NAD+-15-hydroxyprostaglandin dehydrogenase distribution in rat kidney. Rat kidney NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was measured in zones and substructure of the rat kidney nephron. This was accomplished utilizing an assay procedure based upon determining the amount of prostaglandin E1 present before and after the reaction with the 15-hydroxyprostaglandin dehydrogenase contained in the tissue sample. The enzyme activity was assayed in freeze dried, quick frozen rat kidney sections and its distribution within the rat kidney was determined. In kidney zones, it was localized to medullary rays and inner cortex. In kidney substructure, activity was highest in collecting tubule, pars recti tubule, distal convoluted tubule and the ascending limb of Henle (14.2, 11.5, 6.4 and 9.2 mM kg-1hr-1, respectively). Activity in glomeruli, proximal convoluted tubule and small arteries was lower (2.1, 2.8 and 2.1 mM kg-1hr-1, respectively). The assay procedure was verified by established assays (spectrophotometric, fluorometric and radiometric TLC) which are often used in homogenate and purified PGDH preparations.


Light microscope and electron microscope alkaline phosphatase cytochemistry of rat bone marrow leukocytes. An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.


The use of the potassium pyroantimonate-osmium method as a means of identifying and localizing calcium at the ultrastructural level in the cells of calcifying systems. Potassium pyroantimonate-osmium has been used to localize calcium as an electron dense precipitate in the odontoblast, ameloblast and early hypertrophic chondrocytes of the mandibular condylar growth cartilage. The precipitate exhibited a specific association with various cell organelles. The presence of calcium in the precipitates was confirmed using energy dispersive analysis by x-rays. It is suggested that this K-pyroantimonate method provides an accurate technique for the investigation of the subcellular localization of calcium in calcifying systems.


Ferrocyanide staining of transferrin and ferritin-conjugated antibody to transferrin. To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.


Cytochemical properties of mitochondria in the gastric parietal cell. The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.


Spread of Salmonella typhi in a maternity hospital. An Asian patient with undiagnosed typhoid fever was admitted to a maternity hospital and delivered within 10 min. Salmonella typhi (phage type D5) was isolated from her blood and from the faeces of her baby. Another women in a different room of the labour suite at the same time acquired the same organism in her faeces; her brother was admitted to the Infectious Diseases Unit 5 weeks later with typhoid fever. Two babies, born over 60 h after the index case was delivered, became faecal excreters of the same strain and one of them also developed S. typhi osteitis of the hip. These two babies and their mothers were in the same ward as each other, but not that occupied by the infected mother and her baby. Nine other excreters in two of the families involved were identified. The index case and her baby were isolated immediately after delivery, and the relevant rooms in the labour suite were adequately disinfected. No evidence of undisinfected equipment used by the index case and the other infected patients was found, and no spread to staff was detected. The mode of spread remains unknown.


Evaluation of products for treating babies' napkins. A test is described for assessing the sanitizing effect of napkin treatment products on naturally urine-wetted and faecally-contaminated napkins. This test defines in-use conditions which closely resemble typical domestic situations. One napkin treatment product ('Napisan'), tested at two different concentrations and with challenges of different numbers of babies' napkins, performed satisfactorily under the conditions used.


Antibody responses to vaccination with WRL 105 strain live influenza vaccine in previously vaccinated and unvaccinated volunteers. Intranasal vaccination with a single 0.5 ml dose of 10(7.0) EID 50 WRL 105 strain live influenza vaccine elicited four-fold or greater increases in circulating homotypic haemagglutinating inhibiting (HAI) antibody in 60 (64.5%) of 93 volunteers, or in 58 (74.4%) of 78 volunteers with HAI antibody titres before vaccination of less than or equal to 1/20. In comparison, in a group of volunteers vaccinated 9 months previously re-vaccination elicited antibody responses in only 4 (6.9%) of 58 volunteers, or in 3 (14.3%) of 21 volunteers with antibody titres before vaccination of less than or equal to 1/20. Titres of vaccine-induced antibody and antibody resulting from earlier natural infection appeared to fall slowly and at equivalent rates over a 9 month period.


Delayed antimicrobial effects of skin disinfection by alcohol. Aqueous suspensions of Staphylococcus aureus were deposited on a Millipore filter and then exposed for a few seconds to 70% ethyl alcohol. Viable counts of bacteria extracted from the filter immediately after exposure to alcohol, and, in replicate experiments, after a further period of 3 h, showed that the mean immediate reduction of 97.6% in viable counts after treatment with alcohol was followed by a further mean reduction of 67.1% in the further 3 h holding time; the same bacterial suspensions allowed to dry on Millipore filters without exposure to alcohol showed a significantly smaller mean reduction in viable counts (34.3%) during a further 3 h holding time. These findings support the view that the reported further fall in numbers of bacteria on hands while wearing gloves for 3 h after alcohol disinfection can be explained by sublethal damage to some of the bacteria, from which they can recover only if promptly inoculated on culture medium.


Discordance among cell-mediated cytolytic mechanisms in cancer patients: importance of the assay system. ADCC and SCMC directed against Chang cell targets that are mediated by lymphocytes having properties characteristic of K cells were impaired in cancer patients. In contrast, ADCC directed against CRBC targets that is mediated by both K cells and macrophages was normal in cancer patients, whereas SCMC against CRBC that is mediated primarily by macrophages was increased. Thus, there was a discordance among cytotoxic mechanisms in cancer patients with K cell-mediated cytotoxic function being impaired and macrophage-mediated cytotoxicty being enhanced. Regression analysis suggested that these perturbations of cytotoxic function occurred independently. Cancer patients had an increased proportion of circulating macrophages and a decreased proportion of circulating macrophages and a decreased proportion of eosinophils, but these abnormalities did not correlate significantly with ADCC or SCMC.


Identity of C3- and C5-receptors on lymphoid cells. Tannic acid-treated SRBC, incubated with increasing concentrations of C5 (Etan-C5) can be attached to C3 receptor-carrying (Raji) cells. This binding is dependent on the amount of C5 on Etan-C5 and can be inhibited by pretreatment on the Raji cells with either C5 or C3. Similar inhibition by soluble C3 and C5, respectively, is obtained for the interaction between Raji cells and Etan-C3. In addition, the immune adherence reaction between Ehum and EAC1423b could be blocked by previous treatment of Ehum with C5 or C3. These results suggest the presence of binding sites for C5 on lymphoid cells and their identity with C3 receptors.


Immune-type interferon-induced transfer of viral resistance. Mouse immune-type interferon (type II), a lymphokine, caused the transfer of viral resistance from mouse L cells to human WISH cells. The interferon was incapable of protecting WISH cells in the absence of L cells. The transfer of viral resistance occurred with interferon preparations of various specific activities, and was in proportion to the interferon concentration in the preparations. The transferred resistance had the characteristics of an interferon-induced antiviral state in that it was blocked by actinomycin D, effective against different types of viruses, and resulted from an action on the cell rather than on the virus. Mouse immune-type interferon was more efficient than virus-type (type I) at eliciting the transfer of protection. The transfer phenomenon may represent a mechanism for amplification of the interferon system as a host defense against viral infection. Further, it serves as a model for studying the mechanism of lymphokine-induced transfer of information between cells.


Release of E-rosette augmenting factor (E-RAF) after stimulation of human leukocytes with mitogens or antigens. Stimulation of human peripheral blood leukocytes (HPBL) with mitogens such as phytohemagglutinin or concanavalin A increases the total number of lymphocytes that form rosettes with sheep erythrocytes. A similar effect is seen when HPBL from skin test-positive, but not skin test-negative, donors are stimulated by a specific antigen. It was also found that the culture fluids from mitogen-stimulated lymphocytes contained a substance that significantly increased the percentage of E-rosette forming cells (85 to 95%) over that of control culture fluids (40%). A similar phenomenon was also observed with supernatant fluids derived from antigenic stimulation of cells from skin test-positive donors, but not from skin test-negative subjects. The factor that produces this effect has been designated E-rosette augmenting factor (E-RAF). It is nondialyzable; it appears in the supernatants of antigen or mitogen-stimulated cells within 12 hr; and its production is not blocked by mitomycin C. It is produced by cells that do not adhere to glass wool columns and by mitogen-stimulated thymocytes. Sephadex G-100 chromatography showed that mitogen-induced E-RAF eluted from the column in advance of antigen-induced E-RAF. E-RAF has many properties that are characteristic of lymphokines.


Human transfer factors: structural properties suggested by HPRP chromatography and enzymatic sensitivities. Leukocyte extracts containing human transfer factor (TF) were fractionated by exclusion chromatography, and the active fraction (Sephadex G25, Fraction IIIa) was subjected to high pressure, reverse phase (HPRP) chromatography and enzymatic degradation. TF activity was assessed by the systemic transfer of dermal skin test reactivity from KLH-immunized donors to naive recipients. Preparative HPRP chromatography resolved Fraction IIIa into multiple chromophoric regions, two of which demonstrated transfer of KLH reactivity. Alkaline phosphatase treatment of Fraction IIIa converted the major ultraviolet-absorbing component, 5'-inosine monophosphate, to inosine and resulted in TF activity being restricted to one region. This HPRP region (R1A) contained less than 1% of the UV254 active material in Fraction IIIa but greater than 90% of the reactivity. The sensitivity of TF to pronase, proteinase K, phosphodiesterase I, and phosphodiesterase II was evaluated by inhibition of systemic transfer of KLH reactivity. Pronase and proteinase K destroyed systemic transfer activity and the pronase destruction could be inhibited with traysylol. Phosphodiesterase I, a 3' exonuclease, destroyed activity, whereas phosphodiesterase II, a 5' exonuclease, did not. The data are consistent with a phosphodiester-containing polypeptide in the structure of human TF for KLH reactivity.


