Divergent effects of cyclophosphamide administration on mononuclear killer cells: quantitative depletion of cell numbers versus qualitative suppression of functional capabilities.

The effects of various regimens of cyclophosphamide administration on guinea pig peripheral blood leukocytes were studied. Cyclophosphamide-induced immunosuppression was assessed by the effect of drug administration on the proportions and absolute numbers of leukocyte populations, and by the effect on functional capabilities of unfractionated and adherent cell-depleted mononuclear cell suspensions as measured by the PHA-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity assays against chicken erythrocyte targets. Intraperitoneal administration of five daily doses of cyclophosphamide (5 mg/kg) caused a modest absolute leukopenia but no change in cytotoxic effector function of the mononuclear cells remaining in the circulation. As the dosage of cyclophosphamide was increased to 20 mg/kg/day to produce a pronounced leukopenia, a profound neutropenia (less than 300 polymorphonuclear leukocytes/mm3) together with a marked decrease in mononuclear cell effector function was noted. A single i.p. injection of cyclophosphamide (100 mg/kg), which produced identical degrees of leukopenia of each leukocyte class as did daily administration of cyclophosphamide (20 mg/kg/day), caused no change in mononuclear cell effector function when compared to saline controls. Complement receptor-bearing and Fc-receptor bearing mononuclear cells were decreased to the same degree by both regimens of cyclophosphamide administration. Removal of adherent cells from mononuclear cell suspensions by column purification resulted in a marked decrease in cytotoxic effector function at low effector to target ratios. At higher effector to target ratios there was no difference in cytotoxic effector function between unfractionated and column-purified cells. In contrast, the functional defect in mononuclear cell suspensions from animals that received five daily doses of cyclophosphamide (20 mg/kg) could not be compensated for at higher effector to target ratios, indicating that this functional defect was not an artifact of relative depletion of monocytes by cyclophosphamide, but was due to an actual suppression of the effector functional capabilities of the killer cells. This study indicates that, dependent on the particular regimen of drug administration, the quantitative depletion of mononuclear cell populations by cyclophosphamide administration can be clearly distinguished from the qualitative effect on certain functional capabilities of surviving cells.


Hybridization kinetics of ribonucleic acid and deoxyribonucleic acid from antigen-stimulated mouse spleen cells.

In an attempt to explore the mechanism by which antigenic stimulation alters gene expression in lymphoid cells in vivo, three different hybridization techniques have been used to compare the complexity of the genome of lymphoid cells from normal and from immune BALB/c mice. RNA/DNA hybridization experiments at a DNA excess of 1000 demonstrated that normal RNA and immune RNA hybridized identically with DNA extracted from mouse spleen cells before and after immunization and with liver DNA. These findings indicate that DNA sequences complementary to immune RNA or to normal RNA are represented in a number of copies not significantly different in the genome of normal lymphoid cells and in that of immune lymphoid cells. Hybridizations of normal and of immune RNA with normal and immune pulse-labeled DNA, done at RNA excess, detected no differences between these two DNA. However, significant differences were observed in the percentage of DNA hybridized with normal and immune RNA; 3 to 4% of the DNA hybridized with normal RNA and 8 to 9% with immune RNA. This indicates that more DNA sequences are transcribed 48 hr after immunization than before immunization. The RNA exhaustion rates caused by normal and immune DNA were found to be identical, indicating that antigenic stimulation did not induce major changes in the number of DNA base sequences complementary to the RNA tested. However, when normal and immune pulse-labeled RNA were compared by exhaustion with DNA, the immune pulse-labeled RNA obtained 48 hr after immunization displayed a slower exhaustion rate than normal RNA or RNA extracted 72 hr after immunization. These results suggest the temporary synthesis, at a higher frequency, of certain RNA sepcies 48 hr after immunization, as compared to the RNA synthesis in normal, nonimmune cells, or that occurring 72 hr after immunization. Thus, the three experimental approaches used lead to the conclusion that antigenic stimulation does not induce major gene amplification; it does, however, change the transcription rate of certain RNA species.


The ultrastructure of C1t, a subcomponent of the first component of complement: an E.M. and ultracentrifuge study.

C1t is a 9.5 S alpha1 glycoprotein that has been shown to be a fourth subcomponent of the first component of complement. The m.w. of C1t was found to be 233,000 by sedimentation equilibrium in the ultracentrifuge. A subunit m.w. of 23,000 was obtained by sedimentation equilibrium in 5.95 M guanidinium chloride. No change in either m.w. was produced by prior reduction and alkylation. In the electron microscope characteristic pentagonal figures of 85 A diameter were observed together with rod-like figures which appear to be stacked assemblies of the pentagonal figures. These observations lead us to propose that C1t is a noncovalent, decameric protein with the subunits disposed at the vertices of two regular pentagons joined at one of their faces. A possible relationship between C1t and the P-component of amyloid is discussed.


In vitro production of IgE by human peripheral blood lymphocytes: effect of cholera toxin and beta adrenergic stimulation.

Peripheral blood lymphocytes from two human donors with elevated serum IgE concentrations were maintained in short term tissue culture preparations. Repeated culture preparations demonstrated that IgE was produced in vitro in amounts that could be measured by the double antibody radioimmunoassay technique. The amount of IgE produced by replicate cultures of cells from a single bleeding of the donor was similar when the cultures were simultaneously prepared. In contrast, IgE production by the same donor's lymphocytes varied when the culture preparations were initiated from separate bleedings. The results of simultaneous cultures of a single bleeding were sufficiently consistent to provide a means of testing the effect of pharmacologic agents on the in vitro production of IgE. Cholera toxin effected a marked reduction in production of IgE by lymphocytes of both cell donors. Isoproterenol showed marked inhibition of IgE production at 10(-3) M but cell viability studies suggested that this may have been due to decreased cell viability. At lower, nontoxic concentrations of isoproterenol (10(-4) M-10(-6) M) slight but definite inhibition of in vitro IgE production was evident. This inhibition was more pronounced subsequent to the first 24 hr of exposure of the cells to isoproterenol.


Macroscopic cloning assay using complement fixation to isolate secretion variants of myeloma cells.

Mouse myeloma cells were cloned in agar and overlaid in antiserum to a mouse immunoglobulin. The resulting local immune precipitates were used as complement fixing layers to protect macroscopically visible zones of sheep erythrocytes from lysis. The immunological specificity of the assay was confirmed in mixing experiments using two different myeloma cell lines.


A comparison of the direct and indirect leukocyte migration tests in man.

In the Direct Leukocyte Migration Test, as few as 10% mononuclear cells are sufficient to produce antigen-specific inhibition of polymorphonuclear leukocyte migration in the presence of 100 mug of excipient-free PPD per ml. A clear distinction between tuberculin-positive and tuberculin-negative donors can be made. The inhibition of migration is blocked by the presence of actinomycin D. In the Indirect Test, significant inhibition of migration of pure polymorphs is produced by supernatants from stimulated tuberculin-sensitive lymphocyte cultures. Inhibition is maximal at 6 h and is of a similar degree to that obtained in the direct test.


A method for producing anti-DNA antibodies in ascitic fluid of mice.

Immunological methods for the production of antibodies to DNA in ascitic fluid of DDY, A/He, and C57B1/6 mice were investigated. Relatively large volumes of ascitic fluid containing antibodies for DNA in high concentration were obtainable after seven injections of denatured ssDNA-MBSA complexes emulsified with CFA into peritoneal cavities of DDY mice. The quantity of anti-ascitic fluid from 4 to 5 DDY mice was equivalent to that of antiserum from one rabbit. DDY mice accumulation of ascites is much greater than in other strains, and the ascitic fluid contains antibodies of higher titer regardless of sex. The antibody titers in ascitic fluid are not affected by alteration of the ascites volume, and are proportional to those of the corresponding sera. Antibodies to DNA in ascitic fluid of these mice occurred exclusively in IgG as in DNA antisera.


An adjuvant effect of poly A - poly U on production of antibodies to DNA in rabbits.

The effect of polyadenylate-polyuridylate copolymer (poly A-poly U) on the formation of anti-DNA antibodies in rabbits was investigated. 1) Poly A-poly U proved to be an effective adjuvant for production of antibodies to DNA. These were elicited in rabbits at high titer and were formed at an early stage of immunizations. 2) The antiserum obtained reached potently with denatured DNA, but with neither methylated bovine serum albumin (MBSA) used as carrier protein nor poly A-poly U used as adjuvant. This was demonstrable both by C-fixation and double diffusion tests. 3) The antibodies occurred exclusively in the IgG fraction separated by Sephadex G-200 column chromatography.


A micromethod for evaluating the phagocytic activity of human macrophages by ingestion of radio-labelled polystyrene particles.

Studies in vitro of human macrophage function in health and disease have been impeded by the difficulty of obtaining such cells in sufficient number. Unlike animal species, the only readily available source of human macrophages are circulating monocytes. Herein, a method is described whereby the phagocytic rate of small numbers of glass-adherent mononuclear cells can be accurately measured. The method utilizes the ingestion by macrophages of technetium labelled polystyrene particles; both the radiolabel and ingestible substrate are readily available and the labelling process simple and efficient. The phagocytic rate can be expressed as radioactive counts per microgram of cell protein; data is also presented showing that the number of particles ingested per cell can be accurately derived.


Empirical and theoretical relationships between the sedimentation coefficient and molecular weight of various proteins, with particular reference to the immunoglobulins.

The empirical and theoretical relationships between the sedimentation coefficient and molecular weight of proteins of various conformational shapes have been investigated. In each case it was found that the relationship obeys the general formula log S020,W= a log molecular weight +b. The empirical values obtained for the slope and intercept for globular proteins (non-immunoglobulins) and immunoglobulins have been used to calculate the molecular weight from the sedimentation coefficients of other proteins belonging to these two groups. For most proteins and particularly the immunoglobulins the values obtained were found to be similar to those obtained using the classical experimental techniques. The application of this approach to the determination of molecular weights, of proteins and more particularly protein containing complexes is discussed.


A simple numerical method for the construction of isokinetic sucrose density gradients, and their application to the characterisation of immunoglobulin complexes.

