Cross-reactive antigen recognition by an encephalitogenic T cell receptor. Implications for T cell biology and autoimmunity. The dominant immune response to rat myelin basic protein in H-2u mice is directed against the acetylated, N-terminal peptide Ac1-11 (AcASQKR-PSQRHG). This peptide causes encephalomyelitis on injection into mice of the H-2u haplotype. Only two residues of the peptide are required for ligation of the TCR from an Ac1-11-specific T cell hybridoma. Proline at position 6 could not be substituted by any other L-amino acid, whereas glutamine at position 3 could be replaced by phenylalanine, histidine, methionine, or tyrosine. Cross-reactive recognition of these residues appears to be specific, because increasing the affinity of each analogue for its MHC restriction element, by replacing lysine with tyrosine at position 4, did not alter the pattern of cross-reactivity. For the majority of substitutions at this position, a lack of stimulation could not be explained by failure to bind to I-Au. However, competition binding studies showed that introduction of proline at position 3 reduced the efficacy of binding to I-Au. Cross-reactive analogues of Ac1-11 were injected into H-2u mice to test the extent to which cross-reactive T cell activation might lead to autoimmune disease in this model. An analogue containing methionine at position 3 caused clinical experimental autoimmune encephalomyelitis in a small percentage of H-2u mice.


Antibodies to soluble human T cell receptor beta chain recognize multiple epitopes on cell surface TcR. The T cell receptor (TcR) is an integral membrane protein occurring as a disulfide linked heterodimer, non-covalently associated with CD3 on the surface of T lymphocytes. Antibodies to the TcR have been shown to be effective for treating autoimmune disorders in animals. We describe here a method for producing antibodies to cell surface determinants of the human TcR, using a soluble form of the receptor as antigen. Soluble V alpha 1.2, V beta 8.1, V beta 11 TcR chains are expressed from a construct in which the extracellular domains of the TcR are fused to the mouse gamma 2a heavy chain constant region lacking the CH1 domain. These chimeric molecules contain both immunoglobulin and TcR determinants, as revealed by antibody probes. Amino-terminal sequence analysis of a chimeric V beta 8.1 molecule indicates that the TcR leader peptide is correctly processed from the soluble form. Antibodies raised against the soluble human V beta 8.1 molecule recognize the native determinants on Jurkat cells, and on natural T cells derived from resting human peripheral blood lymphocytes. Epitope mapping studies using competitive binding assays suggest that the anti-V beta 8 antibodies produced using soluble antigen recognize multiple overlapping determinants on the cell surface form of the TcR.


Preparative-scale purification and characterization of MHC class II monomers. The MHC class II molecule is a heterodimeric glycoprotein consisting of one alpha and one beta polypeptide chain of almost identical molecular size. Recently it has been shown by others, and confirmed in our laboratory, that isolated monomers of murine MHC II molecules are capable of binding antigenic peptides like the alpha/beta intact heterodimer. In addition, preliminary results from our laboratory indicate that isolated single chain-peptide complexes of murine MHC class II molecules are capable of stimulating cloned T cells in an antigen specific manner. These results prompted us to isolate relatively large quantities of individual alpha and beta subunits of MHC II molecules for further in vitro and in vivo studies. Isolation of alpha and beta monomers proved to be difficult using conventional chromatographic methods. In this report we describe micro-preparative and preparative continuous flow electrophoresis methods by which milligram quantities of MHC II subunits can be purified. An optimal condition for the dissociation of heterodimeric MHC II into alpha and beta monomers was identified, and separation of human HLA DR2 and murine IAs monomers was accomplished. Both methods offer the resolving power of gel electrophoresis with the convenience of continuous sample elution. Purified MHC II subunits obtained by these methods were tested for their ability to bind antigenic peptides. Results presented in this study indicate that monomeric subunits of both human HLA-DR2 and murine IAs are equally active in specific binding of antigenic peptides like the native heterodimer.


An enzyme-linked immunosorbent assay for the quantification of solubilized CD14 in biological fluids. A simple and robust two site-binding ELISA for the quantification of solubilized CD14 in human and animal body fluids is described. The principle of the assay depends on the specific binding of sCD14 to two monoclonal antibodies (MEM-18, RoMo-1) recognizing different epitopes of this glycoprotein. The detection limit for sCD14 was 1 ng/ml. The method was used to quantify sCD14 in different biological fluids, giving an intra-assay coefficient of variation and an interassay coefficient of variation of about 9%. The assay was used to measure sCD14 in human serum and plasma and other body fluids in health and disease, and in cell culture supernatants. With the exception of monkeys there was no reactivity with 29 other species screened. In healthy volunteers the sCD14 serum level had a mean value of 3.98 +/- 0.3 micrograms/ml (mean SEM, n = 102).


