass hepatocytes in light microscopy, as a correlate of HBs-containing hepatocytes; clinically, hepatitis B antigen carrier, or chronic persistent hepatitis. Type IV (HBc+s Equivalence, or Aggressive Type). Spotty expression of both components, especially of core; histologically, periportal hepatitis; clinically, mainly corresponds to chronic aggressive hepatitis and to acute hepatitis with possible transition to chronicity. As a unifying concept for these types, it is suggested that immune responsiveness determines the reaction pattern, the key mechanism being immune elimination of affected cells. Between efficient elimination (type I) and effective immunosuppression (type II), a graded elimination insufficiency is found in chronic forms (types III and IV), explaining the persistence and probably also the aggressiveness of hepatitis B virus infection.


Exocrine pancreatic dysfunction in guinea pigs with diabetes mellitus. In this study, 37 guinea pigs were classified, on the basis of histologic examination of the pancreas, into three categories: nondiabetic, latent diabetic, and overt diabetic. In order to compare the exocrine pancreatic function in these three groups of animals, pancreatic secretion was collected from each animal following an intravenous infusion of secretin and pancreozymin. Pancreatic enzyme activity, bicarbonate concentration, and the total volume of pancreatic secretion were all significantly decreased in guinea pigs with overt diabetes, but not in those with latent diabetes mellitus. Pancreatic histologic changes characteristic of both latent and overt diabetes were beta-cell hyperplasia and generalized fatty degeneration of the acini. Only the animals with overt diabetes showed total degranulation and severe vacuolation of theta-cells. The same type of exocrine pancreatic dysfunction observed in guinea pigs with spontaneous overt diabetes mellitus is found in human diabetics, and is particularly common in the juvenile type. The guinea pig, therefore, appears to be a suitable animal model for the study of human juvenile diabetes mellitus.


Experimental focal glomerular lesions elicited by insoluble immune complexes. Ultrastructural and immunofluorescent studies. Injection of small amounts of ferritin intravenously, into the aorta (above the renal arteries) or into the left renal artery of rabbits hyperimmunized against this protein, results in the formation of circulating, insoluble, antigen-antibody complexes. Some of these complexes localize focally in the renal glomerular capillaries, where they elicit severe lesions. The fate of the complexes and the evolution of the lesions have been followed by immunofluorescent and electron microscopic techniques. Within a few hours, the deposition of complexes in the glomeruli resulted in a massive accumulation of neutrophils platelets, and fibrin, sometimes leading to acute focal necroses of some loops. These lesions were quite similar to those developing in the small dermal vessels during the Arthus reaction. Most of the complexes were rapidly phagocytosed and degraded by neutrophils; swelling and proliferation of endothelial and mesangial cells usually followed the acute damage and contributed to the removal of remaining complexes, cell debris, and fibrin deposits. Later, focal areas of mesangial proliferation and sclerosis were observed, containing large amounts of basement membrane-like material and sometimes of collagen fibrils; synechiae and crescent formation were noted in certain places. These observations suggest that the glomerular localization of very large, poorly soluble or insoluble immune complexes may be responsible for the focal glomerular changes seen in association with subacute bacterial endocarditis, with anaphylactoid purpura, or for some of the most severe lesions developing during chronic immune complex diseases.


Experimental glomerulonephritis in the guinea pig. II. Ultrastructural lesions of the basement membrane associated with proteinuria. Humoral immune processes mediate alterations in glomerular basement membrane (GBM) permeability by two mechanisms. One requires complement and polymorphonuclear leukocytes and the second is complement- and polymorphonuclear leukocyte-independent. The structural basis for enhanced GBM permeability induced by anti-GBM antibody is not clear. Experimental anti-GBM glomerulopathy was induced in guinea pigs by immunization with human GBM in complete Freund's adjuvant. Control animals received injections of complete Freund's adjuvant alone. Light, immunofluorescent, and electron microscopic studies were done on eight heavily proteinuric animals, four immunized nonproteinuric animals, three controls, and two normal animals. All animals that received injections of GBM had intense linear deposits of gamma2 anti-GBM antibody. Complement deposition was not demonstrable in vivo, and anti-GBM antibody deposits did not fix complement in vitro. Histologic abnormalities in proteinuric animals were confined to the GBM, which was of variable density and had a characteristic beaded thickening, with numerous areas of electron lucency most prominent in the outer aspect of GBM in peripheral portions of capillary loops. The inner margin and endothelium were normal. Ultrastructural tracer studies with ferritin demonstrated increased permeability confined to portions of GBM demonstrating ultrastructural lesions. The urine protein excreted by animals with ultrastructural GBM lesions was largely albumin. The absence of complement deposition accompanying anti-GBM antibody deposits in vivo and the unique GBM lesion in this model differ from the findings in nephritis induced by most heterologous nephrotoxic antibodies and suggest that GBM injury in this model is mediated by autologous antibody through complement-independent mechanisms. The selective proteinuria and ultrastructural lesions suggest a derangement in glomerular permeability functionally localized to the epithelial side of the GBM and could reflect an antibody-mediated abnormality in GBM biosynthesis.