A simple numerical method for calculating the parameters required for the production of isokinetic sucrose density gradients is described. Calculation is based on tables of viscosity and density of sucrose solutions at various concentrations. When density gradients prepared according to this method were used for centrifugation of a number of well characterised proteins, it was shown that isokinetic behavior occurs, and that sedimentation coefficients obtained are comparable to those determined by conventional analytical ultracentrifugation. The method was used to characterise polymers of IgM and IgG, and results suggest that IgM polymers up to the tetramer can occur in Waldenstrom's macroglobulinaemic serum, and that IgG polymers up to the pentamer can occur in stored specimens of ethanol precipitated human IgG. The sedimentation behaviour of 125I-IgE was studied by fractionating the gradients and counting the fractions, and it was shown that degradation of the labelled IgE took place during storage. It was also found that labelled IgE induces rapid breakdown of IgG with which it was mixed prior to centrifugation. The method has also been used to study immunoglobulin-containing complexes found in the knee joint fluids of patients with rheumatoid arthritis. The high degree of resolution of this method is demonstrated by the fact that many more of these components were detectable by density gradient centrifugation than by conventional analytical ultracentrifugation.


The use of sucrose as a cryoprotective agent for the labile sheep erythrocyte-complement component--intermediate complex, EAC142.

Sucrose (15% w/v) was found to be a useful agent for cryopreservation of sheep erythrocytes in the intermediate state EAC142. The ordinarilly very labile SAC142, associated with the cells, did not decay to any appreciable degree during periods of storage of 1, 72 or 132 days at -65 to -70 degrees C. Recovery of erythrocytes after freezing and thawing varied. Our best recovery was 76.6% and the poorest was 43.0%. The poorest recoveries were obtained during the latter part of the 132 day experiment after the freezer malfunctioned on day 35 causing a temperature rise to -50 degrees C which lasted for 3 days. The average spontaneous lysis of cells before freezing and thawing was 5.6%. The average spontaneous lysis for cells which had been stored in the frozen state was 6.9%.


Serological techniques for detection of antibody to galactocerebroside.

Two serological techniques were developed for the detection of antibody to galactocerebroside using liposomes as a carrier for the lipid hapten. One assay is a radioimmunoprecipitation test employing [3H] cholesterol as a marker in the galactocerebroside-liposomes. The other is a less sensitive but quick and easy galactocerebroside-liposome agglutination assay. Specificity is demonstrated by comparison of titers when other lipid haptens replace galactocerebroside in the liposomes, and when other anti-glycolipid antisera are reacted with galactocerebroside-liposomes.


Immunology of DNA. IV. Quantitative aspects of the Farr assay.

We have investigated whether the Farr assay measures the primary interaction between DNA and anti-DNA. By the use of zonal centrifugation to analyse DNA/anti-DNA complexes and by a detailed study of the quantitative aspects of the Farr assay we demonstrate that: (a) binding of a single IgG molecule to a PM2 DNA molecule (molecular weight 5.9 X 10(6)) renders the latter precipitable in half-saturated ammonium sulphate; (b) two antibody molecules are required for precipitation of T7 DNA (molecular weight 25 X 10(6)); (c) millipore filtration detects binding of a single antibody molecule per molecule of PM2 DNA; (d) theoretically and experimentally the relation between free DNA (F) and the amount of serum (S) can be described by IfF =-cS where c is a constant; (e) it is feasable to quantitate serum anti-DNA activity in terms of units per ml.


Radioimmunoassay for the aminoterminal peptide of procollagen p alpha 1(I)-chain.

Peptides derived from the aminoterminal portion of the palpha1(I)-chain of calf and sheep procollagen were labeled with iodine-125. Despite changes in electrophoretic homogeneity after labeling, reaction of the labeled peptide with antisera to unlabeled peptide was retained. Antisera to procollagen or the isolated procollagen peptide showed high titers for the native peptide, a much weaker binding with the reduced and alkylated peptide and little or no reaction with collagen. Antisera to collagen showed strong binding with collagen and a weaker but distinct reaction with the procollagen peptide. Evidence was obtained that a minor contaminant of procollagen peptide was present in the acid-extracted collagen and that there were no shared antigenic determinants. Bovine serum and amniotic fluid contained 1-10 mug/ml reactive antigen. These results indicate that the labeled peptides can be used as a specific, sensitive and accurate assay for the amino-terminal portion of procollagen in biological samples.


The use of nylon fibre as a method of separating human lymphocyte subpopulations.

When the mononuclear cell population separated from normal human blood is passed through a nylon fibre column, monocytes and surface Ig bearing cells are retained; the eluted cells are a mixed properties. It appears that a proportion of the T cells eluted form EA-rosettes, and the eluted cell subpopulation includes non-T cells which share with B cells the ability to bind both EA and aggregated human IgG.


Carbodimide coupling to formalin-fixed red blood cells. A direct passive hemagglutination test for anti-horse ferritin.

The water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, has been used to couple horse ferritin covalently to formalin-fixed, human red blood cells. This method has been optimized with respect to sensitivity, range, and reproducibility. The sensitized cells are stable for at least 6 months at 4 degrees C, and they may be stored frozen without additives or special precautions. Titers of 1/216 are routinely obtained with antisera to ferritin.


The use of frozen-stored sheep erythrocytes for demonstrating lymphocytic membrane markers.

The usefulness of cryopreserved sheep erythrocytes (unsensitized or coated with fluorescein, IgG-type antibody or complement) for marker tests on human blood lymphocytes was studied. It appears that the rosetting properties or freshly prepared or frozen-stored erythrocytes are not significantly different. The method may be used to provide homogeneous stocks of sensitized erythrocytes for lymphocyte rosetting technique.


A radioimmunoassay for antibodies against surface membrane antigens using adhering cells.

A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit 125I-anti-mouse-Fab. The bound radioactive material was eluted with glycine-HCI buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolytic or noncyctolytic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface.


A simple method of lymphocyte purification from human peripheral blood.

We present a simple technique for obtaining cell suspension from human peripheral venous blood containing on average 96% lymphocytes, no macrophages and leukocyte red blood cell ratio 1 : 1. Cells are readily prepared under sterile conditions and can be used in culture experiments. The high yield of lymphocytes and the avoidance of Ficoll-Triosil makes the technique especially valuable for micro cytotoxicity tests such as those aimed at measuring tumour immunity.


Countercurrent immunoelectrophoresis on cellulose acetate.

A technique for the detection and demonstration of antigens or antibodies by means of countercurrent immunoelectrophoresis on cellulose acetate (CAM) is described. The method is simple, sensitive and rapid. There is no need for preliminary preparation. The samples are contained in wells punched in the CAM. The equipment required is commonly available in routine laboratories, and the whole procedure can be performed and the results obtained within 90 min.


Inhibition of thymidine uptake by staphylococci, a new method for the investigation of phagocytosis.

A radioassay employing (3H) thymidine, to measure inhibition of growth of Staphylococcus aures by human phagocytes is presented. The principle of this new method is that viable and dividing staphylococci take up thymidine more rapidly than white cells, so that whereas in control cultures containing staphylococci alone high counts per minute (cpm) are obtained within 90 min of incubation in test cultures both leucocytes and plasma from 25 normal subjects reduced the cpm, following ingestion and killing of staphylococci.


Semi-automated method for the enumeration of cytotoxicity results.

This communication provides information on the design and use of a counting machine that enumerates adherent cells in cytotoxicity assays. To date, investigators have relied on inverted microscopes or quantimat automatic image analysis. The former method is tedious and time consuming while the latter method is prohibitively expensive. The counting machine which consists of projection microscope attached to an electronic counter is as accurate as the inverted microscope but is both less tedious and less time consuming.


A modified technique for the radioallergosorbent test.

A modification of the radioallergosorbent test (RAST) is described utilizing a multiple automated sample harvester. The technique increases the efficiency of the procedure by shortening the time required for its performance. A study is reported in which this method is compared to that recommended by the manufacturer.


Common origin and evolution of variable and constant regions of immunoglobulins.

Sequence data show that the immunoglobulins evolved from two sets of paralogous genes: a gene set coding for the V regions and another for the  different C regions. A comparison of sequences from these two gene sets shows homology between the V and C sets of genes: this homology is only significant when VH is compared with Cmu1, Cmu2 and Cgamma1. There is a close agreement between our data drawn from sequence comparisons and the data of Poljak et al. (1974) drawn from crystallographic data. This finding is in agreement with the results of the phylogenetic trees of the C and V gene sets: they suggest that the VH subgroups and the first constant domain of the heavy chains are the most ancient. Moreover homology between the red blood cell glycophorin and Cmu2 suggests that immunoglobulins could have a common origin with some membrane proteins.


Immunochemical observations on the human blood group P system.

Haemagglutination inhibition experiments were carried out with anti-P1, anti-Pk and anti-P sera in an attempt to increase understanding of the chemical, genetical and serological relationships within the P system. The test-substances comprised a glycoprotein with human blood group P1 and Pk activity isolated from sheep hydatid cyst fluid, fragments isolated from the partial acid hydrolysis products of the P1Pk active glycoprotein, glycolipids, monosaccharides and di- and oligo-saccharides of known structure. The trisaccharide alphaGal(1 leads to 4)betaGal(1 leads to 4)GlcNAc isolated from the glycoprotein hydrolysis products, and earlier established as the P1 determinant (Cory et al., 1974), was the only low molecular weight compound that gave strong inhibition with human, rabbit and goat anti-P1 sera. A disaccharide alphaGal(1 leads to 4)Gal, also isolated from the glycoprotein hydrolysis products, failed to react with anti-P1 reagents but inhibited human anti-Pk sera as strongly as the trisaccharide. The glycolipid alphaGal(1 leads to 4)betaGal(1 leads to 4)Glc-Cer (Ceramide trihexoside) and other oligosaccharides containing alphaGal(1 leads to 4)Gal at the non-reducing terminal were also strong inhibitors of anti-Pk sera. Oligosaccharides with terminal alpha-galactosyl residues joined in other positional linkages gave definite, although less strong, inhibition. The inhibition results suggest a close structural relationship between the P1 and Pk determinants and indicate that the specificity of anti-Pk sera is less closely delineated than that of anti-P1. Human anti-P sera differed markedly from anti-P1 and anti-Pk and were not inhibited by any of the compounds containing alpha-galactosyl residues. The glycolipid betaGalNAc(1 leads to 3)alphaGal(1 leads to 4)betaGal(1 leads to 4)Glc-Cer (globoside) strongly inhibited the anti-P sera. The inhibition of anti-Pk and anti-P sera by ceramide trihexoside and globoside, respectively, confirms the observations of Naiki & Marcus (1974) and supports their conclusions that Pk is the precursor of P. The genetic relationship of the P1 antigen to P and Pk is not clear but biosynthetic pathways are discussed that might explain the absence of P1, Pk and P antigens in individuals of the p phenotype.