ELISA for quantitation of L-selectin shed from leukocytes in vivo. L-selectin is a cell surface receptor on granulocytes, lymphocytes and monocytes that is responsible for the initial attachment of leukocytes to endothelium. The extracellular domain of L-selectin is proteolytically shed from leukocytes following cellular activation in vitro. The shed form of L-selectin (SL-selectin) is functionally active and at high concentrations can inhibit leukocyte attachment to endothelium. Therefore, an ELISA was developed to quantitate the levels of SL-selectin in biological fluids, biopsy specimens and during recombinant protein production. This simple, quantitative sandwich ELISA uses two monoclonal antibodies directed against the extracellular domain of SL-selectin. The assay has a detection range of 5-1300 ng/ml, is precise and sensitive. The ability of this assay to detect SL-selectin in serum, plasma, and culture supernatant fluid was demonstrated and it was used to quantitate circulating SL-selectin in normal and patient sera. Patients with sepsis and HIV infection showed markedly elevated SL-selectin levels in serum. Thus, the ELISA should prove useful both for laboratory purposes as well as in the diagnostic evaluation of patients with inflammatory diseases.


A highly sensitive enzyme-linked immunosorbent assay for the measurement of interleukin-8 in biological fluids. Interleukin-8 (IL-8) is a chemotactic and activating cytokine for neutrophils, which plays an important role in acute inflammatory responses. We aimed to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for IL-8 and established 18 clones of anti-IL-8 monoclonal antibodies (mAbs). These mAbs were evaluated in terms of their antigen-binding affinities, and five clones were selected and used for the comparative study of various combinations of antibodies in sandwich ELISA. Affinity purified rabbit polyclonal antibody was also used in this study. One antibody pair, which showed relatively high sensitivity and which was not severely interfered with blood components, was selected and the assay conditions were optimized by choosing the appropriate buffer for sample dilution and by directly labeling the second antibody with enzyme. The finalized ELISA, using polyclonal antibody as first (coated) antibody and horseradish peroxidase-labeled mAb (clone EL139) Fab' fragment as second antibody, could detect as low as 2.5 pg/ml (0.125 pg/well) of IL-8 by in total 2 h incubation, without being affected by body fluid components. The ELISA was specific to IL-8, showing no cross-reactivity with other cytokines or various IL-8 family proteins which share some amino acid sequence homology with IL-8. As an example of its application to clinical specimens, plasma samples from patients with septic shock were measured. The results showed that sepsis patients contain significantly higher levels of plasma IL-8 compared to normal controls. When analyzed by gel-filtration chromatography, IL-8 in sepsis plasma was eluted in a molecular weight (M(r) region corresponding to the monomer form. The ELISA established here is expected to be effectively used for further investigations on the relationship between IL-8 and various diseases.


Detection of human leukemia inhibitory factor by monoclonal antibody based ELISA. Leukemia inhibitory factor (LIF) is known to exhibit multiple functions by regulating the growth and differentiation of multiple normal cell types as well as malignant cells. To have a better understanding of the role of LIF, it is important to determine the level of LIF in various biological samples by developing an easy, sensitive and LIF specific assay. In this study, we have established a double monoclonal antibody (mAb) based ELISA. Four hybridoma cell lines (D3.14.1, D4.16.9, D25.1.4 and D62.3.2) secreting murine monoclonal antibodies (mAbs) against recombinant human leukemia inhibitory factor (rHuLIF) were produced by immunization of BALB/c mice with rHuLIF and by fusing immune spleen cells with P3X63Ag8U.1 myeloma cells. These mAbs each belong to the IgG1 isotype and have unique isoelectrofocusing point patterns. All four mAbs were shown to have high affinities for rHuLIF (Kd = 7 x 10(-10) to 6 x 10(-11) M) and were able to recognize the native as well as the reduced rHuLIF in an immunoblotting assay. All these mAbs showed no cross-reactivities to IL-1, IL-3, IL-6, TNF-alpha, GCSF and GMCSF. MAb D3.14.1 showed a weak binding to Oncostatin M but not to rMuLIF whereas the other three mAbs D4.16.9, D25.1.4 and D62.3.2 showed cross-reactivity to rMuLIF but not to Oncostatin M. Data obtained from a competitive binding enzyme-linked immunosorbent assay (ELISA) suggested that these four mAbs recognized different epitopes on rHuLIF. Using mAb D4.16.9 as coat antibody and horseradish peroxidase (HRP) conjugated mAb D3.14.1 as the conjugate antibody we established a double mAb based ELISA specific for human LIF which could detect as little as 100 pg/ml and 10 pg/ml of rHuLIF in the absence and in the presence of the ELAST ELISA amplification system, respectively. The addition of serum had very minimal effect on this ELISA.