The role of monocytes in human lymphocyte activation by mitogens. Studies were performed to determine the role of monocytes in human lymphocyte activation by mitogens. Velocity sedimentation at 1 x G in a new apparatus was utilized to obtain highly purified lymphocyte fractions (LF) nearly free of monocytes (0.02 to 0.4%) and a fraction (MF) enriched for monocytes (64 to 92%). The average peak responses of the lymphocyte fractions to phytohemagglutinin, concanavalin A, and pokeweed mitogen were 19, 10, and 9% of the responses achieved with unfractionated lymphocyte cultures containing approximately 20% monocytes. These changes were not attributable to altered dose requirements. When mitomycin-C-treated MF cells were used to reconstitute LF cultures, it was found that 4% monocytes fully restored the response to phytohemagglutinin whereas 8 to 16% monocytes were required for a normal response to the other mitogens. Higher numbers of MF cells produced supranormal responses, with 35 to 50% monocytes resulting in the optimal stimulation. Allogeneic monocytes were able to fully reconstitute the response of LF, and 2-mercaptoethanol (50 microM) was only slightly effective. In exploring possible mechanisms by which monocytes potentiate the mitogenic activity of lymphocytes, it was found that the supernatants of MF cultures could partially, but not completely, reconstitute LF responses, suggesting that contact with MF may be required for optimal effectiveness. Addition of graded numbers of monocytes to LF altered both the kinetics of the response and the peak level of proliferation. Monocyte depletion also resulted in markedly decreased survival of cultured unstimulated LF. These observations suggest a variety of possible effects of monocytes in potentiating mitogenic responses, including contact-mediated interactions with lymphocytes (possibly to present the mitogen optimally); enhancement of proliferation kinetics and the size of the responding subpopulation, and maintenance of a requisite growth factor(s) in the culture. Small differences in the monocyte content of cultured lymphocyte preparations may thus account for many of the often observed variations in mitogen responsiveness.


Mechanisms of lipopolysaccharide-initiated rabbit platelet responses. II. Evidence that lipid A is responsible for binding of lipopolysaccharide to the platelet. The mechanism of bacterial lipopolysaccharide-(LPS) initiated, complement-(C) mediated rabbit platelet lysis has been examined. The results of these studies support our previous observations that activation of the alternative C pathway is required for platelet lysis and that preparations of LPS that activate only the classical pathway (e.g., lipid A) do not cause lysis. The temporal relationship of the interaction of the LPS with the platelet before the addition of plasma suggests a time-dependent association of the LPS with the platelet. On the basis of a number of experiments, including inhibition with polymyxin B, treatment of LPS with alkali, and blocking experiments with polysaccharide-free LPS preparations, it is concluded that the lipid A region of the LPS molecule is responsible for attaching the LPS to the platelet. Finally, a comparison of the activity of lipid A-associated protein-LPS complexes with protein-free LPS demonstrated that an equivalent extent of platelet lysis was achieved with one-one hundredth the concentration of the former as that required for protein-free LPS. The data suggest that LAP facilitates attachment of the LPS to the platelet.


Action of the C3b-inactivator on the cell-bound C3b. The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H. The three polypeptides, C3b alpha-60, C3b alpha-40, and C3 beta, are held together as a single unit by disulfide bonds. This unit, referred to as C3b' is covalently bound to cell surfaces via the C3b alpha-60 polypeptide. The conversion of C3b to C3b' by C3bINA and beta 1H abolishes the ability of the C3b-bearing cells to adhere to human erythrocytes as well as the ability to form, on the cell surface, the B, D, and properdin-dependent amplification C3-convertase. However, the agglutinability of the cells with either anti-C3c or anti-C3d is not affected. Treatment of the C3b'-bearing cells with trypsin releases fragments of C3b' into solution, leaving a polypeptide of 32,000 daltons covalently linked to the membrane. Since the trypsinized cells are agglutinable by anti-C3d but not by anti-C3c, the 32,000 dalton polypeptide appears to correspond antigenically to C3d.


Activation of the classical pathway of complement by the C3NeF-stabilized cell-bound amplification convertase. C3 nephritic factor (C3NeF) has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. C3NeF, purified from the sera of eight patients by incorporation of C3NeF into the stabilized fluid phase amplification C3 convertase, C3bBb(C3NeF), followed by its release after decay of convertase function, was investigated for its ability to bind 125I-C1q and to activate 125I-C1. It was found that although fluid phase C3b,Bb(C3NeF) is fully capable of binding 125I-C1q, it is not able to activate 125I-C1 even at concentrations of 1.3 x 10(12) C3bBb(C3NeF) complexs/ml. On the other hand, cell-bound C3bBb(C3NeF) is capable of both binding 125I-C1q and activating 125I-C1. This discrepancy between fluid phase and cell-bound, C3bBb(C3NeF) was found for C3NeF preparations from eight different patients and therefore seems to apply to all C3NeF preparations.


A reexamination of humoral tolerance in chickens congenitally infected with an avian leukosis virus. In a reexamination of congenital infection of chickens with ALV, one of the classical models of immunologic tolerance, we were unable to demonstrate that there was any evidence suggesting humoral immune reactivity to the infecting virus. In ALV-F42 congenitally infected birds that have a persistent viremia and no neutralizing antibody detectable by conventional means, we could find no evidence of host IgG deposits in the kidneys, nor any suggestion of renal pathology, or damage. In addition, attempts to precipitate any putative circulating infections virus-antibody complexes by treatment with a highly specific anti-globulin were negative nor were there titer differences in the viremic plasmas of bursectomized birds compared to nonbursectomized birds, indicating that the chickens were not making either a masked neutralizing or a nonneutralizing antibody response to the infecting virus. The evidence presented indicates that congenital infection of chickens with ALV does, at least at the humoral level, result in a state of immunologic tolerance to the infecting agent.


Some quantitative aspects of the passive sensitization of human basophils. We review the theory for the binding of IgE to Fc receptors on basophil surfaces. We then use the theory to analyze binding experiments of Malveaux et al. and T. Ishizaka on the passive sensitization of basophils from a patient with chronic myelocytic leukemia and 75% basophilia. From their data we determine that the forward rate constant for the binding of human IgE to receptors on these human basophils is (3.0 +/- 1.0) x 10(4) M-1 sec-1.


Intestinal goblet cell mucus release. II. In vivo stimulation by antigen in the immunized rat. We previously reported that the infusion of certain soluble immune complexes stimulated mucus release from the rat small intestine in vivo. The present studies sought to evaluate the response of the intestine of normal and immunized rats to the infusion of antigen alone. One hour after the intraduodenal infusion of antigen, small intestinal washings were obtained and analyzed for the presence of 35S-labeled, high m.w. glycoprotein of goblet cell origin. The amount of goblet cell glycoprotein released was estimated from the radioactivity present in the void volume of a Sepharose 4B gel filtration column. The release of goblet cell mucus was enhanced by antigen stimulation in orally immunized animals. The discharge of goblet cell mucus was not increased after antigen infusion in animals immunized by the i.p. route despite the induction of high levels of serum antibody. The inability to demonstrate release of mucus after antigen challenge in systemically immunized rats suggests that the amount or the type(s) of antibody required at the mucosal surface is produced only after oral immunization.


Turnover and shedding of Ia antigens by murine spleen cells in culture. Lactoperoxidase-catalyzed radioiodination was used to examine the metabolic fate of surface Ia antigens on murine spleen cells in culture. Ia antigens, detected predominately on splenic B lymphocytes, were lost from the cultured cells with biphasic kinetics: a 4 to 6 hr rapid phase, t 1/2 = 5 hr followed by slow release through 20 hr, t 1/2 = 30 hr. The rapid loss of Ia antigens observed was abolished by both harsh iodination conditions and nonphysiologic incubation conditions. The rapid decline in Ia activity was shown to be due to shedding of intact Ia antigens from the cell and to predominant release of IA subregion-coded proteins. Release of Ia antigens from the cell was accomplished by replacement at the cell surface, and thus reflected net membrane Ia turnover. Ia shedding was shown to be extremely temperature dependent, reflecting both a comparatively high activation enthalpy and entropy requirement for turnover.


Monocyte dependence of pokeweed mitogen-induced differentiation of immunoglobulin-secreting cells from human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) when depleted of adherent cells (AC). The diminished responsiveness of the nonadherent cells (NAC) could not be ascribed to cell death, altered PWM dose response characteristics, or a change in the length of incubation required to generate a response. Supplementation with autologous or homologous AC, but not 2-mercaptoethanol, restored the capacity of NAC to generate ISC after PWM stimulation. By standard criteria AC were found to contain 85 to 90% monocytes. Furthermore, the monocytes and not the few lymphocytes contaminating the AC were responsible for restoring PWM responsiveness to the NAC. PWM-induced DNA synthesis of NAC also was markedly reduced compared to PBM. Again, supplementation with monocytes restored responsiveness to NAC. The monocyte dependence of PWM-induced proliferation and generation of ISC was most apparent when cultural conditions were employed that limited cell-to-cell interaction.