Inactivation of HL-A antigens in vitro by action of antibiotics.

The effect of antibiotics on lymphocyte HL-A antigens in vitro is of variable character. All antibiotics under examination suppressed the absorption capacity of HL-A antigens after 2 hr of lymphocyte treatment at 37 degrees C. Ceporin and Kanamytrex inhibited even the cytotoxic reactivity of Hl-A antigens after 15-30 min of lymphocyte treatment. Chloramphenicol, aureomycin, streptomycin and oleandomycin, on the contrary, increased the specific cytotoxic reactivity of HL-A antigens after 15-30 min, after 1 hr they were ineffective for HL-A antigens, and after two or more hours they produced polyreactivity. Penicillin and erythromycin produced polyreactivity after only 15-30 min. The results show that for the follow-up of the drug effect on HL-A antigens the absorption test rather than the cytotoxicity test is of importance. The suppressed absorption capacity of HL-A antigens caused by the action of antibiotics proves their inactivation effect on the lymphocytes. The possibility of an analogous effect of antibiotics on lymphocyte HL-A antigens, even after administration to patients, is discussed.


The component fragments obtained by acid dissociation of papain-solubilized H-2 molecules.

H-2Kk and H-2Dd molecules were specificially purified from a radioiodinated H-2a preparation obtained by papain digestion of spleen cell membranes of A/J strain mice. The molecules were isolated by binding to H-2 alloantisera of the corresponding private specificity followed by precipitation with rabbit anti-mouse IgG antiserum. The specifically precipitated radioiodinated H-2Kk and H-2Dd molecules were dissociated by acid treatment into large and small components of about 37,000 and 11,000 respectively. These were separated by gel filtration at acid pH or by gel isoelectric focusing in the presence of 6 M urea. Each component separated by gel filtration of the acid-dissociated H-2 molecules showed a high degree of size homogeneity as determined by sodium dodecyl sulphate-acrylamide gel electrophoresis. Upon gel isoelectric focusing, however, the small components showed two peaks of radioactivity closely located together at pH 7-8, both of which had a restricted pH range, while the large components gave one peak of a relatively wide pH range of pH 5-6. The H-2Kk and H-2Dd molecules gave essentially the same pattern in terms of the numbers and the positions of the radioactivity bands. Under the iodination conditions used the large components of H-2Kk molecules contained more radioactivity than the small components, while the reverse was true in case of H-2Dd molecules. Such a difference was also found with H-2Kk and H-2Dd molecules isolated by use of alloantisera of the respective public specificity. The assay of binding of the isolated components with H-2 alloantisera of defined specificity revealed that the large components retain most of the allospecificities of the parental H-2 molecules. No H-2 allospecificities were found on the small components. The small components showed extensive binding with rabbit antiserum against mouse beta2-microglobulin. The same antiserum did not show any binding with the large components. On the other hand, both of the components did bind with rabbit antiserum against papain-solubilized H-2 molecules.


Immune response gene (Ir-SRBC) exerts its effect via macrophages in inbred rats.

In the previous report, strain differences of inbred rats were investigated in the antibody response to sheep erythrocytes (SRBC). A low-responder strain (Fischer rats) produced only IgM antibody, but other high-responder strains switched over from IgM to IgG antibody to SRBC. Pretreatment with incomplete Freund's adjuvant made Fischer rats a high-responder. These results seemed to indicate that there are malfunctions of macrophages, together with T cells, in Fischer rat strain. In this report, strain differences in the phagocytic activity of macrophages were examined using radiolabelled SRBC. High uptake of SRBC by the spleen in the low responder rats, and vice versa, was confirmed in various experimental conditions. Genetic analyses were made of the specific spleen uptake of the radiolabelled SRBC in backcrossed rats. The result clearly showed that the degree of spleen uptake of the radiolabelled SRBC is genetically determined by a single gene and this property itself has a close negative correlation with the ability of each rat to produce haemolysin against SRBC. These results strongly suggest that the Ir-SRBC gene exerts its effect, at least in part, via macrophages. The relationship between the function of the macrophage and the antibody response is discussed.


Juvenile diabetes mellitus: HLA-antigen frequencies dependent on the age of onset of the disease.

A close correlation between juvenile mellitus and HLA-B8, HLA-BW15 and HLA-CW3 was found. Association of these antigens with juvenile diabetes mellitus was closely dependent on the ages of onset of the disease. Frequencies of BW15 and CW3 showed a remarkably low incidence in the childhood diabetics (0-15 years old) and were found increased in the patients with later (16 years or older) onset diabetes. HLA-B8 frequencies were found increased in all the groups of diabetics with different ages of onset. These findings point to the importance of HLA-B8 in childhood and HLA-B8, BW15 and CW3 in the later onset juvenile onset diabetes mellitus (JOD).


Effect of antibiotics and chemotherapeutics on the cytotoxic reaction between HL-A antigens and corresponding sera.

Effects of antibiotics and chemotherapeutics on the interaction between the HL-A antigens and the corresponding lymphocytotoxic sera vary from case to case. Streptomycin, oleandomycin, cephaloridine and kanamycin completely inhibit the cytotoxic reaction when added in the first or second stage of the test. Cytembena weakens the reaction while chlortetracyclin exhibits various effects. The rest of the drugs under investigation proved ineffective.


Genetic studies in inbred rats. VII. Tentative model for the major histocompatibility complex.

Several lines of evidence are presented which support a tentative model of the major histocompatibility complex (MHC) of the rat. The serologically defined (SD), lymphocyte defined (LD) and immune response (Ir) loci are separate but linked, and there are probably two loci controlling the serologically defined antigens. The MHC in the rat appears to resemble that in the human more than that in the mouse.


Synthesis of DNA in isolated nuclei from differentiated mammalin epidermal cells.

Epidermal spinous and granular cells from the newborn rat neither replicate their nuclear DNA nor proliferate in vitro under conditions which support both processes in basal cells. However, as shown by autoradiography, (3H)thymidine and (14C)bromodeoxyuridine do label nuclei removed from spinous cells but not from granular cells. CsCl density gradient centrifugation of DNA obtained from early differentiated nuclei which had been exposed in vitro to (14C)bromodeoxyuridine, indicated that a considerable level of the tracer was present in the nucleic acid and suggested that replication of the genome had occurred. Therefore, spinous cells appear to retain the capability of reproducing nuclear DNA. Since differentiated cells appear to have the "diploid" level of DNA, these observations point to the replication of DNA as a possible locus of the mitotic inhibition which is coincident with epidermal differentiation.


The effect of MSH on thymidine incorporation by keratinocytes in the epidermal melanin unit.

Epidermal melanocytes were observed in the black but not in the white skin of black-and-white spotted guinea pigs. In experiments designed to determine whether melanocyte-stimulating hormone (MSH) affects the incorporation of thymidine by kerationcyte nuclei of the epidermal melanin unit, the labeling index was the same in all skin before MSH administration. After MSH injections, the level of (3H)thymidine incorporation in keratinocytes increased significantly in black skin but not in white. We suggest that through the mediation of melanocytes MSH indirectly afffects keratinocytes in the epidermal melanin unit.


Trace-element concentrations in human hair measured by proton-induced x-ray emission.

Proton-induced x-ray spectroscopy has been used to measure trace elements concentration ratios for 10 elements relative to Zn on a group of the population in the harbor city of Rijeka, Yugoslavia. The average biologic levels of elemental ratios for the inhabitants of this region have been established. The presence of pollutants has been confirmed by studying the variation of elemental concentration ratios along single samples of hair.


Percutaneous absorption of dexamethasone estimated by a plasma radioimmunoassay.

Precutaneous absorption of dexamethasone and its effect on the pituitary adrenal axis were measured in vivo in normal human subjects after application to skin. Specific plasma dexamethasone and cortisol radioimmunoassays were used. Following application of 1% dexamethasone on 500 cm2 of normal skin, the plasma dexamethasone concentration was maximal at 2 hr, and the average absorption was 0.25% over 8 hr; significant cortisol suppression occurred at 2, 4, and 8 hr. This technique: (1) provides an accurate assessment of the in vivo absorption of dexamethasone applied to human skin, (2) avoids exposure of the subjects to radioactive steroids, (3)permits estimation of the quantity of unmetabolized steroids absorbed, and (4) serves as a possible model for the development of similar assays for other topical steroids.


Percutaneous penetration and metabolism of topical (14C)flutamide in men.

This study was designed to determine the fate of the nonsteroid antiandrogen flutamide in men following a single 6-hr topical application of 5 mg 14C-labeled drug dissolved in 50% ethanol/50% propylene glycol. Analysis of 0-120 hr urine shows at least 16% of the applied flutamide is absorbed. Fifty-six percent of the dose is recovered from the site of application with cotton swabs moistened with 50% ethanol/50% propylene glycol. Flutamide plasma levels peak in 4 to 6 hr at about 1.3 ng/ml and then decline rapidly to about 0.08 ng/ml 24 hr after application. Only 13% of plasma 14C is associated with flutamide 6 hr after drug application. There are at least 10 plasma metabolites, of which 6 have been tentatively identified. These are alpha, alpha, alpha-trifluoro-4'-amino-m-acetotoluidide (A); alpha, alpha, alpha-trifluoro-4'-amino-2-methyl-m-lactotoluidide (B); alpha, alpha, alpha-trifluoro-4'-nitro-m-acetotoluidide (C); alpha, alpha, alpha-trifluoro-2-methyl-4'-nitro-m-lactotoluidide (D); alpha, alpha, alpha-trifluoro-4'-amino-2-methyl-m-propionotoluidide (E); and alpha, alpha, alpha-trifluoro-6-nitro-m-toluidine (F). (D) is the major plasma metabolite, and its concentration exceeds flutamide's between 8 and 24 hr after drug. All the plasma metabolites are found in 0-24 hr urine in minor amounts. An additional metabolite, alpha, alpha, alpha-trifluoro-amino-5-nitro-p-cresol (G), accounts for 27% of urine 14C.


Epilogue as prologue. The skin: continual source of biologic problems and discoveries.