Homogeneity of lipopolysaccharide antigens in Pseudomonas pseudomallei. An antiserum raised against a single strain of Pseudomonas pseudomallei reacted equally in a whole cell agglutination test, an indirect haemagglutination (IHA) test and an ELISA with a panel of 12 strains of the species which had been isolated from human beings and animals in various parts of the Far East and Australia between 1923 and 1990. Absorption of the serum with either of two strains removed all reactivity of the serum with other strains. Phenol-water extracted lipopolysaccharide (LPS) from a single strain blocked the reactivity of the serum with red cells sensitised with crude extracts of any of the panel of strains, thereby suggesting that the 'common' antigen was LPS. This antigen was not detected in other Pseudomonas species with the exception of Pseudomonas mallei. Protease K-digested extracts of the 12 strains gave highly similar silver-stained LPS banding patterns in gel electrophoresis. Furthermore, immunoblots of LPS with either rabbit or a patient's serum showed identical ladder profiles for each strain. The results suggest that the LPS antigen is highly conserved throughout P. pseudomallei and that this antigen is detected by the IHA test.


IgE-positive Langerhans cells and Th2 allergen-specific T cells in atopic dermatitis. In atopic dermatitis (AD) IgE-positive Langerhans cells (LC) may be present in the epidermis. These LC are able to capture allergens by means of their specific IgE, inducing an allergen-specific T-cell response in autologous peripheral blood T cells. Epicutaneous patch testing (EPT) may induce an eczematous reaction when IgE is present on the LC. Thus, both in vivo and in vitro, it appears that IgE may be crucial for induction of allergen-specific T-cell responses. Indeed, the cloning of infiltrating T cells from a positive 12-h EPT produced allergen-specific T cells, wheras no in vivo activated bystander T cells have yet been cloned. Moreover, greater than 85% of the T cells cloned were of Th2 phenotype after anti-CD3 and phorbol myristate acetate stimulation, whereas all clones were Th2 after allergen-specific stimulation, and they were able to induce IgE production in normal B cells. This completes the circle of events, because IgE produced by peripheral B cells may bind to LC and facilitate new allergen-specific reactions in the skin.


Epitope mapping of CD1a, CD1b, and CD1c antigens in human skin: differential localization on Langerhans cells, keratinocytes, and basement membrane zone. CD1 antigens are classified into at least three groups, CD1a, CD1b, and CD1c. In order to delineate the localization of epitopes of CD1 antigens in human skin, we examined the immunoreactivity of fourteen different CD1 antibodies (seven CD1a, five CD1b, and two CD1c antibodies). The epitopes for CD1a, CD1b, and CD1c are differentially localized on epidermal Langerhans cells, dermal dendritic cells, keratinocytes, the luminal portion of eccrine gland ducts, and the basement membrane zone in normal human skin.


Early events in the induction phase of contact sensitivity. After allergen application to skin, there is enhanced class II major histocompatibility complex antigen expression, as well as enhanced T-cell-stimulatory function by epidermal Langerhans cells (LC). In this study, we investigated the early changes in the epidermal cytokine profile using a sensitive reverse transcriptase PCR technique to determine whether cytokines may be related to LC activation. We found that, on the mRNA and protein level, changes in the epidermal cytokine pattern caused by allergens in the induction phase of contact sensitivity are distinct from those caused by irritants or tolerogens. The earliest of the changes is the LC-derived interleukin (IL) 1 beta mRNA signal strength that is increased within 15 min of allergen painting.


Linkage of the epidermolytic hyperkeratosis phenotype and the region of the type II keratin gene cluster on chromosome 12. Bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis) is a severe, generalized, lifelong disease of the skin. As in epidermolysis bullosa simplex, intraepidermal blisters and clumping of keratin intermediate filaments are characteristic. We report here linkage of the inheritance of this disease to the region of chromosome 12q containing the genes encoding type II keratins. This suggests that keratin gene mutations may underlie this complex hyperproliferative and hyperkeratotic phenotype.