Interactions of C-reactive protein and complement with liposomes. II. Influence of membrane composition. We found previoulsy that interaction of C-reactive protein (CRP) with liposomal model membranes resulted in complement(C)-dependent membrane damage. In the present study, we investigated the influence of membrane composition on the interactions of CRP and C with liposomes. Adsorption experiments showed that binding of CRP was greatest to strongly positive liposomes. A lesser, but still substantial, extent of CRP binding also was observed with negative liposomes, but negligible amounts of CRP bound to neutral or weakly positive liposomes. CRP-mediated consumption of hemolytic C, and C-dependent glucose release from liposomes both were strongly influenced by liposomal charge, positive being superior to negative. Glucose release and, to a lesser extent, consumption of hemolytic C were inversely related to phospholipid fatty acyl chain length. Phospholipid fatty acyl unsaturation and liposomal cholesterol concentration both had strong influences on C consumption and glucose release. The data suggest that CRP-mediated C consumption and membrane damage require an optimum membrane fluidity. Complement damage in the presence of CRP was enhanced by certain sphingolipids and also by digalactosyl diglyceride, but not by sphingomyelin. Our results thus demonstrate that CRP-mediated C consumption and C-dependent membrane damage both are influenced by the liposomal membrane composition.


Studies on the terminal stages of immune hemolysis. IV. Effect of metal salts. The inhibitory effect of metal salts on the formation and transformation of E to ghosts was studied. Ferrous and ferric salts inhibited the binding of C9 to EAC1-8 cells but had no other inhibitory role in the reaction. Uranly, copper, and zinc salts inhibited the transformation of the EAC1-9inserted intermediate to E ghosts. This inhibition occurred at a stage prior to detectable damage as measured by 86Rb or hemoglobin release and at a step prior to that inhibitable by high molarity EDTA. Consequently a further step in the reaction sequence of E to ghosts was identified. The reaction sequence leading from EAC1-9 to ghosts can be summarized as follows: formula: (see text).


A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of Schistosoma mansoni. Centrifugation of human white blood cells over either Ficoll-Hypaque or slightly hypertonic Metrizamide discontinuous gradients reliably produces separate fractions that are enriched for either neutrophilic or eosinophilic granulocytes. This single step purification routinely yields 90 to 100% pure neutrophils and 85 to 100% pure eosinophils. Metrizamide gradients, in particular, reproducibly provide high yields of 90 to 100% pure eosinophils from normal subjects with 2 to 3% eosinophils in their peripheral blood. The method does not damage cells as judged by morphologic or functional criteria. The purified cell populations were tested for their ability to damage antibody-coated schistosomula either by the measurement of 51Cr release from labeled organisms, or by direct morphologic assessment. Neutrophils were superior in their ability to release 51Cr from labeled organisms, but eosinophils adhered to the organisms to a greater extent and induced microscopically detectable damage.


Activation of human complement by liposomes: a model for membrane activation of the alternative pathway. Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.


Isolation of a sperm membrane sialoglycoprotein autoantigen from rabbit testes. A single rabbit sperm membrane autoantigen has been isolated from rabbit testes by immunoadsorbent chromatography, agarose chromatography, and electrophoresis. This rabbit sperm autoantigen (RSA-1) is located in the sperm plasma membrane and blocks the sperm cytotoxic (immobilizing) activity of autoantisera. RSA-1 is a sialoglycoprotein, approximately 40% carbohydrate and containing 0.2 micrograms sialic acid/microgram protein. Its m.w. is estimated at 13,000 +/- 1200 by SDA-PAGE. RSA-1 can self-aggregate into higher m.w. forms. Approximately 0.6 mg of RSA-1 can be isolated from 100 rabbits testes. The isolated RSA-1 shows a 300-fold increase in specific cytotoxic (immobilizing) inhibitory activity over the starting testis pellet material.


Two spontaneous BALB/c lymphomas synthesize IgM: monomers and half molecules are isolated and characterized whereas another molecule resembles IgD. Spontaneous lymphomas of BALB/c mice, both in vivo tumors and cell lines established in long term tissue cultures, were investigated for their ability to synthesize IgM by using radiolabeled amino acid precursors. Immunoglobulins manufactured by lymphomas K46 and L10A had the m.w. of monomeric IgM and IgM half molecule. Both of these molecules could be immunoprecipitated with class-specific anti-IgM but not anti-IgA or anti-IgG. When precipitated with polyvalent anti-Ig L10A synthesized monomeric immunoglobulins that migrated as two peaks in contrast to their single counterpart precipitated with anti-IgM. The second peak migrated in the region expected for IgD. Monomer and half molecules were composed of similar ratios of mu-chains to light chains linked by disulfide bonds. The mu2L2 monomer of these B cell lines migrated slightly slower in SDS PAGE than a mu2L2 secreted by a myeloma. Thus, these lymphomas synthesize immunoglobulins with the chemical and antigenic characteristics typical of monomeric membrane-attached IgM and IgM half molecules, plus a molecule resembling IgD on L10A only. Lymphoma assembly of monomeric IgM may follow the same initial biosynthetic sequence as myeloma assembly.


Genetic study of mouse sensitivity to MHV3 infection: influence of the H-2 complex. Genetic study of acute and chronic mouse hepatitis virus type 3 disease was carried out in segregating generations of a cross involving a susceptible (C57BL/6) and a resistant (A/J) mouse strain. The data obtained indicate that one or two recessive genes may be involved in resistance of acute and chronic diseases but suggest that the genes involved in both diseases are different. In this cross, no correlation was observed between H-2 and acute or chronic disease. In mice of congenic lines, however, A/Sn (H-2a), A.SW (H-2s), A.BY (H-2b), and A.CA (H-2f), it appeared that the presence of the H-2f allele conferred to heterozygote as well as to homozygote animals the capacity to resist the development of chronic disease. It seems, therefore, that MHV3 sensitivity in mice is under the influence of at least two major genes: one for the acute disease and the other, H-2 linked, for the chronic disease.


Immunoregulatory properties of human esophageal tumor extract. Noncytotoxic aqueous extracts of esophageal tumors (TEx) inhibit the spontaneous uptake of 3H-thymidine by human peripheral blood mononuclear cells (PBM). TEx also inhibits mitogen- and antigen-induced PBM blastogenesis, mitogenesis, 3H-thymidine uptake, and 3H-leucine incorporation. Inhibition is reversible and is mediated by a heat-labile protein with a m.w. of approximately 70 to 80,000 daltons. Inhibitory activity is not due to trivial binding of mitogen or neucleotide by the inhibitor, nor is it entirely tumor specific since similar but quantitatively less activity is extractable from adjacent normal esophageal mucosa. Assorted cell surface membrane phenomena such as contact inhibition of in vitro endothelial cell proliferation, lymphocyte E rosette formation, and cap formation are unaffected by TEx. In contrast, immunoglobulin synthesis by pokeweed mitogen-stimulated PBM is enhanced by TEx. The potential importance of the local production of immunoregulatory agents like TEx in the immunologic control of tumor cell growth is discussed.


Association between H-2 and vaccinia virus-induced antigens on the surface of infected cells. The relationship between H-2 molecules and vaccinia virus-induced antigens on the surface of H-2d infected cells was investigated by the differential redistribution method and by the blocking capacity of monospecific anti-H-2 sera on an anti-vaccinia cell-mediated cytotoxicity (CMC). Capping of either H-2K or H-2D molecules upon addition of monospecific and anti-H-2 sera was followed by the complete redistribution of viral antigens, suggesting the formation, on the cel membrane, of complexes of H-2K, H-2D molecules and vaccinia virus-induced antigens. However, not all H-2 molecules were involved in this association since i) free H-2K and H-2D molecules still moved independently on the cell surface, and ii) capping of vaccinia virus-induced antigens failed to induce the redistribution of all the H-2K and H-2D molecules. In addition, either monospecific anti-H-2K or anti-H-2D antiserum was found to exert potent blocking activity on anti-vaccinia CMC, indicating also a close topographical relationship between H-2K, H-2D molecules and vaccinia virus-induced antigens.


Hybrid resistance to BALB/c plasmacytomas. I. Resistance to MPC-11 controlled by a locus not linked to H-2. Genetic control of hybrid resistance to the BALB/c plasmacytoma MPC-11 was investigated. The results indicate that a single dominant autosomal gene or gene complex, which segregates independently of H-2 and the coat color c and b-loci, controls resistance to this tumor. This gene has the same strain distribution pattern in the CXB Bailey recombinant inbred strains as three unlinked genes, H-2, Ly-4, and Ea-4. It is possible, therefore, that it could be linked to either of the latter two loci. Strains that carry a positive allele for resistance are C57BL/10 and all of its congenic resistant partners tested, C57BL/6, C57L, C57BL/Ks, AKR, and DBA/1. BALB/c and its congenic resistant partners are presumed to carry a negative allele of the gene for resistance to MPC-11. Strains such as SJL, DBA/2, and A and its congenic resistant partners, which form susceptible hybrids with BALB/c, could carry either the negative allele of the gene for resistance, like BALB/c, or could carry both a positive allele of the gene and some other gene conferring susceptibility on the hybrids. Heterozygosity within the H-2 complex increases resistance only in the presence of this non-H-2 linked gene for resistance, and the effect maps to the left of the H-2D region.


Effects of arachidonic acid and other unsaturated fatty acids on mitogenesis in human lymphocytes. The effect of fatty acids and other lipids on mitogenic responses in cultured human peripheral blood lymphocytes was studied. Several-fold enhancement of tritiated thymidine incorporation was observed at 0.1 to 5.0 micrograms/ml concentrations of arachidonic acid. Other unsaturated fatty acids produced less marked changes. Increased responsiveness was demonstrable in a variety of media including RPMI 1640 supplemented with 10% fetal calf serum. Changes were also observed in uridine incorporation, total cell number, and blast transformation, indicating that the effect was not on thymidine transport or pool size per se. Arachidonic acid failed to affect PHA binding, indicating that the lectin-cell interaction was not altered. Higher concentrations of fatty acids were inhibitory.