In much the same way as the survival of many species depends upon their abundant production of such genetic materials as pollens and spermatozoa, so does scientific progress depend upon a continual, lavish production of basic scientific works. In stimulating assembling, and disseminating various scientific studies related to the skin, the Montagna Symposia on the Biology of Skin have ensured progress not only in the understanding and management of skin was, is, and probably will always will be one of the richest sources of basic biologic information. No scientific discovery is wasted, no accurate clinical observation useless. No one can foresee when or how some seemingly irrelevant discovery in pure science or some seemingly insignificant clinical observation will yield fruits of incalculable practicable value. We cite here a few examples of some of the best results from apparently unrelated research and isolated clinical findings. In dermatologic research, the circular rhythm from patient with cutaneous disease to laboratory and from laboratory back to patient creates a centrifugal force that often spins off valuable discoveries.


Percutaneous absorption after twenty-five years: or "old wine in new wineskins".

Two developments have greatly added to our knowledge of percutaneous absorption over the past quarter century: (1) proof that the entire stratum corneum is the effective barrier layer of the skin, and (2) the advent of highly sensitive analytical techniques. The first enabled us to measure permeation rates on excised skin and the second greatly extended the kinds of substances that could be studied in vivo and in vitro. The process of percutaneous absorption is beginning to be understood more quantitatively. New light has been shed on the elementary mechanisms of permeation, the site of diffusional resistance at the molecular level, the effect of specific functional groups on solute permeation, the role of skin appendages, and the effects of vehicles.


Clearance of gentamicin during hemodialysis: comparison of four artificial kidneys.

The effects of four types of artificial kidney on dialyzer clearance rates and serum pharmacokinetics of gentamicin were compared. In 19 patients undergoing chronic hemodialysis, the mean (+/-SE) interdialysis half-life of gentamicin in serum was 49.3 +/- 3.5 hr, whereas during dialysis this value was reduced to 10.0 +/- 0.7 hr. The mean half-life of gentamicin in serum at conventional flow rates for the Hollow Fiber Kidney, Kiil, Gambro, and Coil dialyzers was 11.3, 10.9, 8.2, and 7.4 hr, respectively, and the mean values for clearance of gentamicin were 26,28,42, and 48 ml per min, respectively. For all dialyzers, rates of clearance of gentamicin increased linearly with plasma flow rate over the flow range studied. The Gambro and Coil dialyzers had significantly higher rates of clearance of gentamicin from serum (P less than 0.05) than the Hollow Fiber Kidney and Kiil dialyzers over a wide range of clinically useful plasma flow rates (119-300 ml per min), whereas the Kiil dialyzer cleared gentamicin more effectively (P less than 0.05) than the Hollow Fiber Kidney dialyzer over a more limited interval (117-177 ml per min). Therapeutic recommendations for patients undergoing hemodialysis were made in light of current findings.


Immunity to intranasal challenge with Rubella virus two years after vaccination: comparison of three vaccines.

The persistence of antibodies was studied in 143 women two years after postpartum rubella vaccination. In addition, the effect of intranasal revaccination with the RA 27/3 strain was studied in 99 women after two years. All women had responded to primary immunization with the development of hemagglutination-inhibiting antibodies. Intranasal revaccination resulted in a significant rise in titer of hemagglutination-inhibiting antibodies in 9% of the vaccinees who received the RA 27/3 strain compared with 53% and 24% of those who received the Cendehill and HPV-77 DE-5 vaccines, respectively. The difference between the response in the RA 27/3 group and that in the C Cendehill group was statistically significant (P less than 0.001), as was the difference between the response of the latter group and that of the HPV-77 DE-5 group (0.03 greater than P greater than 0.01). The susceptibility to intranasal challenge was found to be related to low levels of serum hemagglutination-inhibiting antibodies. The titers of hemagglutination-inhibiting antibody were well maintained during the follow-up period, but three of the 52 Cendehill vaccinees had titers of less than 1:10 after two years.


Protein binding and concentrations of cephaloridine and cefazolin in serum and interstitial fluid of dogs.

Percentages of free and protein-bound cefazolin and cephaloridine in serum and interstitial fluid of dogs were determined by ultrafiltration and microbiologic assay. The percentages of cephaloridine and cefazolin bound to protein in serum were 10% and 80%, respectively. In interstitial fluid accumulating within tissue-embedded polypropylene capsules, 29% of cefazolin was bound to protein, and cephaloridine was unbound. Both antibiotics rapidly penetrated the interstitial fluid and attained measurable levels 5 min after intravenous administration. Levels of total cefazolin in the interstitial fluid were generally higher than those of cephaloridine; however, concentrations of free cephaloridine in the fluid exceeded the levels of free cefazolin after the first 15 min. Binding of anitbiotics by serum proteins does not restrict such agents to the intravascular space, since a highly protein-bound compound has been shown to penetrate interstitial fluid as readily as one that is minimally bound. It should be noted, however, that this penetration may be due primarily to the slow rate of binding of cefazolin to serum proteins.


Infection of hypercholesterolemic mice with Coxsackievirus B.

Adult male mice were made hypercholesterolemic by a diet high in cholesterol, cholic acid, animal fat, and sucrose. After three months on this diet, animals were infected with 5 X 10(9) plaque-forming units of coxsackievirus B5. Control groups consisted of uninfected hypercholesterolemic mice and infected mice maintained on a standard laboratory diet. Infection in the hypercholesterolemic animals was associated with leukopenia, severe fatty metamorphosis and focal necrosis in the liver, cholelithiasis, ileus, cardiomyolysis, and lack of inflammatory response. These mice died within seven to 14 days. Uninfected hypercholesterolemic animals had lesser degrees of fatty liver and cholelithiasis, and all survived. Infected mice maintained on a standard diet also survived. Titers of virus in representative tissues were lower in the hypercholesterolemic than in the normal mice, an indication that replication of virus was not solely responsible for the lethal outcome of the infections. These experiments demonstrate that hypercholesterolemia may alter host defenses against group B coxsackievirus in the mouse.


Enhanced efficacy of amphotericin B and rifampicin combined in treatment of murine histoplasmosis and blastomycosis.

Amphotericin B in combination with rifampicin was more effective in the treatment of murine histoplasmosis and blastomycosis than either agent alone. The increased therapeutic effects were not accompanied by an increase in toxicity.


Clinical pathology of the Jarisch-Herxheimer reaction.

The Jarisch-Herxheimer reaction is a complication that can follow treatment of several infectious diseases. Its most severe form is in louse-borne relapsing fever; in this syndrome the reaction can cause death. Information from studies in Ethiopia during the past eight years is presented, and clinical, physiological, pathological, and immunological features of the reaction are described. Possible causative mechanisms of the reaction are discussed, especially in relation to the role of endotoxin, and an attempt is made to consider this reaction in relation to other endotoxin-associated states.


Optimal schedules for use of interferon in the corneas of rabbits with herpes simplex keratitis.

The 50% infectious dose of a preparation of herpes simplex virus was measured in eyes of rabbits by a multiple corneal inoculation method. One hour after inoculation of virus, one eye was treated with drops of human leukocyte interferon, and the other was treated with saline or with a different dose of interferon. Results from groups of three to four rabbits were combined for analysis. Treatment reduced the 50% infectious dose of virus in proportion to the concentration of interferon applied (within the range of 6.5 X 10(4)-1.3 X 10(6) units/ml) and not according to the total number of units instilled. Different treatment schedules were tried. Two applications of interferon each day were as effective as eight applications at intervals of 15 min or 1 hr. One application produced near-maximal antiviral effects for 18-24 hr. Thus, in human herpetic keratitis, a single daily application of the most concentrated available preparation of human interferon might be the most efficient schedule of treatment.


Small-particle aerosols of antiviral compounds in treatment of type A influenza pneumonia in mice.

Three chemotherapeutic drugs active against type A influenza virus (amantadine, rimantadine, and ribavirin) were tested as therapeutic agents against established infections with influenza virus in mice. The drugs were administered intraperitoneally or as aerosols either from 6 hr to four days or from three to seven days after infection. Small-particle aerosols were administered continuously 24 hr per day. Continuous dissemination of aerosols was superior to intraperitoneal administration, as evidenced by higher survival rates at 21 days. Rimantadine, amantadine, and ribavirin were effective when treatment was delayed for three days. Ribavirin was the most efficacious if therapy was initiated as an aerosol 6 hr after infection. In contrast to amantadine, ribavirin given in samll-particle aerosols at 6 hr prevented the development of pneumonia and decreased titers of virus in lung.


Comparison of rates of clearance of human fibroblast and leukocyte interferon from the circulatory system of rabbits.

In view of the lower physical stability of human fibroblast interferon than of human leukocyte interferon, the rates of clearance of the two types of interferon were compared in vivo. Both types were injected intravenously and intramuscularly into rabbits, and serum titers were determined at various times after injection. The rates of clearance of the two types of interferon were very similar after both intravenous and intramuscular injection, a finding which indicated that, at least in the circulatory system of rabbits, human fibroblast interferon is not less stable than human leukocyte interferon. After intravenous injection of either type, very little interferon was found in soluble extracts of the rabbit liver. This result implies that heterologous interferon is broken down rapidly in the liver, that it is bound to some particulate fraction, or that it does not accumulate in the liver.


Paradoxical effects of immunopotentiators on tumors and tumor viruses.

Growth of tumors was inhibited or enhanced in mice by a synthetic (pyran) or a biologic (corynebacterium parvum) immunopotentiator. Marked inhibition of leukemogenesis induced by Friend leukemia virus was produced by prophylactic intraperitoneal treatment with pyran, while intravenous treatment with pyran (in the same dose and regimen) significantly enhanced growth of tumor virus. Paradoxical effects were also seen with the biologic immunopotentiator C. parvum in solid tumor systems. Treatment with C. parvum either potentiated disease or had no effect on the life span of most mice bearing the Lewis lung carcinoma. In contrast, the same treatment could produce a high percentage of tumor regressions in mice bearing the MCA 2182 sarcoma, although the effect was somewhat variable. These data, which show that a change in route of drug administration or in the type of tumor treated may reverse the effect of treatment, emphasize that the mechanism of action of immunopotentiators must be elucidated before consistent beneficial treatment of tumor viruses or tumors can be achieved.


Clinical use of human globulin immune to rabies virus.