Langerhans cells are the major source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated mouse epidermal cells. Immunocompetent cells of the epidermis can interact by the elaboration and recognition of cytokines. Although much new information has been reported concerning the cytokines secreted by keratinocytes, little is known about cytokines derived from Langerhans cells (LC). To address this deficiency, we examined cytokine mRNA profiles in different epidermal preparations from BALB/c mice, taking advantage of the sensitive technique of polymerase chain reaction (PCR), after reverse transcription of mRNA. In assays of epidermal sheets separated from dermis by ammonium thiocyanate, mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-7, tumor necrosis factor alpha (TNF alpha), TNF beta, granulocyte macrophage/colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) were unequivocally present. By contrast, faint bands were detected for IL-4, IL-5, and interferon gamma (IFN gamma), and no PCR signal was detected for IL-2. Importantly, assays of epidermal cells (EC) dissociated with trypsin revealed similar mRNA profiles. To determine the effects of cell isolation, fluorescence-activated cell sorter (FACS)-purified Ia- EC were first analyzed; all of the previously cited cytokine mRNA were present except for IL-1 beta and MIP-1 alpha. EC depleted of LC by a second technique, lysis using anti-Ia monoclonal antibody and complement, revealed similar profiles, with substantially reduced PCR signals for IL-1 beta and MIP-1 alpha. Finally, FACS-purified LC (Ia+ EC) clearly expressed IL-1 beta and MIP-1 alpha mRNA, a finding that was verified by Southern blotting using internal oligo probes. We conclude that these cell-isolation procedures did not produce substantial alterations in basal mRNA profiles and that LC are the principal source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated EC in mice.


Functional role of adhesion molecules LFA-3 and ICAM-1 on cultured human epidermal Langerhans cells in antigen-specific T-cell activation. Interaction of T-cell antigen receptors with antigen/major histocompatibility complex (MHC) molecule complexes on antigen-presenting cells (APC) leads to T-cell activation. Additional interaction between adhesion molecules on the APC and their ligands on T cells is required for optimal T-cell activation. In vitro cultured human epidermal Langerhans cells (cLC) are powerful APC that express adhesion molecules LFA-3 and ICAM-1. Both molecules on cLC serve a functional role in the generation of a T-cell response.


Recovery from cytomegalovirus infection is associated with activation of peripheral blood lymphocytes. The number of CD8bright and CD56+ lymphocytes in the peripheral blood and their activation status were monitored by flow cytometry in 23 renal transplant recipients with cytomegalovirus (CMV) infection and were correlated with the virus load (as determined by CMV antigenemia) and clinical symptoms. Recovery from CMV infection coincided with expansion of the CD8bright and CD56+ subsets and with increased expression of the activation marker HLA-DR. Primary infection was associated with activation of both subsets, whereas during secondary infection, mainly CD8bright cells responded. Progressive CMV disease (requiring antiviral treatment) and relapse occurred in association with low numbers of activated CD8bright and CD56+ cells. Lymphocyte activation and antibody responses against CMV often occurred simultaneously, but different kinetics of these responses in some patients indicated that cellular responses are necessary to control viral replication, whereas humoral responses alone may be insufficient. Monitoring of lymphocyte activation may provide clinically useful information during CMV infection.


Hepatitis C virus is detected in a monocyte/macrophage subpopulation of peripheral blood mononuclear cells of infected patients. Hepatitis C virus (HCV) is the primary agent of posttransfusion non-A, non-B hepatitis. HCV RNA was detected in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction in 17 of 24 HCV-infected patients with chronic hepatitis with or without cirrhosis. One of 5 patients whose PBMC contained HCV RNA also had negative-stranded HCV RNA in the PBMC. In 3 of 11 patients whose PBMC contained HCV RNA, flow cytometry with a murine monoclonal antibody to HCV core epitope revealed cytoplasmic staining of peripheral blood monocytes. The monocyte surface and the peripheral blood lymphocytes did not stain for HCV core epitopes. No correlation could be made between the presence of HCV RNA or antigen in PBMC and any serologic markers of HCV infection. These results indicate that monocyte uptake of HCV by either phagocytosis or infection may be part of the pathophysiology of this chronic disease.