Formation of thromboxane B2 and hydroxyarachidonic acids in purified human lymphocytes in the presence and absence of PHA. The metabolism of exogenous and endogenous [14C] arachidonc acid was studied in purified human peripheral blood lymphocytes carefully freed of contaminating platelets. Formation of products co-migrating in a number of different solvent systems with 5-hydroxyarachidonic acid (5-HETE), thromboxane B2 (TB2), prostaglandins and probably 12-hydroxyarachidonic acid (12-HETE) was demonstrated. In cells prelabeled with [14C] arachidonic acid, phytohemagglutinin (PHA) produced substantial (3.5- to 12-fold) increases in 5-HETE, 12-HETE, and TB2 radiolabeling. The metabolism of exogenous [14C] arachidonic acid was much less affected by PHA. Since PHA releases cell-bound arachidonic acid, it appears that the response involving endogenous label is due to increased availability of free arachidonic acid rather than induction of arachidonic acid-metabolizing enzymes. Various inhibitors of arachidonic acid metabolism exerted similar effects in lymphocytes to those described previously in other tissues providing a possible basis for interpreting their inhibitory effects on mitogenesis, described in the preceding paper.


Anti-DNA activity of IgG F(ab')2 from normal human serum. The components of normal human serum (NHS) which bound DNA in a standard assay for anti-DNA antibody were characterized. IgG was the major detectable protein isolated from NHS by affinity chromatography on DNA-cellulose. A second adsorption of the whole serum IgG with DNA-cellulose did not remove additional gamma-globulin indicating that only a very small fraction of the IgG was capable of binding DNA. This binding activity was largely restricted to denatured DNA. IgG (Fab')2 bound DNA as well as the intact molecules demonstrating the antibody-like nature of the IgG binding. These results suggest that IgG antibody to denatured DNA is a normal component of human serum.


Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. VII. Genetically restricted and nonrestricted physical interactions. We have assessed the genetic restrictions on physical interactions between macrophages and  central lymphocytes and between central and peripheral lymphocytes in antigen-specific macrophage-lymphocyte clusters with respect to I-region differences of inbred strains 2 and 13 guinea pigs. When using lymphocytes from guinea pigs immunized with DNP-OVA or DNP-GL in CFA, the antigen-specific interaction between central lymphocyte and macrophage requires that both cells be derived from animals syngeneic at the I-region of the major histocompatibility complex. In studies using antigens, the responses to which is under the control of MHC-linked Ir genes, macrophages from the responder, but not from the nonresponder parental strain support cluster formation with responder x nonresponder F1(2 X 13) T cells. In contrast, the physical interactions between central and peripheral T lymphocytes are not restricted by the I-region of the MHC and the peripheral lymphocyte need not be from an animal immune to the antigen used to drive macrophage central lymphocyte interactions.


Selective adherence of lymphocytes to myelinated areas of rat brain. An in vitro system developed for studying lymphocyte binding to high endothelial venules (HEV) of lymph nodes was used to determine if there are similar binding sites in other organs of the rat. Thoracic duct lymphocytes (TDL) adhered selectively and uniformly to white matter when overlaid onto glutaraldehyde-fixed tissue sections of cerebellum and cerebrum. The pattern of TDL adherence to cerebellar sections showed that binding to nonmyelinated areas was negligible. Comparison of TDL-white matter to TDL-HEV binding demonstrated that the density of adherence to each site was quantitatively similar. In contrast, lymphocytes exhibited little tendency to bind to tissue sections of liver, spleen, heart, thymus, and salivary glands. TDL adherence to cerebellar white matter occurred rapidly, was cell dose dependent and optimal at 7 degrees C. White matter binding was also a property of spleen lymphocytes but the thymus was deficient in cells with this capability. The affinity of TDL and spleen lymphocytes for myelinated areas of the brain suggests the presence of myelin binding receptors on these cells.


Enhanced uptake of 3H-glucosamine by macrophages stimulated by macrophage-activating factor. The enhanced uptake and incorporation of 3H-glucosamine by guinea pig peritoneal exudate macrophages stimulated by macrophage-activating factor (MAF) were studied. MAF was produced by concanavalin A- (Con A) stimulated lymphocytes. Two systems were developed to assay this phenomenon, one using microtiter plates, the other using suspension cultures. Enhanced uptake of 3H-glucosamine by macrophages was found after 48-hr incubation in microtiter plates and after 24 hr in suspension cultures. Increased uptake of 14C-glucose and 3H-galactose was also observed under these conditions. The enhanced uptake of 3H-glucosamine was markedly reduced or abolished by the addition of glucose to the cell cultures or by replacement of the medium with fresh medium before pulsing with labeled sugar. These observations indicate that the mechanism of enhanced uptake reflects in part increased glucose utilization by the stimulated macrophages.


Thermal injury-associated immunosuppression: occurrence and in vitro blocking effect of post recovery serum. Sera from 38 of 72 burn patients have been found to be significantly suppressive to the PHA-induced blastogenesis of normal human lymphocytes in culture. In many of these patients, we have observed that suppression levels decline with recovery. In a study of eight of these patients, we have found that the addition of post recovery serum to cultures of normal lymphoyctes blocked the suppressive effect of autologous serum obtained earlier. Blocking appears to be achieved through the formation of antibodies since: a) IgG levels are greatly elevated in serum samples having blocking activity, b) the time of appearance of blocking substances in the serum is compatible with the generation of antibody, and c) blocking activity is contained in the protein-A isolated IgG fraction of such post recovery serum.


Immunologic vasculitis in beige mice with deficiency of leukocytic neutral protease. Immune complex vasculitis has been induced in normal mice and in mice with features of the Chediak-Higashi syndrome ("beige" mice). The accumulation of neutrophils in peritoneal exudates after the injection of C5a is not quantitatively depressed in beige as compared with normal mice. Immune complex-induced vasculitis in these two strains of mice is not quantitatively different, as assessed by vascular damage (vasopermeability changes and histologic criteria). Measurements of leukocyte enzymes confirm the findings of Vassalli et al. that leukocytes of beige mice lack neutral protease activity. The data suggest that the neutral protease of murine leukocytes does not account for the vascular damage of immune complex vasculitis.


Evidence for a common mucosal immunologic system. I. Migration of B immunoblasts into intestinal, respiratory, and genital tissues. The origins of immunoglobulin-containing cells in intestinal, respiratory, mammary, and genital tissues were studied in CBA/J female mice by using an adoptive lymphocyte transfer method. Within 24 hr after transfer, [3H]thymidine-labeled donor mesenteric lymph node (MLN) cells were observed in recipient gut, cervix and vagina, uterus, mammary glands, and MLN, where approximately 60% contained IgA and 25% IgG. In peripheral lymph nodes (PLN), 44% of the labeled cells after MLN transfer contained IgG, whereas only 8% were of the IgA isotype. The preference of the MLN to populate mucosal sites was clear from the results. Labeled PLN cells were transferred and the majority of these returned to their sites of origin and contained IgG. Of the small number of labeled PLN cells found in mucosal tissues, approximately equal percentages (30%) of IgA- anti IgG-containing cells were seen. Dividing cells prepared from mediastinal (bronchial) lymph nodes (BLN) showed a propensity to localize in the lungs rather than the intestine. However, the predominant immunoglobulin content of these donor cells in gut, lungs, and MLN was IgA. In recipient PLN, most labeled BLN cells contained IgG. These data support the concept of a common mucosal immunologic system.


Shared chemical properties of different murine thymus-leukemia antigens. Immunochemical studies of murine thymus-leukemia antigens (TLA) have confirmed that the subunit structure consists of a 45,000-dalton heavy chain and a beta 2 microglobulin (beta 2m) light chain. Similar structural features are exhibited by the TLA from thymocytes of Tlaa, Tlac, Tlad, and a leukemia cell derived from C57BL/6, a Tlab strain. In addition to the similar subunit structure from the four haplotypes, each TLA shows a similar pattern of trypsin proteolysis. This procedure yields a major heavy chain cleavage product of approximately 37,000 daltons that remains associated with beta 2m and retains most or all of the antigenic determinants of the intact TLA. Evidence is presented that TLA do not exhibit Fc receptor properties, nor do they adsorb to murine leukemia virus antigens under the conditions of isolation for analysis on polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Taken together these findings strongly support the hypothesis that TLA comprise a family of chemically similar antigens belonging to a structurally and genetically related group that includes H-2D, H-2K, and Qa-2,3.


Humoral and formed elements of blood modulate the response of peripheral blood monocytes. I. Plasma and serum inhibit and platelets enhance monocyte adherence. Human and rabbit peripheral blood monocytes normally adhere to plastic tissue culture plates in vitro when they are suspended in Hanks' media. Increasing amounts of autologous serum or heat-inactivated plasma in the cell suspensions prevented the adherence of both monocytes and lymphocytes. The inhibitory effect of plasma was separated into three areas of activity by chromatography on Sephacryl S-200. The profile of inhibitory activity did not coincide with the protein elution profile, suggesting that inhibition was not a nonspecific protein effect. A layer of adherent platelets overcame the inhibitory effect of plasma on monocyte adherence. Platelets selectively increased monocyte as opposed to lymphocyte adherence and this was specific for platelets in that neither neutrophils nor fibroblasts could substitute for platelets. Both plasma and platelets acted directly on monocytes.


Opposing effects of cryostat sections of preneoplastic and neoplastic mouse mammary lesions on in vitro migration of peritoneal exudate cells. Cryostat sections of normal mouse tissues and of preneoplastic HAN and neoplastic mammary tumors were used as "antigens" in MMI tests. Nonspecific inhibition of normal and sensitized PEC migration was induced by HAN and some normal tissues, including normal mammary gland. This inhibition did not require the presence of lymphocytes, was not species specific, and could be blocked by sera from HAN-bearing mice. Cryostat sections of mammary tumors did not inhibit, indeed occasionally enhanced PEC migration. Further, the presence of tumor cryostats and eluates interfered with inhibition induced by HAN cryostats and by PPD with PEC from donors sensitized to that antigen. Histologic examination of HAN and of mammary tumor tissue revealed inflammatory cells to be distributed diffusely in the former and localized peripherally around the latter type of lesion.