Studies of human globulin immune to rabies virus before licensure showed that it suppressed active antibody responses when individuals received 16 doses of duck embryo vaccine but not when they received 23 doses of duck embryo vaccine. Prospective surveillance of use of human globulin immune to rabies virus since licensure in 1974 has revealed that 40% of persons who receive 14-16 doses of duck embryo vaccine have low or undetectable antibody responses 30-90 days after initiation of the series. Ten percent of individuals receiving 21-23 doses of duck embryo vaccine have inadequate antibody responses, a percentage not significantly different from that found in recipients of 14 doses of vaccine alone. Human globulin immune to rabies virus has also been used for treatment of one case of clinical human rabies; this use was based on the observation that antibodies to rabies virus in serum do not develop until after the seventh day of clinical illness, and antibodies are absent from spinal fluid as long as 19 days after onset of symptoms. Intracranial pressure and neurological function remained stable after administration of human globulin immune to rabies virus, but two days after initiation of therapy, the patient died of progressive pulmonary dysfunction and tension pneumothorax.


Target cells for interferon induction by BL-20803.

The role of certain cells of the reticuloendothelial system in the interferon response of mice to the low-molecular-weight compound 1,3-dimethyl-4-(3-dimethylaminopropylamino)-1H-pyrazola-3,4-b-quinoline dihydrochloride (BL-20803) was studied. Mouse spleen adherent cells cultured in vitro with subtoxic concentrations of BL-20803 produced interferon. Adherent cells isolated from peritoneal washes were unresponsive to the compound. Treatment of mice with rabbit antiserum to mouse adherent cells suppressed interferon production by at least 65%, wheras antisera to thymocytes or to nonadherent spleen cells failed to suppress the interferon response. Pretreatment of mice with zymosan resulted in the appearance of circulating interferon in mice given 100 mg of the inducer/kg (a dosage to which they are normally unresponsive). No potentiation of the interferon response to the drug occured in immunized mice actively making a humoral or delayed-type hypersensitivity immune response. It is concluded that a specific type of cell in the mouse, the so-called "fixed macrophage," may be a major target for interferon induction by BL 20803.


Pharmacokinetics of human leukocyte interferon.

Most of the human leukocyte interferon or rabbit serum interferon administered intravenously to rabbits was detectabble in their plasma 1 min after injection. An increase in the dose did not affect the early clearance of human interferon but did prolong its persistence in the serum. Repeated intramuscular injections had no effect on the kinetics of circulating interferon. The purity of the preparation influenced the rate at which interferon entered the blood after intramuscular or subcutaneous injection but did not affect clearance from the blood after intravenous injection. The circulating interferons obtained after intravenous or intramuscular administration cleared at similar rates. During the first 1-2 hr after intravenous or intramuscular injection into rats, the interferon levels were lower in lymph than in serum; after 2 hr lymph and serum samples contained similar amounts of interferon.


Interferon and delayed-type hypersensitivity to a viral antigen.

Delayed hypersensitivity to Newcastle disease virus was measured with use of a footpad test in mice. When tissue culture interferon was administered 24 hr before sensitization, footpad swelling upon subsequent challenge with the virus was significantly decreased. In animals already sensitized to the virus, footpad swelling was similarly decreased if interferon was administered a few hours before footpad challenge. This result indicates that interferon can affect both the afferent and the efferent pathways of delayed hypersensitivity to Newcastle disease virus and is in agreement with previous results obtained with nonviral antigens.


Changing ideas on antiviral agents for respiratory infection.

Since the early 1950s it has been shown that some nontoxic antiviral substances, given either orally or intransally, can inhibit the growth of respiratory viruses and their clinical effects. The next targets of research are the improvement of present methods of drug administration and the exploration of the possibility of giving drugs in combination. The most potent antiviral agents available should be used to establish whether drugs given when symptoms commence can have a significant effect, i.e., whether antiviral therapy is possible.


The need for antiviral therapy and prophylaxis of viral ocular disease.

The cost of development of a compound with clinical potential as an antiviral agent is so great that it cannot be undertaken unless there is likely to be a market that will return the investment. The current practice, assessment of the prospect on the basis of the present market for idoxuridine and calculation of a likely percentage of capture by a potentially superior compound, is basically erroneous and may lead to gross underestimates of the market. In addition to the market for agents effective in treatment of dendritic and amoeboid ulcers, there is a potentially much greater market, first, for somewhat less toxic compounds that may be used more or less continuously over very long periods for prevention of recurrences. Substances with promise of filling each of these requirements are already under investigation. There is also a potential market for therapeutic preparations with activities against a range of agents, particularly herpesviruses, adenoviruses, and chlamydiae, that commonly cause follicular conjunctivitis or keratoconjunctivitis.


Superoxide dismutase in red blood cells: method of assay and enzyme content in normal subjects and in patients with beta-thalassemia (major and intermedia).

The applicability to red blood cells of two widely used methods for spectrophotometric assay of superoxide dismutase activity has been tested, in view of the demand for routine screening of the level of this enzyme in various circumstances of hematological interest. The nitro blue tetrazolium reduction method was found to be inapplicable, even after removal of hemoglobin by chloroform-ethanol extraction. On the other hand, the epinephrine oxidation method gave reliable values after chloroform-ethanol extraction. The enzyme content in normal adults was found to be 6.2 +/- 1.4 X 10(15) gr. per red blood cell. No significant change in the enzyme content was found in 19 patients with beta-thalassemia major and 5 patients with beta-thalassemia intermedia, indicating no influence on the level of this enzyme by the increased flux of oxygen radicals which is to be expected in thalassemic red blood cells.


A new test for both neutrophil marrow reserves and intravascular survival.

Reinfusion of a patient's own blood through a hemodialysis coil causes neutrophilia, reaching a peak less than 1 hour after starting the blood reinfusion and returning to baseline count over the next few hours, resulting from a transient release of neutrophils from the bone marrow. This report gives data regarding the double clinical utility of so stimulating a patient's marrow. First, we found that the magnitude of this neutrophilia was a good measure of marrow neutrophil reserves, as determined by comparison with the marrow mature neutrophil cellularity. Second, the falloff from peak neutrophilia to baseline count was a good estimate of neutrophil intravascular survival, as determined by comparison with the standard, in vitro, diisopropylfluorophosphate (DF32P) survival procedure. Thus, this one test could detect abnormalities both of marrow neutrophil production (release) and of peripheral destruction. The coil test requires no radioactive isotope, utilizes commonly available equipment, is apparently harmless and acceptable to the patients, and can be greatly simplified by the development of a blood bag containing sterile cellophane. Even in its present form, it offers considerable clinical and economic advantages in the assessment of pathophysiology of neutropenia in individual patients.


Thrombocytopoiesis in W/Wv mice.

W/Wv mice are genetically anemic, but usually have normal numbers of circulating granulocytes and platelets. An intrinsic stem cell defect has been suggested by numerous experiments, and a reduced number of megakaryocytes has been found in the bone marrow. Platelet production in W/Wv mice and +/+ litter mate controls was studied by measurement of the incorporation of 75Se-methionine (SeM) into platelets after intravenous injection of 2 muCi of SeM. Mean incorporation of SeM into platelets of W/Wv mice was 17 per cent greater than that of +/+ controls at 66 hours. Mean platelet counts of W/Wv mice were not different from controls. Recovery from thrombocytopenia resulted in a greater proportion of labeled platelets in W/Wv mice, and a slight delay in recovery of the platelet count. These results suggest that W/Wv mice have a normal overall rate of thrombopoiesis despite having a decreased megakaryocyte mass.


The roles of renal catabolism and uremia in modifying the clearance of fibrinogen and its degradative fragments D and E.

Elevated levels of fibrinogen/fibrin degradation products (FDP) occur in uremia, and have been thought to be in part related to intravascular coagulation in the kidney. More recent data indicated that delayed catabolism of fibrinogen fragment D occurred in anephric animals. To further evaluate FDP catabolism in the kidney, turnover studies of purified dog 131I-Fg-D and 125I-Fg-E were performed on dogs before and after acute subtotal nephrectomies, and later during chronic uremia. 131I-fibrinogen clearances were also perfomed. Slowed catabolism of Fg-D and Fg-E was observed in both the acute and chronic uremic stages. Altered urinary excretion was not a factor as only minimal amounts of Fg-D and Fg-E were excreted in the urine of the control animals. In the 131I-fibrinogen studies, there were significant changes in plasma volume, fibrinogen t 1/2, and intravascular/extravascular distribution, but not in fractional catabolic rate. To differentiate fully, the effects of uremia from those of loss of catabolic renal tissue, the Fg-D and Fg-E turnover studies were repeated on other animals with intact kidneys whose ureters were diverted into the peritoneum and compared to subsequent studies after total nephrectomy. The control and ureter-severed studies had the same clearance pattern, whereas decreased catabolism occurred in the nephrectomized dogs. The results demonstrate uremia per se does not have a major effect upon the catabolism of fibrinogen, Fg-D, and Fg-E. Loss of renal tissue does impair the clearance of Fg-D and Fg-E, indicating these proteins are normally catabolized in part by the kidneys. Thus elevated plasma FRA in uremic patients may reflect decreased Fg-D and Fg-E catabolism rather than increased FDP production from primary or secondary fibrinolysis.


Effect of aging on renin activity and aldosterone excretion.

Twenty or more normal subjects (half of whom were males) in each decade of life from age 20 to 80 years were included in the study. Plasma renin activity (PRA) was measured with the subjects on a normal sodium diet and after sodium restriction and standing. Aldosterone and sodium excretion rate was measured on unrestricted sodium intake and on day 3 of sodium restriction. Plasma renin activity was quantitated by both bioassay and radioimmunoassay of angiotensin-I. Plasma renin activity under both test conditions declined with increasing age, especially those over 40. Values of those in the seventh decade averaged 60 per cent of the value of that of the younger age group. Response of renin activity to sodium restriction and standing was 73 per cent of that of the younger age group. Older females tended to be less responsive to sodium restriction and standing. Aldosterone excretion rate declined with increasing age so that by the seventh decade the values averaged 55 per cent of those in the second decade of life. Aldosterone excretion rate in response to sodium restriction also declined with increasing age, particularly above the fourth decade, so that the values on day 3 of sodium restriction in the older subjects averaged 33 per cent of that of the subjects in the second decade of life. Renin substrate did not decline with increasing age. The decline in renin activity and aldosterone values in the older subjects appeared to be due to factors other than sodium or potassium intake.


Inhibitory effect of furosemide on glucose transport.