Human monoclonal antibodies against an epitope on the class 5c outer membrane protein common to many pathogenic strains of Neisseria meningitidis. Neisseria meningitidis is a causative agent of meningitis. Despite vaccination programs, it still causes a large number of deaths in young children. Early diagnosis followed by passive immunization with human monoclonal antibodies could be an approach to effective therapy. Peripheral blood lymphocytes from normal, healthy blood donors and from vaccinated individuals were immunized in vitro, using outer membrane proteins purified from N. meningitidis B:4:P1.15. The immunized human B cells were Epstein-Barr virus transformed and fused to a heteromyeloma. Several stable human hybridoma cell lines were established and two, secreting antibodies against the 31-kDa class 5c outer membrane protein, were characterized further. The human antibodies were of IgG1 and IgG3 isotypes, with kappa light chains. The recognized epitope was commonly found among pathogenic strains of N. meningitidis; thus, these human monoclonal antibodies may be important in the evaluation of N. meningitidis infections.


Binding of plasminogen to Neisseria meningitidis and Neisseria gonorrhoeae and formation of surface-associated plasmin. Forty-two strains of Neisseria meningitidis and 17 of Neisseria gonorrhoeae were tested for their ability to interact with 125I-labeled Glu-plasminogen. All strains tested reacted substantially with plasminogen, resulting in uptake values of 20%-48%. Scatchard analysis with selected N. meningitidis strains demonstrated a dual-phase receptor interaction, one more avid receptor with a Kd of 50 nM and 3000-6000 receptors per bacterium and a second receptor with a Kd of 200 nM and 10,000-20,000 receptors per bacterium. Plasminogen uptake could be completely eliminated by low concentrations of epsilon-aminocaproic acid, suggesting that the lysine binding sites on the plasminogen molecule are involved in the receptor-ligand interaction. The binding of plasminogen to the bacterial receptor facilitates the tissue-type plasminogen activator-mediated conversion to Glu-plasmin, which also modifies itself to the Lys form. Receptor-associated plasmin is enzymatically active, monitored as a breakdown of the chromogenic substrate S-2251, and retains its activity in the presence of naturally occurring inhibitors in plasma.


[Clinical studies on maternal serum alpha-fetoprotein (MSAFP) of 532 cases--the median value, pregnancy outcomes and clinical evaluation]. MSAFP screening program has gradually become widespread. Prenatal diagnosis of fetal anomalies by means of the MSAFP level has become increasingly integrated into the routine components of obstetric management. During the period from June, 1990 through January, 1991, 516 pregnant women between 12 and 19 weeks' gestation participated in MSAFP screening and there were 532 MSAFP cases including repeat 16 cases for which follow-up data have been completed. 1. Of this population, 5 cases (0.9%) had increased MSAFP (> or = 2.5MOM). One case was IUFD, one case was twin, one case was Breus' mole, two cases had normal repeat MSAFP levels. Thirty-three cases (6.2%) had low MSAFP (0.5 < or = MOM). One abnormal karyotype (46,XY,16p+) was discovered among twenty-six cases by undergoing amniocentesis (3.8%). 2. Of the four hundred ninety-four cases (92.8%) with a normal MSAFP level, thirty-three (6.9%) had an adverse pregnant outcome. Eight cases were prematurity, eight cases were low birth weight infants, six cases were large-for-date infants, two cases were IUFD, one case was Klinefelter's syndrome, one case had cystic hygroma and one case was had a tracheo-esophageal fistula. 3. A woman with an extremely high MSAFP level, with normal ultrasonography and with normal amniocentesis results should be regarded as a high-risk pregnancy and need further close follow-up to detect placental abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)


Marked increase in plasma interleukin-6 in burn patients. We measured the levels of interleukin-6 in plasma samples from 18 consecutive burn patients, including three lethal cases, during the early postburn period. In survivors burn injury caused initial increases in interleukin-6 levels that peaked at 6 hours after burn; this was significantly higher than interleukin-6 levels in normal controls (718 +/- 216 vs 70 +/- 4 pg/ml; p < 0.01). The increment in nonsurvivors was even more prominent (11,554 +/- 4,407 pg/ml; p < 0.01). The peak interleukin-6 levels at 6 hours correlated with total burn surface area (r = 0.65, p < 0.025), and tended to be higher in patients with inhalation injury. These data provide evidence that burn injury causes rapid release of interleukin-6 according to the severity of the injury. We also measured acute-phase reactants including fibrinogen, alpha 1-antitrypsin, C1 inhibitor, and alpha 2-plasmin inhibitor. After initial declines, these four proteins increased rapidly in survivors. In addition, the peak interleukin-6 levels correlated well with the increases in fibrinogen (p < 0.025), alpha 1-antitrypsin (p < 0.01), C1 inhibitor (p < 0.01), and alpha 2-plasmin inhibitor (p < 0.0001). In contrast, despite the marked increase in interleukin-6, the levels of acute phase proteins in nonsurvivors remained low. Based on these observations, we suggest that interleukin-6 is released as an alarm signal and has a role for the wound healing in burn patients, and that the levels of interleukin-6 after injury is an indicator of the severity of burn.