IgE mediated triggering of rat basophil leukemia cells: lack of evidence for serine esterase activation. Inhibition of mediator release from mast cells and basophils by diisopropylfluorophosphate (DFP) and other organophosphorus compounds known to inhibit serine esterases has in the past led to the hypothesis that immunologic triggering of these cells involves an activatable serine esterase. In this study we have shown that two nonphosphorylating or poorly phosphorylating structural analogs of two potent phosphorylators inhibit release of incorporated serotonin from cultured rat basophil leukemia cells. We conclude that, by itself, inhibition of immunologic mast cell triggering by phosphorylating organophosphorus compounds can no longer be considered evidence for involvement of an activatable serine esterase in mast cell triggering.


In situ lymphoid cells of mouse mammary tumors. IV. Comparison of functional activity of lymphoid cells separated from mammary tumors to that of spleen and lymph node cells of tumor-sensitized mice. Lymphoid cells isolated form several types of mouse mammary tumors are capable of stimulating tumor cell growth or survival in MCT assays. Lymph node and spleen cells of mice bearing such a tumor are specifically cytotoxic to the tumor cells. Surgical removal of the tumor is followed in 4 to 7 days by the appearance of stimulatory capacity in spleens and lymph nodes. By day 10, cytotoxic cells specific for the sensitizing tumor are again detected. These reach a peak on day 13. By day 17 no reactivity is detectable. The functional distribution of tumor-reactive lymphoid cells is different between tumor masses and peripheral lymphoid organs.


Regulation of IgE antibody production by serum molecules. IV. Complete Freund's adjuvant induces both enhancing and suppressive activities detectable in the serum of low and high responder mice. Two biologically active serum molecules manifesting precisely opposite biologic effects, both of which are selective for IgE antibody synthesis, can be detected in the serum and ascites fluids of CFA-immune mice. One activity, described previously, is suppressive and hence termed suppressive factor of allergy (SFA); the other, reported for the first time herein, is enhancing and has been termed enhancing factor of allergy (EFA). The ability to detect one vs the other activity requires certain special manipulations such as different doses of low dose x-irradiation. Conclusive evidence for the existence of two distinct factors mediating these two opposing biologic effects was obtained in studies demonstrating that affinity chromatography on concanavalin A-Sepharose segregated the two molecular entities. Thus, SFA binds poorly or not at all to Con A-Sepharose, whereas EFA binds to Con A and can be recovered in the eluate eluted with the competitive sugar alpha-methyl-D-glucopyranoside.


Structural studies on the murine Ia alloantigens. IV. NH2-terminal sequence analysis of allelic products of the I-A and I-E/C subregions. Murine Ia alloantigens encoded by the I-A and I-E/C subregions were isolated from radiolabeled splenic lysates and examined by NH2-terminal sequence analysis. Haplotype-associated sequence variation was detected in the beta, but not alpha, subunits of both the A and E/C alloantigens. The A beta-polypeptides from k and b haplotypes show four differences in the 12 positions compared, whereas the E/C beta-polypeptides from k and r haplotypes show two differences in the 13 positions compared. No sequence variation was detected between the Ak and Ab  alpha-chains (six positions compared) or between the E/Ck and E/Cr alpha-chains (11 positions compared). Homology relationships between these murine Ia alloantigens and the human Ia (DR) alloantigens are also presented.


Tumor-basophil interactions in vitro--a scanning and transmission electron microscopic study. Purified guinea pig basophils, or basophils either specifically degranulated with antigen or nonspecifically degranulated with lectin, were cultured with guinea pig line 1 hepatoma cells for 1 to 24 hr and studied ultrastructurally. As early as 1 hr of culture, degranulated or nongranulated basophils and tumor cells formed close contacts by mutually intertwined elongated cell processes and also in cultures containing degranulated basophils, extruded membrane-free basophil cytoplasmic granules became firmly attached to tumor cells. At later intervals, some tumor cells cultured with basophils exhibited cytostatic and cytopathic changes, including dense mitochondria, centralization of organelles, dilated perinuclear and rough endoplasmic cisternae, cell swelling and cytoplasmic lucency, disrupted cytoplasmic organelle and plasma membranes, nuclear pyknosis and fragmentation. Some tumor cell specialized surface attachments were either disrupted or damaged at points of basophil or basophil granule adhesion. Tumor damage was most extensive in cultures containing degranulated basophils, although only a minority of tumor cells (less than 10%) was affected. Tumor injury was seen much less frequently in the presence of nondegranulated basophils, and was absent in control cultures of tumor alone. The occasional viable tumor cells that phagocytosed basophil granules were apparently unharmed, suggesting that internalization of basophil granules by tumor cells is not cytotoxic.


An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade. A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.


Respective influence of extrinsic and intrinsic factors on the age-related decrease of thymic secretion. The serum level of a circulating thymic factor (FTS) described in our laboratory diminishes in mice after adult thymectomy and with age. However, thymuses from old mice, when grafted into young adult thymectomized recipients lacking circulating FTS, can still partially restore the circulating FTS level of the recipients, whereas newborn thymuses are less efficient in restoring the serum level of FTS in old recipients than in young adult thymectomized recipients. Taken together, our results suggests that in addition to an intrinsic deficiency of the thymic secretion, "environmental" factors play a role in the disappearance of circulating FTS with age, the more so since we observed in old mouse sera factors inhibiting the in vitro biologic activity of FTS, factors that are absent from young mouse sera.


Low molecular weight eosinophil chemotactic factor in granulomatous liver of murine schistosomiasis. An acidic peptide, preferentially chemotactic for eosinophils, was extracted from livers of mice infected with Schistosoma mansoni. Sephadex G-25 column chromatography showed that the majority of the eosinophil chemotactic activity was detected in the fractions just after elution of the molecular marker vitamin B12 (m.w. 1355.4). This activity began to appear in the livers of some mice 5 weeks after infection. Peak activity was detected at 8 to 12 weeks after infection and persisted at least until 16 weeks. It was sensitive to carboxypeptidase-A. By Dowex-1 anion exchange chromatography, the activity eluted as a narrow peak at pH 3.1 TO 2.6 as shown for eosinophil chemotactic factor of anaphylaxis (ECF-A). The activity was also detected in a broad peak at pH 6.3 to 3.7. Unlike ECF-A, the activity was stable to boiling in both acid and alkali. These findings suggest that granulomatous liver of murine schistosomiasis-derived eosinophil chemotactic factor (ECF-G) may play a specific role in eosinophil accumulation in this chronic inflammation.


Requirement for a bacterial flora before mice generate cells capable of mediating the delayed hypersensitivity reaction to sheep red blood cells. A delayed-type hypersensitivity (DTH) reaction can be elicited by an injection of 10(8) sheep red blood cells (SRBC) into a rear footpad of conventional (CV) mice previously immunized with small doses of SRBC. In contrast, immunization of germ-free (GF) mice with the same doses of SRBC produced no DTH when immunization was by the intravenous (i.v.) route, and only weak reactions when immunization was by the subcutaneous (footpad) route. Varying the immunizing dose of SRBC, or the time at which DTH was elicited, did not produce a state of DTH responsiveness in i.v. immunized GF mice. However, the transfer of lymphocytes from CV mice, immunized 4 to 5 days previously with SRBC, into GF mice, conferred on GF mice the capacity to express DTH. Although DTH was not readily demonstrable in GF mice immunized with SRBC, they nevertheless produced normal levels of hemagglutinating antibody to SRBC. Finally, it was shown that GF mice could generate a normal DTH response to SRBC if they were first monoassociated with a Gram-negative bacterial flora.


Immunoprophylaxis of respiratory syncytial virus infection in the infant ferret. Infant ferrets can be protected from respiratory syncytical virus challenge at 3 days of age by gestational infection of their mothers. Ferrets acquire their immunity to respiratory syncytial virus postpartum via immunizing products of lactation. The level of protection against viral replication correlates with the maternal serum neutralizing titer or a concomitant factor. Passive administration of adult ferret serum with a neutralizing titer of 1:1024 or greater, either i.p. or orally does not confer immunity. A nonantibody-mediated protective mechanism appears to play an important role in protecting the infant ferret from respiratory syncytial virus replication. Our findings allow the testing of the efficacy of future human vaccines before human clinical trial.


Increased prostaglandin production by human monocytes after membrane receptor activation. Human monocytes in culture release small amounts of prostaglandin E (PGE) into the medium. Addition of Fc fragments of IgG to human monocyte monolayer cultures results in a marked increase in PGE release; Fab fragments, monomeric IgG, and human serum albumin have no effect. An IgG1 myeloma has no effect on PGE levels but addition of the heat aggreagted protein results in a marked increase of PGE secretion. Exposure of the cells to Con A, which binds to a specific monocyte plasma membrane receptor, also results in a large increase in PGE release. The magnitude of the increase in PGE secretion produced by exposure of the monocytes to these ligands greatly exceeds the stimulation observed after the addition of antigen-activated mononuclear cell supernatants, zymosan, Sephadex beads, or endotoxin, to monocyte cultures. Prostaglandin E2 (PGE2) accounts for approximately 70% of the total prostaglandins released by stimulated cells. After addition of Indomethacin to monocyte cultures, the stimulatory effects of the ligands on PGE release are inhibited. Addition of Con A to monocyte cultures results in an increased incorporation of [3H]-arachidonic acid into PGE2. These results suggest that this ligand stimulates synthesis as well as release of this prostaglandin.