The carrier-mediated glucose transport function in the intact human erythrocytes and also that of their isolated membranes is markedly inhibited in the presence of furosemide in vitro. A noncompetitive process is believed to be involved since the transport inhibition was not affected by the presence of high concentrations of glucose in the medium. The inhibition developed slowly over 2 hours. Most of the inhibition was slowly reversed during a 3-hour incubation in medium free of the drug. It is concluded that furosemide inhibits the glucose transport function directly in absence of insulin-mediated transduction mechanism and independently of any interference with intracellular glycolysis.


Changes in serum potassium and blood glucose concentrations after trimethaphan administration in man.

Blood glucose and serum potassium (K+) concentrations were measured before, during, and 60 minutes after operation in two groups of 10 patients during nitrous oxide/halothane/d-tubocurarine anesthesia for major orthopedic surgery. In the control group, arterial blood pressure was maintained within normal range, while in the study group trimethaphan camsylate was administered as an intravenous infusion (average, 218 mg.) to maintain a systolic blood pressure of 60 to 65 torr. In the normotensive group, blood glucose rose significantly during operation and early postoperatively and serum K+ was essentially unchanged. In the hypotensive group, trimethaphan caused a striking modification of surgically induced hyperglycemia, together with a small significant decrease in serum K+ intraoperatively. The observed increase in blood glucose is part of the autonomic response to surgical stress. Hormonal factors (growth hormone, cortisol and glucagon) may conceivably be involved. The decrease in serum K+ is probably caused by decreased hepatic glycogenolysis and attenuation of the suppressive effect of catecholamines on insulin release, both effects being secondary to the ganglionic blocking property of trimethaphan. These results indicate that trimethaphan, in contrast to other ganglionic blocking drugs, does not cause hypoglycemia and suggest that serum K+ concentration should be monitored whenever these drugs are used.


The prevalence of hepatitis B surface antigen in commercially prepared plasma products.

Commercially available lots of plasma derivatives prepared between 1957 and 1975 were tested for hepatitis B surface antigen (HBsAg) by radioimmunoassay. In all, 69 per cent of lots of plasma protein fraction, 40 per cent of factor IX concentrate, 20 per cent of normal serum albumin, 13 per cent of antihemophilic factor, 3 per cent of fibrinogen, and 0.7 per cent of immune serum globulin lots tested were HBsAg-positive. There was great variation in the prevalence of HBsAg-positive lots of each product among the different manufacturers, reflecting not only differences in methods of processing plasma, but also differences in donor populations. Those manufacturers relying upon volunteer donor plasma or placental source material demonstrated lower rates of HBsAg-positive lots of final products than those relying upon commercial donor plasma. There was a marked decrease in the prevalence of positive lots during the period 1971 to 1973, coincident with the onset of routine plasma donor screening for HBsAg. However, current requirements for plasma screening have not resulted in totally HBsAg-free plasma products. Use of more sensitive and more reliable tests for HBsAg will probably reduce contamination of plasma pools with HBsAg to undetectable levels. Despite HBsAg-status, however, the "high-risk" plasma products (fibrinogen, antihemophilic factor, factor IX concentrate) must still be considered capable of transmitting hepatitis and used only with the strictest indications.


Composition of nasal secretion in patients with cystic fibrosis.

Quantitative examination of nasal secretion in patients with cystic fibrosis revealed a significantly greater than normal concentration of calcium, a finding in keeping with the hypothesized importance of this ion in the pathophysiology of the disease.


Hemoglobin Hope: studies of oxygen equilibrium in heterozygotes, hemoglobin S-Hope disease, and isolated hemoglobin Hope.

Hemoglobin Hope (beta(H14)136gly leads to asp), a mildly unstable variant, was found to have decreased oxygen affinity, a normal Bohr effect and diminished cooperativity. Decreased oxygen affinity of hemoglobin Hope may explain the previous failure to find an appropriate response to hemolysis in individuals studied who were heterozygous for both hemoglobin Hope and sickle hemoglobin. Salt bridge formation between NA1 valine and H14 aspartic acid may stabilize the beta Hope subunit in its deoxy form thus producing intrinsically low oxygen affinity and reduced cooperativity.


131I-asialo-alpha 1-acid glycoprotein. Investigation of its use for liver function tests; metabolism in the rat.

On intravenous injection into rats, 131I-asialo-alpha 1-acid glycoprotein was rapidly removed from the circulation. Five minutes after injection only a few per cent of the injected dose remained in the plasma and 75 per cent was found to be in the liver, where it could be localized in the hepatocytes. Fifteen minutes after injection, however, the 131I reappeared in the plasma after the labeled protein had been metabolized by the liver. One hour after injection less than 5 per cent of the injected dose remained in the liver. Studies with this material in a series of rats using a gammacamera showed the reproducibility of the the time-activity curves of the liver. In contrast, the unmodified radioactive alpha 1-acid glycoprotein disappeared from the blood at a much slower rate. The disappearance curve of the unmodified alpha1-acid glycoprotein could be fitted with the sum of two exponential curves with half-times of 2.88 H. and 27.3 H., respectively. The 131I-alpha1-acid glycoprotein did not accumulate in any tissue except the kidneys and some of the radioactivity was excreted in the urine. The possible application of 131I-asialo-alpha1-acid glycoprotein and the comparison with other radiopharmaceuticals for liver studies in nuclear medicine are discussed.


Malondialdehyde formation as an indicator of prostaglandin production by human platelets.

The formation of malondialdehyde, a metabolite of prostaglandin endoperoxides, has been studied in platelet-rich plasma. Small amounts were produced in response to ADP, epinephrine, or collagen and larger amounts in response to thrombin. Arachidonic acid induced platelets to produce large amounts of malondialdehyde. Malondialdehyde formation by platelets was prevented by aspirin or indomethacin. The measurement of malondialdehyde in platelet-rich plasma may be used as an indicator of platelet prostaglandin synthesis, and is simpler than the measurement of prostaglandins.


The rheological factor in mucociliary clearance.

The viscoelastic properties of sputum samples obtained from bronchitic patients were measured and correlated with the speed at which the samples were transported by the ciliary action of isolated pharyngeal palates of bull frogs maintained in amphibian Ringers solution at 28 degrees C. The viscoelastic properties were measured at very low shear stress which allowed to express them in terms of Newtonian viscosity and linear elastic modulus, which greatly minimized the variations in such measurements previously experienced with sputum. Samples with lower values of viscosity and moduli (less than 10,000 poises and 15 dynes per square centimeter, respectively) showed faster transport rates in a system where ciliary beat frequency and volume of added mucus were kept relatively constant.


Chromosomal breakage and scleroderma: studies in family members.

Chromosome studies were performed on 54 apparently healthy relatives of 29 scleroderma patients and on 40 controls. Increased chromosomal breakage is observed in 86 per cent of brothers and sisters and in 68 per cent of children of patients. If the findings in relatives are compared to those obtained in the 29 scleroderma patients of these families, the percentage of abnormal cells does not differ significantly between the two groups (25.8 per cent and 29.1 per cent respectively), and there is no difference for the frequencies of gaps and open breaks. However acentric fragments, "minutes" and morphologically abnormal chromosomes are significantly increased in patients as compared to their asymptomatic relatives with increased breakage. The distribution of breaks is found to be random. The presence of structural chromosome aberrations in relatives of scleroderma patients may have a special importance with regard to the concept of familial autoimmune disease.


Hemodialysis and chloroquine poisoning.

The use of hemodialysis in the treatment of chloroquine intoxication was investigated. The in vitro and in vivo clearances of chloroquine were adequate (in vitro 53.4 ml. per minute, in vivo 57 ml. per minute). However, dialysis did not reduce the mortality in dogs. Eight milligrams per kilogram were uniformly fatal with or without dialysis whereas 5 mg. per kilogram were not fatal in any dog. Less than 5 per cent of administered chloroquine was removed during 6 hours of dialysis. Red cell chloroquine averaged 4 times serum values. Hemodialysis is ineffective in the treatment of chloroquine intoxication probably because chloroquine rapidly becomes intracellular after administration.


Removal of hepatitis B surface antigen (HBsAg) from plasma fractions.

Endogenous or deliberately added hepatitis B antigen was removed and concentrated for assay from albumin, and from coagulation factor II, VII, IX, and X concentrates as model plasma fractions. The concentrates carry considerable risk of causing hepatitis in transfused patients. The amount of antigen remaining in the fraction was estimated to be less than 1/10,000 of that detectable by the Ausria II radioimmunoassay and 1/100 of that found to be infectious when highly contaminated human sera were diluted and injected in chimpanzees. Batch fractionation methods with polyethylene glycol were used. The yield of albumin was 96 per cent and of the coagulation factors about 90 per cent.


The occurrence of the Tullio phenomenon in congenitally deaf children.

One hundred and fifty deaf children between 5 and 16 years old of the 'Rudolf Mees' Institute were investigated in search of the Tullio phenomenon with nystagmus as a criterion. Seventy-six out of those 300 ears were positive. A functioning (calorically excitable) pars superior of the labyrinth was a 'conditio sine qua non' for this reflex. We may assume that deaf ears which show a positive Tullio phenomenon have a labyrinthine pathology of the Mondini-Alexander type and that this symptom is a pathologic one.


The differential diagnosis of the orbital manifestations of paranasal disease.

The paper attempts to summarize the orbital conditions likely to be referred to the ear, nose and throat surgeon and to outline the pathological classifications in current use. Particular attention is paid to the various named syndromes in an effort to clarify their meanings. A series of twenty seven cases referred to the Ear, Nose and Throat Department at the National Hospital for Nervous Diseases, Queen Square is presented and analysed in the light of the classifications given. The value of radiological investigation in the determination of the site of the lesion in these cases is stressed.


Deafness in sarcoidosis.

A case of sarcoidosis and sensori-neural deafness in which the lesion was thought to be cochlear in position is presented and the audiometric findings are discussed in detail. A review of the reported cases shows that sarcoid lesions may involve the hearing mechanism at any point along its neural axis.


Personality measures in E.N.T. outpatients.

Middlesex Hospital Questionnaires were given to 170 new adult outpatients at Lymington Hospital, the Royal South Hants Hospital, Southampton, and the Royal Northern Hospital, London. The examiners were asked to give their comments on the patients personality and a tentative clinical diagnosis. The significantly older population of Lymington Hospital was found to have lower hysteria and phobic anxiety scores. High hysteria and anxiety scores were found in a group of patients with rhinitis. As a whole there seemed to be little relation between the examiner's impression of the patient and their personality scores.