Evidence for the involvement of tyrosine kinases in the locomotory responses of human neutrophils. Erbstatin, a recently described inhibitor of tyrosine kinases, has been used to examine the potential role of tyrosine phosphorylation in human neutrophil locomotion. Preincubation of human neutrophils with erbstatin inhibited both spontaneous and directed migration induced by chemotactic factors such as formylmethionylleucylphenylalanine (fMet-Leu-Phe) and leukotriene B4. The decreased migratory responses were correlated with an inhibition of adherence of neutrophils to serum-coated surfaces. Erbstatin did not, however, affect the adherence of human neutrophils to uncoated surfaces. These results indicated that the inhibitory effects of erbstatin were specific and not due to a generalized alteration of the surface of human neutrophils. To elucidate the mechanism of the inhibitory effect of erbstatin on adherence properties, the expression of the leukocyte integrin Mo1 was studied. Erbstatin induced a small but significant increase in the expression of Mo1, but decreased the stimulation of the expression of Mo1 elicited by fMet-Leu-Phe. These results suggest that mechanisms in addition to alteration of the number of surface integrins are involved in the inhibition of neutrophil adherence and locomotion by erbstatin.


Functional alterations of swine peripheral blood mononuclear cells by methadone. In vitro exposure of the synthetic opiate drug methadone allowed evaluation of putative immunomodulatory activities of swine peripheral blood mononuclear cells. Respiratory burst, an index of microbicidal activity, was suppressed by methadone in a dose-dependent manner following exposure for 48 h. The suppression was blocked by the opiate antagonist naloxone. Another macrophage function phagosome-lysosome fusion was impaired by exposure to methadone. A primary lymphocyte-mediated function natural killer cell activity was also affected. In contrast, the macrophage function antibody-mediated phagocytosis was not affected. Because the functions affected by methadone are critical to host defenses against pathogenic organisms, our findings suggest that opiate-mediated immunomodulation merits further study. Moreover, our studies suggest that swine may provide an ideal model for the investigation of opiate-mediated suppression of immune cell functions.


Interleukin-2 signal transduction in human NK cells: multisite phosphorylation and activation of the tyrosine kinase p56lck. Interleukin-2 (IL-2) potently stimulates natural killer (NK) cell proliferation and cytotoxic function. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the electrophoretic mobility of p56lck. The present studies indicate that IL-2 induces a rapid (< or = 1 min) increase in the catalytic activity of p56lck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56lck itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorylation of the NH2-terminal region of p56lck containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p56lck. These results suggest that p56lck serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56lck.


Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I. Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)


Phytanic acid alpha-oxidation: accumulation of 2-hydroxyphytanic acid and absence of 2-oxophytanic acid in plasma from patients with peroxisomal disorders. A stable isotope dilution method was developed for the measurement of 2-hydroxyphytanic acid and 2-oxophytanic acid in plasma. In plasma from healthy individuals and from patients with Refsum's disease, 2-hydroxyphytanic acid was found at levels less than 0.2 mumol/l, whereas the acid accumulated in plasma from patients with rhizomelic chondrodysplasia punctata, generalized peroxisomal dysfunction, and a single peroxisomal beta-oxidation enzyme deficiency. In plasma from both healthy controls and patients with peroxisomal disorders, 2-oxophytanic acid was undetectable. Four different groups of diseases were characterized with a defective phytanic acid alpha-oxidation and/or pristanic acid beta-oxidation: 1) Refsum's disease, with a defect at phytanic acid alpha-hydroxylation; 2) rhizomelic chondrodysplasia punctata, with a defect at 2-hydroxyphytanic acid decarboxylation; 3) generalized peroxisomal disorders, with defects at 2-hydroxyphytanic acid decarboxylation and at pristanic acid beta-oxidation; 4) single peroxisomal beta-oxidation enzyme deficiencies, with a defect at pristanic acid beta-oxidation, resulting in an impaired phytanic acid alpha-oxidation by inhibition. The results indicate that 2-hydroxyphytanic acid decarboxylation and pristanic acid beta-oxidation take place in peroxisomes.