Defective resistance to Plasmodium yoelii in CBA/N mice. The role of B lymphocytes in resistance to malaria was studied in defective and normal F1 mice derived from CBA/N mice, a strain with an X-linked B cell defect. When infected with normally nonlethal Plasmodium yoelii, immune defective F1 male mice had higher parasitemias and more prolonged infections than normal F1 mice, as well as a 50% mortality rate. Before infection the plasma levels of IgM and IgG were lower in defective F1 males than normal F1 mice. The polyclonal IgM and IgG responses of infected abnormal F1 mice were delayed and lower in absolute magnitude than those of normal F1 mice. Furthermore, specific IgM and IgG anti-plasmodial antibody titers, as determined by radioimmunoassay, were depressed on day 12 in the defective F1 males. Although IgG titers approached those of the normal F1 mice on day 19, defective F1 male IgM titers remained depressed. These data demonstrate that an X-linked gene that affects B cell function influences malarial resistance in mice, presumably via a decreased specific IgM response, and the slow development of a specific IgG response to P. yoelii infection.


Isolation of functional subsets of human peripheral blood monocytes. Monocytes were isolated by counterflow centrifugation of Ficoll-Hypaque separated peripheral blood mononuclear cells. The monocytes formed a bimodal volume distribution of "large" and "small" phagocytic esterase-positive, peroxidase-positive cells with peaks at 470 and 410 mu3, respectively. The large monocytes were predominately Fc receptor positive, and were able to lyse both sensitized human and chicken erythrocyte targets in ADCC assays, whereas the small monocytes were largely FcR negative and were inactive against sensitized human erythrocyte targets. However, ADCC against chicken erythrocyte targets was seen in some fractions containing small monocytes and was probably due to FcR+ lymphocytes (K cells) in those fractions. These experiments establish that monocytes are effectors of ADCC against both human and chicken erythrocyte targets and that the peripheral blood monocyte is heterogeneous in size, function, and surface receptor distribution.


Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells. Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.


Human leukocyte Fc (IgG) receptors: quantitation and affinity with radiolabeled affinity cross-linked rabbit IgG. Quantitative studies have been made of Fc receptors on human leukocytes derived from peripheral blood, thymus, tonsil, and spleen. The relative affinities and average numbers of receptors per cell were determined by measuring the binding of 125I-labeled, affinity cross-linked trimers of rabbit IgG to various populations of cells. In parallel, the sizes of receptor-bearing populations were determined by fluorescence microscopy. Fc receptors could be detected on leukocytes from peripheral blood and spleen, but not from tonsil or thymus. In the peripheral blood, the highest density of receptors was found on polymorphonuclear leukocytes; a subpopulation of lymphocytes had somewhat fewer receptors per cell, and circulating monocytes had the lowest receptor density. Among splenocytes, most of the receptors were found on myeloid cells and monocytes. In all populations, the affinity of Fc receptors for the trimer was about the same. At 0 degrees C the average value for the association constant was 5 x 10(7) M-1.


Lack of correlation between neutralizing antibody production and suppression of coxsackievirus B-3 replication in target organs: evidence for involvement of mononuclear inflammatory cells in host defense. CD-1 mice inoculated with coxsackievirus B-3 i.p. developed a generalized infection involving the heart, pancreas, and liver. The disease was nonlethal and viral growth in the target organs was terminated in about a week. Administration of cortisone acetate 30 min to 2 hr before infection markedly enhanced the severity of disesae. Abnormally high titers of virus were found in the target organs between days 3 and 7 with persistence of infectious virus in the heart for at least 2 weeks. In addition the extent of necrosis of myofibers, pancreatic acini, and hepatic parenchyma was increased and a high percentage of the animals died. There was no evidence that the anti-viral antibody response was impaired in steroid-treated mice since concentrations of neutralizing antibody in the circulation were normal. In contrast, immigration of mononuclear inflammatory cells into the hearts of these animals was depressed and when present, foci of inflammation contained some polymorphonuclear leukocytes. The data indicate that inhibition of coxsackieviral growth cannot be attributed to the sole effects of neutralizing antibody and suggest that mononuclear inflammatory cells infiltrating the heart play a role in primary host defense.


Re-expression of nonglycosylated surface IgA in trypsin-treated MOPC 315 plasmacytoma cells. The importance of glycosylation for the re-expression of surface immunoglobulin in trypsin-treated MOPC 315 plasmacytoma cells was examined by using tunicamycin, an antibiotic that prevents glycosylation by inhibiting the formation of N-acetylglucosamine-lipid intermediates. Tunicamycin greatly inhibited the secretion of nonglycosylated MOPC 315 IgA in trypsin-treated cells. Two hours after trypsin treatment, there was an 80% inhibition of secretion as measured by immunoprecipitation assays of biosynthetically labeled immunoglobulin. However, tunicamycin had no effect on the time course of re-expression of surface IgA in these cells as measured by TNP-sheep erythrocyte rosette formation and [125I] TNP-albumin binding to the plasmacytoma cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-labeled cell surface IgA re-expressed in the presence of tunicamycin revealed a protein with an apparent m.w. identical to nonglycosylated MOPC 315 alpha-chains, further suggesting that nonglycosylated surface IgA was being inserted into the plasma membrane. This protein did not bind to concanavalin A-Sepharose. These data suggest that in MOPC 315 plasmacytoma cells, glycosylation is necessary for immunoglobulin secretion but not for immunoglobulin expression at the cell surface.


Studies on in vivo priming of the TNP-reactive cytotoxic effector cell system. I. Comparison of the effects of intravenous inoculation with TNP-conjugated cells on the development of contact sensitivity and cell-mediated lympholysis. A comparison was made of the effects of i.v. inoculation of trinitrophenyl-(TNP) conjugated syngeneic cells on the subsequent in vitro generation of TNP-reactive effector cell activity and on the in vivo development of TNP-contact sensitivity. The administration of syngeneic TNP-conjugated spleen cells before 2,4,6-trinitro-1-chlorobenzene (TNCB) painting abolished the capability of animals to develop TNP-contact sensitivity. In contrast, the same treatment resulted in an appreciable augmentation in the generation of TNP-reactive cytotoxic effector cell activity as measured by subsequent in vitro sensitization with TNP-conjugated cells. The possible mechanisms by which enhanced TNP-reactive cytotoxic effector cell activity was elicited under conditions identical to those that induced unresponsiveness for TNP-contact sensitivity are discussed.


Glucocorticoids inhibit trypsin-induced DNA release from phytohemagglutinin-stimulated blood lymphocytes. Trypsin-induced DNA release from phytohemagglutinin (PHA)-stimulated human lymphocytes is inhibited by different glucocorticosteroid compounds at low pharmacologic concentrations, in a dose-dependent manner, and in order of the known anti-inflammatory potency of the different preparations. In contrast, PHA-stimulated cell growth is 100- to 1000-fold less sensitive to inhibition by the same glucocorticoids. Nonglucocorticoid steroids have little effect on either DNA release or cell growth except at high concentrations. Inhibition of DNA release appears to be mediated through glucocorticoid receptors since progesterone, which is ineffective alone, competitively inhibits the effect of dexamethasone. The glucocorticoid effect on DNA release is tightly coupled to the initial, PHA-induced stimulus. Glucocorticoids are maximally effective when added to cultures 1 hr before PHA. When added 6 hr after PHA, their effect is minimal or absent, even though they are then continuously present until DNA release is measured 5 days later. Lymphocytes from certain donors in these studies were resistant to glucocorticoids; these individuals all had allergies, including asthma, allergic rhinitis, and bee sting hypersensitivity.


Ontogeny of the ferret humoral immune response. Infant ferrets are born with nearly undetectable immunoglobulin levels, but by 9 days of age the infant ferret serum contains 77, 29, and 13% of adult mean serum levels of IgG, IgA, and IgM. Transmucosal uptake of IgG by the infant ferret occurred for the first 30 days of life. The specific anti-respiratory syncytial virus neutralizing titer of whole milk was 5.5 times higher than maternal serum despite a lower concentration of immunoglobulins in the milk.


Effect of dicarboxylic acids on lentigo maligna. Dicarboxylic acids from C8 to C14 are competitive inhibitors of tyrosinase in vitro, and here, the effect of a cream containing 15% azelaic acid (C9) on 3 cases of lentigo maligna is described. The lesions were treated for 90 days, with remarkable clinical and histological effect, maintained for up to 2 yr after cessation of treatment. Progress during treatment of one case was additionally monitored by electron microscopy, which revealed progressive elimination of abnormal melanocytes both basally and suprabasally, and their replacement by essentially normal cells engaged in normal melanogenesis. There was also progressive diminution in the general disorganization of the epidermis, and disappearance of lymphocyte response. It is concluded that dicarboxylic acids have a direct inhibitory and cytotoxic effect on abnormally active or structurally disordered melanocytes in lentigo maligna, but further investigations are required to establish their precise mode of action. Similar application of dicarboxylic acids to normal skin affects only a small proportion of melanocytes, suggesting that some phasic factor, or individual states of activity, may be concerned in their susceptibility.


Heterotransplantation of human basal cell carcinomas in "nude" mice. Human basal cell carcinomas, obtained from 10 subjects were transplanted to 25 "nude" mice. Two methods of transplantation were used and compared. Grafting gave better results than subcutaneous implantation. Tumor cells were identified in 5 mice, however only 2 of them developed lesions with histology similar to human basal cell carcinoma. Grafts, in which tumors developed, were obtained from superficial basal cell carcinoma. No reasons for the cause of the low percentage of successful transplantation and slow rates of growth of the transplants are found; however, possible immunological, vascular and environmental factors are discussed.