Estimation of surface area and counterion binding characteristics in fatty amine monolayers from desorption kinetics.

The surface area per molecule of an un-ionized fatty amine is very similar to the surface area per molecule of an un-ionized fatty acid. Surface area increases with ionization in both fatty amine and fatty acid films. However, fatty amino cations have much smaller surface areas than the corresponding fatty acid anions. Thus counterion binding is stronger with fatty amine cations than with fatty acid anions. Surface area data show that counterion binding affinities for fatty amine cations decrease in the strong field sequence Cl- greater than Br- greater than I- greater than SCN-. Furthermore, surface areas in the presence of the most strongly bound counterions, Cl- and Br-, increase significantly with an increase in subphase ionic strength. These data are consistent with the formation of strong ion-pair bonds and their disruption with an increase in ionic strength. Fatty amine cations desorb as micelles with much lower relative diffusion coefficients than the corresponding fatty acid anions. Furthermore, relative diffusion coefficients for fatty amine cations are strongly dependent on the specific cation. These data show that fatty amine cations form larger micelles when they desorb in the presence of strongly bound counterions. Anions enhance the solubility of a fatty acid anion in the sequence Cl- less than I- less than SCN-, which is characteristic of chaotropic anions that disrupt water structure.


Effects of fasting on bile acid metabolism and biliary lipid composition in man.

The effects of a four to six day fast on gallbladder bile lipid composition, bile acid pool size, bile acid composition, and cholic acid metabolism have been determined in normal human subjects. Total bile acid pool size and cholic acid pool size were measured before and after fasting by a one-sample technique previously validated in our laboratory. The rate of synthesis of cholic acid and its fractional turnover rate before fasting were measured using standard techniques. Estimates of fasting cholic acid synthesis rate and fractional turnover rate were calculated as daily averages from the change in cholic acid pool size, in combination with the change in cholic acid specific activity, during the fasting period. Since these estimates are approximate, a maximum value for cholic acid synthesis rate during fasting was also determined by assuming that the entire change in cholic acid specific activity during the fasting period occurred instantaneously. The molar percent of cholesterol in gallbladder bile was reduced in eight of nine subjects after a four to six day fast (p less than .01; mean reduction 30.5%). The molar percents of bile acid and phospholipid were not significantly altered by fasting. The cholesterol saturation index, calculated on the basis of these data, was reduced by an average of 31.0% after a four to six day fast (p less than .02). The average daily cholic acid synthesis rate and the fractional turnover rate were reduced in all six subjects on whom isotope kinetic studies were carried out. The mean decrease in synthesis rate was 68.5% (p less than .05; range 55.2-79.8%) while the mean decrease in fractional turnover rate was 64.4% (p less than .05; range 30.2-100%). Reduction in synthesis rate was confirmed by the determination of maximum fasting synthesis of cholic acid, which averaged 61.1% lower than synthesis in the fed period. Fasting had no consistent effect on total bile acid pool size, cholic acid pool size, or bile acid species composition.


Isolation, characterization, and biological activity of retinyl phosphate from hamster intestinal epithelium.

Epithelial cells from hamster small intestine, in short term culture, incorporated [carbinol-14C]retinol into a compound that is identical to synthetic retinyl phosphate, as judged by chromatography on DEAE-cellulose, silicic acid, and thin layers of silica gel. The biological compound displays the same absorption spectrum as does synthetic retinyl phosphate with a maximum at 325 nm. Hydrolysis with mild alkali yields anhydroretinol, as it does for synthetic retinyl phosphate, with absorption maxima at 388, 368, and 346 nm. Enzymic hydrolysis by alkaline phosphatase releases 9% of the radioactivity as [14C]retinol. Under the same conditions, 9% of synthetic retinyl phosphate is hydrolyzed to retinol. The biological compound was tested for biological activity. At a concentration of 5.5 x 10-8 M it was as active as retinol and retinyl phosphate in reversing keratinization induced in hamster tracheal epithelium by vitamin A deficiency. It is concluded that hamster intestinal cells synthesize retinyl phosphate.


Synthesis of 11, 12-2H2- and 11, 12-3H2-labeled chenodeoxycholic and lithocholic acids.

Deuterium- and tritium-labeled chenodeoxycholic acid and lithocholic were prepared by catalytic reduction of their respective delta11 derivatives. Structures of the intermediates and their isotopic purity were verified by chemical ionization and electron impact mass spectrometry and by nuclear magnetic resonance spectroscopy. Experimental conditions for reductive deuteration were defined which gave complete reduction of the olefin and a product of high isotopic purity. Conditions for optimal tritiation were developed with which little exchange of protons with the solvent occurred; the product had high specific activity. To test biological stability of the label, the 3H-labeled chenodeoxycholic acid was administered simultaneously with 14C-labeled chenodeoxycholic acid to two healthy subjects and the 3H/14C ratio in bile was determined daily for several days. The ratio remained identical to that administered, suggesting that the 11,12-3Hlabel in chenodeoxycholic acid is stable during enterohepatic cycling and can be used for valid estimates fo bile acid kinetics in many by the isotope dilution technique.


Side chain hydroxylation of cholesterol, campesterol and beta-sitosterol in rat liver mitochondria.

The extent of the side chain hydroxylation of cholesterol, campesterol (24 alpha-methylcholesterol), and beta-sitosterol (24 alpha-ethylcholesterol) in rat liver mitochondria has been compared. Two beta-sitosterol metabolites, tentatively identified by liquid chromatography, thin-layer chromatography, gas-liquid chromatography, combined with radioactivity detection, and gas-liquid chromatography-mass spectrometry as the 26- and 29-hydroxy derivatives, were formed in the proportion 1:1. The sum of 26-hydroxy- and 29-hydroxy-beta-sitosterol obtained amounted only to about one-fourth of the yield of 26-hydroxycholesterol. Campersterol appeared to give rise only to 26-hydroxycampesterol (tentatively identified), which was formed in similar yields as 26-hydroxycholesterol (0.2-0.4%). The formation of 29-hydroxy-beta-sitosterol but not of 28-hydroxycampesterol indicates that the omega-hydroxylation of the steroid side chain is dependent on the length of the side chain. Cholesterol gave rise to identifiable amounts of a 25-hydroxy derivative but the formation of 25-hydroxy derivatives of beta-sitosterol and campesterol could not be established with certainty. 24-hydroxycholesterol was also found to be formed in the mitochondrial system. The ratio between the yields of 26- and 25-hydroxychoelsterol ranged between 2 and 3, and that between 26- and 24-hydroxycholesterol was about 10.


An improved and simplified radioisotopic assay for the determination of free and esterified carnitine.

The radioisotopic assay for carnitine first described by Cederblad and Lindstedt (Clin. Chim. Acta. 37:235-543, 1972) and modified by Bohmer et al (Clin. Chim. Acta. 57:55-61, 1974) has been improved and simplified. As a result, the assay yields a linear response over a wide range of carnitine concentrations without the need for excessive amounts of labeled acetyl-CoA. In addition, it will measure very small quantities of carnitine even in the presence of excess acylcarnitine. The method allows rapid determination of free and esterified carnitine in small volumes of plasma (50 mul is sufficient) without the need for prior deproteinization of the samples.


Isolation of disaturated phosphatidylcholine with osmium tetroxide.

A simple, rapid, inexpensive method for isolating disaturated phosphatidylcholine from adult rat lung has been devised. Total lipids are reacted with osmium tetroxide dissolved in carbon tetrachloride, and the disaturated phosphatidylcholine is isolated on a column of neutral alumina. More than 99% of the fatty acids in the phosphatidylcholine fraction are saturated and 94% of this material migrates as phosphatidylcholine on subsequent thin-layer chromatography.


Rapid screening of lipid metabolism in monolayer cell cultures.

Monolayer cell cultures grown on coverslips in the presence of radioactive lipid precursors were embedded in silica gel layers for extraction and resolution of the labelled products directly by thin-layer chromatography. The method permits rapid screening of lipid metabolism in tissue cultures with a small number of cells.


Preparative isolation of cerebrosides (galactosyl and glucosyl ceramide).

An improved method for isolating cerebrosides from natural sources is described. The method is particularly suited to large scale work and can be adapted to the isolation of sphingolipids that are less polar than the gangliosides. It is based on the use of sodium sulfate to absorb the water from chloroform-methanol tissue extracts, the use of triiodide to cleave the ether linkage of plasmalogens, and the use of alkaline methanolysis to cleave the ester linkages of the glycerolipids. The final separation of the lipids is done with a silica gel column.


Sarcomere length changes during stimulation of frog semitendinosus muscle.

A method is described for detecting rapid changes in sarcomere length at different points along a muscle specimen during stimulation. Reproducible differences in behaviour are observed at different positions along the length of the muscle both during contraction and during relaxation. By summing individual observations made at points closely spaced along the specimen it is found that in tetanus at 6 degrees C the average sarcomere contraction is about 5% for an initial length of 2.6 mum; a maximum rate of contraction of about 4 nm.ms-1 (0.16% ms-1) is reached and maintained for some 10 ms during the development of tension. In twitch the corresponding values are 4% and 3 nm.ms-1 (0.12% ms-1).


Polymerization polarity of actin.

The actin polymer, like the thin filaments of muscle, is known to be polarized as demonstrated by the well known "HMM decoration" technique to give a "herringbone" pattern pointing in one direction. The question "Does polymer formation proceed unidirectionally, or bidirectionally?" was raised and tested experimentally. Short fragments of actin polymers were prepared, fully decorated with HMM and these decorated fragments were used as initiation centers for further actin polymerization without HMM. The resultant polymers showing both decorated and undecorated portions were examined and found to consist of a large majority of "spears" i.e., the added undecorated polymer extended in the direction opposite to that direction pointed by the "herringbone" pattern. However, a few cases of polymers indicating the opposite direction of polymerization were also found. Analysis leads to the conclusion that actin polymerization is unidirectional, although further experimentation is necessary to establish this completely.


Cinemicrographic analysis of the movement of flagellated bacteria. II. The ratio of the propulsive velocity to the frequency of the wave propagation along flagellar tail.