Two-year follow-up after laser thermal balloon angioplasty (LTBA) in lower extremities: initial experience. We describe the results obtained with the use of laser thermal balloon angioplasty (LTBA) in the treatment of atherosclerosis obliterans of the lower limbs in 37 patients (34 males, 3 females, mean age 58 +/- 9 years) with occlusive arterial disease (Fontaine stages II-IV) presenting 39 significant lesions. Immediate results and two years of clinical follow-up are analyzed. Initial ankle/brachial Doppler index was 0.51 +/- 0.17. Eighteen lesions were located in the iliac area (13 stenoses 2.3 +/- 1 cm and 5 occlusions 4.2 +/- 3 cm) and 21 lesions in the femoropopliteal area (5 stenoses 2.6 +/- 2 cm and 16 occlusions 5.7 +/- 3 cm). A percutaneous procedure was used in 38 cases. In only one case was femoral dissection needed. The laser source was argon in 26 cases and Nd-YAG in 13. Initial success was 85% (89% in iliac lesions and 81% in femoropopliteal lesions; 100% in stenoses and 70% in occlusions). The presence of occlusion (p less than 0.01) and/or calcium (p less than 0.05) negatively influenced the immediate results. No major complications were observed; seven (17%) minor complications occurred. Ankle/brachial Doppler index after treatment was 0.82 +/- 0.21. Cumulative clinical patency for successfully treated patients after two-year follow-up was 91%. LTBA thus represents an effective and less aggressive way of treating peripheral atherosclerosis obliterans. In spite of some limitations, it is useful in selected patients. The results of this study are very much like those in the literature for similar series and early experience.


Atrial natriuretic factor in essential hypertension: echocardiographic and humoral correlates. Aim of this study was to assess the relationship between plasma concentration of atrial natriuretic factor (ANF) and its two-dimensional echocardiographic (left ventricular mass, left atrium diameter) and humoral (plasma renin and aldosterone) variables in essential hypertension (EH). We evaluated 32 patients with uncomplicated mild to moderate EH and 10 controls. They were studied in the supine position after 7 days of constant dietary sodium intake and were off therapy since at least 3 weeks. ANF values overlapped between EH patients and controls (27.8 +/- 11.5 vs. 19.5 +/- 7.4 pg/ml, p = NS). In EH, no significant correlation was found between ANF values and left ventricular mass (r = 0.29), left atrial diameter (r = 0.04), mean arterial blood pressure (r = 0.26), plasma renin activity (r = 0.00), and aldosterone (r = 0.26). In EH, ANF values overlapped between the 15 patients with hypertrophy and the 17 patients with normal ventricular mass: 30.3 +/- 17 vs. 25.6 +/- 10.6 pg/ms (p = NS). We conclude that there is a substantial overlap in plasma ANF values between mild to moderate uncomplicated EH and controls, and left ventricular hypertrophy is not a major independent stimulus to ANF release in EH.


The effect of the plasma levels of proteins C and S on the prediction of warfarin maintenance dose requirements. Determination of the effects of the vitamin K-dependent anticoagulants, proteins C and S, on warfarin dose requirements and on the prediction error of a bayesian warfarin dose predicting program. Patients in the study were consecutive inpatients (n = 18) starting treatment with warfarin who were monitored as outpatients for 4 weeks. The following measurements were taken: repeated (n = 8) prothrombin times, expressed as the international normalized ratio (INR), plasma protein C and S antigen levels (percentage of pooled normal plasma), demographic, clinical and biochemical variables. Maintenance doses (adjusted to INR 2.5) were 6.7 +/- 3.4 mg/day. Protein C decreased to 56.9% +/- 15.3%, protein S to 63.7% +/- 17.3%, INR increased to 2.46 +/- 0.14. Prediction error decreased from 2.84 +/- 2.0 mg/day to 0.95 +/- 0.78 mg/day. Protein C accounted for only 4.2% of the mean maintenance dose but protein C and S levels accounted for 31% of the mean dose prediction error. Protein C and S levels affect warfarin doses and predictions significantly but not to a clinically meaningful degree.