Evaporation of diethyltoluamide from human skin in vivo and in vitro. Relative evaporation and penetration of the insect repellent, N,N-diethyl-m-toluamide, has been measured by 3 methods, 2 in vivo and 1 in vitro. The evaporation rate 30 min after application was found to be similar by all 3 methods. At an applied dose of 0.25 microgram/cm2, 9.6% in vivo and 9.7% in vitro evaporated from the skin in the first hour after application. Although the techniques used produced similar results in vivo and in vitro for diethyltoluamide, studies are being conducted to further elucidate kinetics of loss by evaporation.


Tumor-susceptibility generated in mice treated with subcarcinogenic doses of 8-methoxypsoralen and long-wave ultraviolet light. Evidence is presented to show that mice treated with various regimens of 8-methoxypsoralen followed by exposure to long-wave ultraviolet light (PUVA) are rendered tumor-susceptible when challenged with short-wave ultraviolet light (UVB) induced regressor tumors. These same tumors are readily rejected when implanted into normal syngeneic animals. Similar observations have been made in mice treated with subcarcinogenic doses of UVB, where it was shown that the tumor-susceptible state is mediated by suppressor T lymphocytes. These suppressor T-cells may be generated in response to antigens expressed by UVB damaged skin cells. It is now known that suppressor T-cells generated in mice treated with UVB are Ia-positive and have specificities for cross-reacting tumor antigens shared by all UVB-induced tumors, which have been tested to date. Our data suggest that, like UVB treated mice, treatment of mice with PUVA results in tumor-susceptibility mediated through the generation of Ia+ suppressor cells. Since PUVA treatments appear to generate a suppressor cell response in mice, a possible mechanism by which these treatments act to manage autoimmune type skin diseases, such as vitiligo, is discussed.


Effects of neutron radiation on mouse hair matrix cell populations. We have investigated the effects of a single low dose of 42 rads neutron radiation on hair matrix cell kinetics. Anagen hair skin sites in 3-mo-old Carworth Farms no. 1 female mice were exposed to a fast neutron beam yielding a modal energy of 3.6 MeV. In contrast to low LET (linear energy transfer) radiation at a 2 1/2 fold higher dose, neutrons induced a markedly prolonged depression (greater than 60%) in the mitotic index for more than 18 hr postradiation. In addition, from 10--18 hr after neutron irradiation sharp elevations (greater than 80%) in the labeling indices apparently reflected significant impairment in cell maturation rates and/or slowed exit rates from the proliferative compartments. Postradiation reductions of up to 20% in the size of proliferating matrix cell populations were found. At the cellular level, this study confirms previous findings of a disproportionately high relative biological effectiveness for neutron radiation following low dose level exposure.


Experimental tinea versicolor in rabbits and humans with Pityrosporum orbiculare. The purpose of this investigation was to produce experimental tinea versicolor in rabbits and humans with Pityrosporum orbiculare. Inoculation with P. orbiculare under plastic occlusion on the glabrous follicle-rich inside of the rabbit ear resulted in a tinea versicolor-like lesion after 1 week in 3 of 4 animals. One week after inoculation with P. orbiculare under plastic occlusion, experimental infections similar to those found clinically in tinea versicolor were seen in 10 of 12 patients with a history of tinea versicolor and in 3 of 6 normal volunteers. It was not possible to produce experimental infections without occlusion. Spontaneous healing usually occurred.


DNA repair processes protect human beings from premature solar skin damage: evidence from studies on xeroderma pigmentosum. The repair of DNA damage by ultraviolet light is defective in the hereditary disease xeroderma pigmentosum. A deoxyribonucleotide excision-proficient form and several excision-deficient forms of xeroderma pigmentosum have been identified. Premature solar skin damage develops in all xeroderma pigmentosum patients. Some patients also have neurological abnormalities caused by premature death of nerve cells. This abnormal aging of the central nervous system and of sun-exposed skin appears to be the result of the abnormal DNA repair processes. Clinical, biological, and physicochemical studies on DNA-repair-dependent processes and on the DNA repair defects in xeroderma pigmentosum are elucidating the mechanisms by which such abnormal aging is prevented in normal human beings.


Age-related changes in sebaceous gland activity. The sebaceous glands of man show age-related differences in their activity as determined by quantitative and qualitative examination of sebum. Sebaceous secretion is low in children and begins to increase in mid- to late childhood under the influence of androgens. This rise continues until the late teens, after which no further significant change takes place until late in life. In elderly men, sebum levels remain essentially unchanged from those of younger adults until the age of 80. In women, sebaceous secretion decreases gradually after menopause and shows no significant change after the 7th decade. The most likely explanation for the decrease in sebaceous gland secretion with age in both men and women is a concomitant decrease in the endogenous production of androgens. Although surface lipid levels fall with age, paradoxically the sebaceous glands become larger, rather than smaller, as a result of decreased cellular turnover. Nonetheless, as the higher surface lipid levels after administration of fluoxymesterone (a synthetic testosterone derivative) indicate, the glands have the capacity to respond to androgens.


The effect of aging on the rate of linear nail growth. The linear nail growth rate is a simple, inexpensive, noninvasive technique for the measurement of aging. When the many endogenous and exogenous factors known to influence this rate are either controlled or considered, the measurement of the rate for 1 year gives data that are both age-related and age-caused. The rate of linear nail growth decreases 50% over the life spans of both dogs and humans. In the beagle, which has a life span 20% that of man, the rate of decrease is 5 times faster than in man. There are circadian and multiple-year biorhythms of the rate of linear nail growth. There are approximately 7-yr periods of slow decline that alternate with 7-yr periods of more rapid decline.


Aging, environmental influences, and photocarcinogenesis. Repeated exposure of human skin to solar ultraviolet radiation (UVR) over a period of many years is responsible for the induction of most nonmelanoma skin cancers in man. The tumors are progressively more common in chronologically older people. Is this fact purely a function of adequate dose accumulation and development time, or is tumor expression influenced by "physiological age"? The answer to this question influences risk estimates of the results of atmosphere modification. Data from animal studies indicate that the tumor incidence is affected by dose-delivery factors and not just by the accumulated lifetime dose. In addition, young mice are more prone to tumor induction by a given UVR dose than are older animals. Because the quanlity and quantity of the stimulus (UVR) can be readily manipulated and accurately described, studies on photocarcinogenesis offer distinct possibilities for untangling some of the interactive variables in the aging process.


Aging and cultured human skin fibroblasts. We have studied aging at the cellular level by examining skin fibroblast cultures derived from skin biopsy samples obtained from old and young members of a longitudinal study on human aging. Results from a number of studies already completed indicate that cell cultures from old human donors, unlike cultures from young donors, have impaired cell proliferation and reduced induction of sister chromatid exchanges. A number of other parameters, however, are not affected by the age of the human fibroblast donor. These include cell protein and RNA content and the rate of cellular macromolecular synthesis. Studying cell cultures derived from members of an ongoing longitudinal study permits examination of correlations between in vivo and in vitro measurements on the same people. In addition, it allows for future longitudinal studies on human cellular aging.


Aging research: current and future.

Aging research: current and future. The key events that lead to organismic senescence appear to occur in cells that have been genetically programmed to arrest their own proliferation. After arrest, a variety of subcellular events occur, among the more important of which are the accumulation of waste product pigments, an increase in the fraction of inactive enzymes, and (probably of central importance) the loss of key genes, specifically those that code for rRNA. An understanding of the senescence of the epidermis and dermis would almost certainly provide answers to related problems in those organ systems whose sudden or slow failure leads to individual death.


Cellular immunity in aging.

Cellular immunity in aging. Our study of the aging process in human beings and in mice is complicated by our need to know whether we are observing diseases of aging or natural nondisease state processes. Results from studies on inbred strains of mice and retrospective studies on HLA types in aging human populations suggest that genetic effects play a significant role in predetermining the life span of an individual. It is clear that in such mouse strains genetic defects that affect cell regulatory mechanisms result in the production of autoimmune reactivity, tumor development, and a shortened life span. In human beings, although results are less clear-cut, strong associations exist between some disease states and the HLA type. Also, the disappearance of HLA-B8 from older women suggests that this HLA type does not confer longevity. Cellular immune reactivity declines with age in all populations studied to date, and cell cooperative or regulatory mechanisms function less well. We need to characterize the specific nature of the cells directly responsible for these alterations and to attempt to correct deficiencies by dietary manipulation or transfer techniques.


Studies on age-related diseases in cultured skin fibroblasts. In order to trace the origins of age-dependent diseases to the cellular level, we studied cultured human fibroblasts from subjects with 3 discrete inherited disorders and normal controls of various ages. Skin fibroblasts from subjects with progeria and Werner syndrome had a moderate to severe reduction in growth capacity, whereas cells from subjects with diabetes mellitus had a more subtle growth impairment. There was a decreased response of progeric fibroblasts to insulin-like hormones, and in normal cells the response decreased as a function of the passage level and donor age. Tissue factor, a procoagulant, was more abundant in progeric and Werner fibroblasts. An understanding of fibroblast aging in vitro may help us explain various concomitant phenomena of organismic aging such as diabetes mellitus, cell dropout, impaired hormone responsiveness, and increased atherothrombosis.


Structural changes in aging human skin.

Structural changes in aging human skin. In both exposed and protected areas of progressively older people the underside of the epidermis becomes increasingly flattened out. This flattening is accompanied by a comparable rarefaction of the superficial blood vessels. In this article we review the architecture of the elastic fiber framework in the papillary dermis of skin protected from the sun; aging changes that occur are similar to, but less severe than, those in sun-exposed areas. Most cutaneous sensory end organs are little affected by aging; those in the external genitalia, however, and particularly those underneath the vaginal epithelium become smaller and some disappear.