We took cinemicrographs of the movement of the flagellar tail of the peritrichously flagellated bacterium, Salmonella, swimming in a medium containing methylcellulose under a dark-ground microscope. By analysing the film, the velocity of translation u, the frequency of the propagation of helical waves along the tail fF and the frequency of the induced rotation of bacterial body fB of individual organism were measured and the experimental values of the ratios u/fF, u/fB and fF/fB were obtained. On the other hand, the theoretical values of these ratios were calculated by inserting the geometrical parameters describing the shapes and the sizes of the body and the tail of individual organism into the equations previously derived for the hydrodynamic model of the propulsion of flagellated bacteria (Holwill and Burge, 1963; Chwang and Wu, 1971). For four bacterial specimens presently analysed, the experimental values of u/fF ranged from 0.5 to 0.9, whereas the theoretical values were about 0.3. As reported by the preceding paper, such a tendency for the experimental values to exceed the theoretical ones by two or three times was also seen in u/fB and, consequently, the experimental and the theoretical values of fF/fB showed good agreement. From the results of these quantitative analyses of the movements of flagellated bacteria, it was concluded that the validity of the hydrodynamic model was further supported experimentally.


Conformational changes induced in plasmodium actin polymer by Ca2+ in the presence of muscle native tropomysin.

The flexible polymer of plasmodium actin (Mg-polymer) combines with muscle native tropomyosin up to a weight ratio of actin: tropomyosin:troponin = 6:1:1. Mg-polymer which combined with muscle native tropomyosin undergoes a conformational change when the Ca2+ concentration of the solution is decreased by adding 0.5 mM ethylene glycol bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA): the viscosity of the solution increases by 50% and its ATPase activity decreases to about 20% of the original value, which suggests that the structure of Mg-polymer becomes more rigid. This change is reversible with respect to the Ca2+ concentration in the solution. The threshold concentration of Ca2+ for the conformational change is about 10(-6) M. The possible role of this transformation in vivo is discussed.


Incorporation of rat prolactin into rat milk in vivo and in vitro.

Rat prolactin (11 i.u./mg) was continuously infused into the circulation of urethane-anaesthetized lactating rats for 35 min at doses of either 200 or 472 ng/min. The immunoreactive prolactin in both milk and plasma rose quickly during the first 20-25 min of infusion, then stabilized at similar levels over baseline (68 and 98 ng/ml for milk and plasma, respectively, with the 200 ng/min dose and 250 and 230 ng/ml, respectively, with the 472 ng/min dose). The concentration of prolactin in plasma fell after the infusion was stopped, whereas that in the milk either did not fall at all or fell slightly to a new stabilized level. There was a rapid and extensive loss in the immunoreactivity of prolactin added to milk when rat milk was incubated in vitro (37 degrees C for 1-120 min) with 600 ng/ml of extracted pituitary prolactin (NIAMDD RP-1) or unit equivalent amounts of prolactin obtained from pituitary culture media (secreted prolactin, supplied by C.S. Nicoll). Significantly greater amounts of added RP-1 prolactin were lost when it was incubated with milk obtained after 4 h than after 18 h of non-suckling. There was, however, no drop in endogenous immunoreactive milk prolactin levels (350-400 ng/ml) when rat milk was incubated with saline for 30 min. This suggests that milk prolactin obtained as a result of plasma transfer is different chemically from the milk prolactin resulting from the addition of either RP-1 or secreted prolactin to milk in vitro. Approximately 90% of 131I-labelled rat prolactin appeared in the trichloroacetic acid precipitable fraction after incubation (37 degrees C for 120 min) with milk obtained after 4 h of non-suckling in either the presence or absence  of thiouracil (added to prevent binding of 131I or 131I-labelled fragments to milk protein). The recovery was slightly less when 131I-labelled prolactin was incubated with milk obtained after 18 h of non-suckling. These data suggest that prolactin is quickly transferred from plasma into milk in direct relation to the plasma concentration. Once there, much of it appears to be retained by the milk perhaps chemically or physically bound; there is little, if any, degradation of the hormone. We conclude that the lactating the mammary gland may function normally as an excretory organ for prolactin.


Regulation by testosterone and serum protein of DNA synthesis in the developing epididymis of the rat.

Rates of DNA synthesis were measured as an index of cellular proliferation during the pubertal development of the rat epididymis. A highly reproducible pattern of DNA synthesis was defined by (1) a prepubertal, testosterone-insensitive peak of DNA synthesis at 25 days; (2) a dramatic decrease in DNA synthesis with the onset of puberty; (3) a major burst of testosterone-dependent synthesis peaking at 40 days in the head of the epididymis and at 40-60 days in the tail; (4) a fall to low levels as the adult organ weight was attained. An organ culture system was defined and utilized to analyse further the hormonal dependence of DNA synthesis in the epididymis. Testosterone and dihydrotestosterone failed to activate DNA synthesis at any stage of development in vitro. DNA synthesis was stimulated 100-300% by insulin at supra-physiological concentrations and by protein serum factor(s) at physiological concentrations. The serum activity was stable to heat treatment at 60 degrees C, destroyed by heating at 70 degrees C, and was present in the sera of hypophysectomized animals. These results indicate a primarily 'permissive' role for the action of testerone on DNA synthesis in the epididymis: testosterone acts to permit the expression of a developmental 'programme' of cell proliferation which is activated by specific protein(s) in serum.


Corticosteroid production by the human foetus: evidence from analysis of urine from women pregnant with a normal or an anencephalic foetus.

The quantities of nine corticosteroids in 24 h urine samples collected by pregnant women (nine with normal foetuses and nine with anencephalic foetuses) were measured after hydrolysis with beta-glucuronidase and separation by paper chromatography. The excretion (mumol/24 h, mean +/- S.D.) of prenanetriol (0-85 +/- 0-17), 3 alpha, 17 alpha-dihydroxy-5 beta-pregnan-20-one (17 alpha-hydroxypregnanolone, 0-55 +/- 0-17), 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnan-20-one (tetrahydro-11-deoxycortisol, 0-17 +/- 0-14) and tetrahydrocorticosterone (0-65 +/- 0-26) by women with an anencephalic foetus was significantly lower (P less than 0-01 or less than 0-05) than the excretion of these compounds by women with a normal foetus (pregnanetriol, 2-42 +/- 0-62; 17 alpha-hydroxypregnanolone, 2-72 +/- 0-69; tetrahydro-11-deoxycortisol, 0-56 +/- 0-37; tetrahydrocorticosterone, 1-95 +/- 0-94). These differences suggest that the adrenal of the normal foetus contributes to the quantity of pregnanetriol, 17alpha-hydroxypregnanolone, tetrahydro-11-deoxycortisol and tetrahydrocorticosterone in maternal urine. The excretion of tetrahydrocortisol, tetrahydrocortisone, tetrahydrodeoxycorticosterone, cortol and cortolone were similar in both groups of subjects. No evidence was obtained therefore to indicate the secretion of cortisol or deoxycorticosterone by the foetal zone of the adrenal of the undisturbed human foetus in late gestation.


Effect of aldosterone on lithium permeability of rat colon mucosa.

The effect of aldosterone on the ionic composition of colonic epithelial cells and on lithium absorption rate was studied by experiments on rats in vivo. Aldosterone considerably increased the rate of sodium absorption without measurably altering the sodium and potassium content of the epithelium. Aldosterone did not alter the electrical resistance of the tissue. With lithium in the lumen, the net sodium fluxes in the colon were similar in normal and aldosterone-stimulated rats but the rate of diffusion of lithium across the epithelium was greater in the aldosterone-stimulated group. The amount of lithium accumulating in the epithelial layer was also considerably increased by aldosterone stimulation. Aldosterone appears to increase the permeability of the mucosal (luminal) barrier allowing increased entry of lithium into the colonic epithelial cells.


Adsorption of free steroids with charcoal-gelatin disks in radioimmunoassays.

The preparation of charcoal-gelatin disks and their use for removing free steroid in radioimmunoassays are described. Plasma concentrations of several steroids were determined using charcoal-gelatin disks or charcoal suspension and the results are compared. The advantages of using charcoal-gelatin disks are discussed.


Suppression of luteinizing hormone release by 5alpha-androstane-3alpha, 17 beta-diol and its 3 beta epimer in immature ovariectomized rats.

The effect of 5alpha-androstane-3alpha, 17 beta-diol, its 3 beta epimer and oestradiol benzoate on suppression of LH release after ovariectomy was studied in immature rats. At doses of 50 and 100 mug/100 g body weight/day the 3alpha compound suppressed LH release after ovariectomy to the same extent as 0-1 mug oestradiol benzoate/100 g/day. 3alpha-Androstanediol at a dose of 25 mug/100 g/day suppressed LH release only up to day 45 of life, while the same dose of the 3beta epimer had no effect on LH suppression. The effect of 3beta-androstanediol on inducing precocious vaginal opening was found to be mediated by the ovaries, since it was eliminated by ovariectomy. These results confirm our previous findings on the participation of androstanediol in the normal regulation of LH and in the mechanism of onset of puberty in the rat.


Studies on the uptake and binding of 5alpha-androstane-3alpha, 17alpha-diol by canine prostatic nuclei.

Nuclei have been isolated from canine prostatic tissue previously incubated with tritiated testosterone or tritiated 5alpha-androstane-3alpha, 17alpha-diol. The protein-bound radioactivity has, in both cases, been shown to be associated with 5alpha-androstane-3alpha, 17alpha-diol. The formation and translocation of this steroid to the nucleus supports previous evidence that it has an active part to play in prostatic function in the dog. The formation from testosterone of another titriated steroid, possibly 5alpha-dihydrotestosterone, was also demonstrated although this product did not enter the nucleus and was apparently bound to the nuclear membrane.


Renin, cortisol and plasma volume in marine teleost fishes adapted to dilute media.

The renin-angiotensin system has been found in teleost fishes from both marine and fresh-water environments. In an attempt to define whether activity of the renin-angiotensin system is related to sodium balance in fishes, we transferred two euryhaline teleosts from seawater to hypo-osmotic media. Plasma renin activity decreased in American eels, Anguilla rostrata, after they were transferred from seawater to fresh water, and it did not change in the aglomerular toadfish, Opsanus tau, after transfer from 50% seawater to 5% seawater. Plasma sodium concentrations decreased significantly in toadfish in 5% seawater and in one group of eels in fresh water. Plasma levels of cortisol, a major mineralocorticoid in teleosts, and plasma volume, measured in eels, remained relatively constant. There are no clear correlations between plasma renin levels and those of plasma sodium or plasma cortisol. These results provide no evidence that the need of these fishes to conserve sodium when in hypo-osmotic media stimulates the renin-angiotensin system.