The cell biology of aging.

The cell biology of aging. Cultured normal human and animal cells are predestinued to undergo irreversible functional decrements that mimick age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 +/- 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occur in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.


Aging of collagen.

Aging of collagen. Current knowledge of the structure and the mechanism of formation of the covalent cross-links that fuse individual collagen molecules into a stable fiber is reviewed. Some of the mechanical properties of dermal connective tissue and the way in which these change with age can be correlated with the types of cross-link present in the tissue. Cross-links are routinely detected by treatment of a tissue sample with tritium-labeled borohydride and subsequent isolation and quantification of the cross-linked compound, which has been rendered radioactive by reaction with this reducing compound. After maturity, the number of detectable cross-links decreases even though the mechanical stability of the tissue increases. This anomaly is examined in the light of recent data suggesting that cross-links may be oxidized in vivo and thus become undetectable since they can no longer react with borohydride.


Age-related changes in the mechanical properties of human skin. The mechanical properties of skin have been studied both in vivo and in vitro by a variety of test methods. These properties are well matched to the function of the skin, and they depend on the geometry of the collagen and elastin networks of the dermis. The time dependence of these properties is thought to be related to the "ground substance" components of the dermis. Age-related changes in the mechanical properties are a function of the degradation of the elastin network and of some as yet undefined changes in the viscoelastic properties of the "ground substance."


Wound healing and aging.

Wound healing and aging. There are "natural delays" in the healing of older individuals. Open wounds contract more slowly and incised wounds gain strength more slowly. Experimental studies indicate that cellular proliferation, wound metabolism, and collagen remodeling occur later in old animals. Clinical studies show, however, that operations can be performed safely in elderly patients and that the major increased risk to these patients is of nonwound medical complications that affect the wound.


Cellular aging in Werner's syndrome: a unique phenotype? Werner's syndrome is commonly regarded as a model for the study of premature aging. There are, however, a variety of clinical and pathologic anatomical features that clearly distinguish it from aging in normal individuals. In this paper we report on in vitro cytogenetic and cell fusion studies that indicate cultured fibroblast-like cells derived from Werner patients differ from cells of normal donors. Despite these discordances with "natural" aging, however, Werner's syndrome, like several other "segmental progeroid syndromes," may prove useful for the investigation of selected aspects of the aging process and of age-related diseases.


Psoralen-DNA cross-linking photoadducts in dyskeratosis congenita: delay in excision and promotion of sister chromatid exchange. Dyskeratosis congenita is a rare X-linked recessive disease, characterized by mucosal leukokeratosis, nail dystrophy, telangiectasia, reticulated hyperpigmentation, pancytopenia, and a heightened susceptibility to infection and malignancy. We exposed cultured fibroblasts and peripheral leukocytes from normal persons and from 2 unrelated young adult men with dyskeratosis congenita to 4,5',8-trimethylpsoralen and ultraviolet light. We than compared certain of their responses. Labeled DNA from fibroblasts exposed to 4,5',8-trimethylpsoralen and ultraviolet light showed fast-sedimenting DNA, a pattern we interpreted as evidence that cross-linking, psoralen-DNA photoadducts had been formed by the treatment. Fast-sedimenting DNA persisted for 24 hr in dyskeratosis congenita cells but disappeared from normal cells during a 24-hr repair period. Cultured peripheral blood leukocytes from persons with this syndrome similarly exposed to 4,5',8-trimethylpsoralen and ultraviolet light developed more sister chromatid exchanges than did cells from normal persons. These data suggest that a heightened susceptibility in DNA cross-links may be of fundamental importance in the etiology of dyskeratosis congenita.


Potentiation of experimental allergic encephalomyelitis in hamsters with persistent encephalitis due to measles virus. To clarify mechanisms underlying acute disseminated encephalomyelitis (ADE) in patients with infection due to measles or other viruses, a new animal model was devised. Adult hamsters that had clinically recovered from acute encephalitis induced by prior intracerebral injection of the HBS strain of measles virus were challenged with neuroantigen plus adjuvant. Such hamsters, which had a high likelihood of carrying persistent HBS measles virus in the central nervous system (CNS), exhibited a significantly higher incidence of experimental allergic encephalomyelitis (EAE) following challenge as compared with simultaneously challenged but previously uninfected littermates. Occurrence of EAE in hamsters previously injected with heat-inactivated virus was not potentiated, a finding suggesting that persistence of the virus in the CNS renders that organ system more vulnerable to immunologic attack. This new model has promise for the probing of relationships between persistent viral infections of the CNS and host autoimmune responses directed against that target organ system.


Eosinophilia following treatment of patients with schistosomiasis mansoni and Bancroft's filariasis. Eosinophilia is frequently observed after treatment of patients with infections due to parasitic helminths. For definition of the characteristics and causes of post-treatment eosinophilic responses, 47 patients with Bancroft's filariasis, who were treated with diethylcarbamazine, and eight patients with schistosomiasis mansoni, who were treated with niridazole, were studied. After therapy for eight days, both groups developed significant (P less than 0.05) increases in their levels of eosinophilia, which peaked in two to four weeks. Maximal levels averaged two and one-half to three time the levels before treatment. Before therapy the number of circulating eosinophils was unrelated to intensity of infection in both groups, but after treatment, when the same relationship was examined in the larger group (the patients with filariasis), the degree of post-treatment eosinophilia was significantly correlated with the patients' worm burdens before treatment (r = 0.727; P less than 0.001). Thus, treatment of helminthic infections can provide a unique opportunity for studying eosinophil responses of humans to graded doses of antigen and has shown that acute responses are determined quantitatively by the amount of antigen initially presented to the host.


Dinitrochlorobenzene responsivity: difference between patients with severe pulmonary coccidioidomycosis and patients with disseminated coccidioidomycosis. Immunologic responses in 15 patients with severe pulmonary coccidioidomycosis and in 50 patients with disseminated coccidioidomycosis were measured by determination of complement-fixing (CF) antibody titers to coccidioidin in serum, coccidioidin (1:100) skin tests, and sensitization to dinitrochlorobenzene. Among the patients with desseminated coccidioidomycosis, the nine with CF antibody titers of less than or equal to 1:8 had normal responses to dinitrochlorobenzene, but the 41 with titers of greater than or equal to 1:16 had responses that were significantly lower than those of controls (P less than 0.001). In contrast, all patients with severe pulmonary coccidioidomycosis had CF antibody titers of greater than or equal to 1:16 and had responses to dinitrochlorobenzene that were greater (but not significantly greater) than those of controls. Among subjected with antibody titers of greater than or equal to 1:16, responsiveness to coccidioidin was found in 27% of those with severe pulmonary disease and in 39% of those with disseminated disease. Thus impaired responsivity to dinitrochlorobenzene in coccidioidomycosis is restricted to patients who have disseminated illness and high titers of CF antibody and is separable from lack of responsiveness to coccidioidin.


Chlamydia trachomatis in women: antibody in cervical secretions as a possible indicator of genital infection. One hundred eighty-five women college students were examined for genital infection with Chlamydia trachomatis. This organism was isolated from nine (5%) of the 185 women. Antibody was demonstrated in the genital secretions of 26 (14%) and in the serum of 70 (38%) of the women. None of the sexually inexperienced women was infected. Among those sexually experienced, the prevalence of isolation of C. trachomatis and of detection of local antibodies in cervical secretions and serum antibodies to C. trachomatis increased in relation to the number of life-time sexual partners. Local antibody appeared to be a more reliable indicator of infection with C. trachomatis than serum antibody in this college population.


Early-appearing antiviral activity in human tears during a case of picornavirus epidemic conjunctivitis. The clinical, virological, and immunological courses of a laboratory-acquired hemorrhagic conjunctivitis were followed. Coxsackievirus type A24 (CA24) was isolated from tears for only two days but from sputum and fecal samples for 20 days. Maximal levels of virus were detected in the right eye (10(5.1) 50% tissue culture infective doses/ml) 5-7 hr before conjunctivitis in the right eye, and in feces (10(2.8) 50% tissue culture infective doses/g) and sputum (10(2.5) 50% tissue culture infective doses/ml) five and eight days after onset of conjunctivitis, respectively. Specific neutralizing activity against CA24 was detected in tear samples 48-56 hr after exposure, and elimination of virus was associated with the rise in the neutralizing activity in tears. This activity was detected earlier than previously reported antibody responses of the eye. The absence of interferon and the association of this early neutralizing activity in tears with early elimination of virus from the eye suggest that neutralizing activity may play a major role in the clinical and pathological manifestations of this infection.


Factors in the pathogenesis of acute schistosomiasis mansoni. Acute schistosomiasis mansoni was studied in 26 Puerto Rican patients whose clinical presentations differed widely in severity. Severity of illness was found to be positively correlated (r = 0.79) with the intensity of infection as measured by the concentration of eggs of Schistosoma mansoni in stool specimens. However, some patients had severe illness but relatively light infections. The disappearnace of symptoms and return toward normal of laboratory measures of disease activity were not associated with any diminution in the fecal egg count. Elevations of IgG, IgM, IgE, and of titers of anibody in serum indicated that the illness is associated with intense immune activity. The magnitude of the IgG and IgE responses was related to intensity of infection. The fact that incubation periods were shorter than the time needed for schistosomes to reach adulthood and lay eggs suggests that the syndrome can be initiated by parasite stages present before oviposition. No marked changes in complement (C3, C3p, C4, and CH50) and no signs of renal disease were noted in any of these patients.