Antihypertensive mechanism of amlodipine in essential hypertension: role of pressor reactivity to norepinephrine and angiotensin II. Calcium entry blockade may affect the pressor reactivity to vasoconstrictors. The pressor response to norepinephrine and angiotensin II, as well as several other blood pressure modulating factors, were studied in normal subjects (n = 9) and patients with essential hypertension (n = 10) before and after 8 weeks of treatment with the long-acting dihydropyridine amlodipine. In control subjects, calcium entry blockade did not modify blood pressure, the pressor and aldosterone response to angiotensin II, the activity of the renin-angiotensin and sympathetic nervous systems, or urinary dinoprostone (prostaglandin E2) excretion; however, the pressor response to norepinephrine was significantly decreased (p less than 0.01). In patients with hypertension, amlodipine decreased blood pressure (p less than 0.01) and the pressor response to both norepinephrine and angiotensin II (p less than 0.01), without changes in body weight, plasma renin, angiotensin II and catecholamine levels, dinoprostone excretion, or aldosterone responsiveness to angiotensin II. These findings suggest that calcium entry blockade modifies sympathetic-dependent vasoconstriction in both normal subjects and in patients with hypertension. Angiotensin II pressor response may be selectively decreased in essential hypertension.


Plasma concentration--effect relationships for felodipine: a meta analysis. The plasma concentration versus antihypertensive effect relationship for the calcium antagonist felodipine was investigated in 67 patients with hypertension and 21 healthy subjects by use of the Emax model. No consistent effect of felodipine on blood pressure was observed in the healthy subjects. In patients with hypertension the plasma drug concentration and blood pressure versus time curves mirrored each other, indicating a close relationship between concentration and effect. The maximum effect (Emax) model fitted the diastolic blood pressure data of most patients, but the model was less often applicable in patients with low initial diastolic blood pressure levels. The average Emax values and the plasma felodipine concentration needed to obtain 50% of Emax for the patients with hypertension were 29 mm Hg and 8 nmol/L, respectively. The Emax values increased with increasing initial diastolic blood pressure levels but were similar in patients with high and low plasma felodipine concentrations. Age had negligible influence on the antihypertensive response to felodipine when compensation was made for the plasma concentrations.


Ondansetron is effective in decreasing postoperative nausea and vomiting. The efficacy of ondansetron, a selective 5-HT3 receptor antagonist, in preventing postoperative nausea and vomiting in surgical patients was studied. Fifty women were randomized in a double-blind manner to receive either two 8 mg doses of intravenous ondansetron or two doses of placebo vehicle: the first given just before general anesthesia induction and the second 8 hours later. During the first 24 postoperative hours, the number of emetic episodes was recorded and the subjects rated their nausea on a scale from 0 to 10. Ondansetron-treated subjects had fewer emetic episodes (p less than 0.001) and lower subjective nausea scores (p less than 0.001). The number of complete responders (no emetic episodes and no rescue therapy) was 1 of 24 (4%) and 15 of 26 (58%) in the placebo and ondansetron groups, respectively (p less than 0.001). Ondansetron is clearly more effective than placebo in the prophylaxis of postoperative nausea and vomiting. The adverse event profile for ondansetron was similar to that of placebo.


Increased capillary permeability for plasma proteins in oral contraceptive users. The transcapillary fluid balance was examined in eleven women before administration of a monophasic oral contraceptive (desogestrel 0.15 mg, ethinylestradiol 0.03 mg), and after three and six months of use. The interstitial colloid osmotic pressure was measured by the "wick" method, and the interstitial hydrostatic pressure by the "wick-in-needle" method in subcutaneous tissue on thorax and leg. During the six-month observation period, the following changes were observed: Plasma colloid osmotic pressure decreased (mean 1.8 mmHg, p = 0.047), as well as serum albumin (mean 5.1 g/l, p = 0.0006), total protein concentration (mean 2.8 g/l, p = 0.0006), hemoglobin (mean 0.5 g/dl, p = 0.014) and hematocrit (mean 1.8%, p = 0.047). Blood pressure and body weight remained unchanged, but foot volume showed a significant increase. The colloid osmotic pressure gradient (plasma-interstitium) was significantly reduced. The results indicate an increase in plasma volume in addition to an increased capillary permeability to plasma proteins during oral contraceptive use. We suggest that the observed changes in transcapillary fluid balance is caused by the estrogen component of the oral contraceptive pill.


Increased proportions of peripheral blood gamma delta T cells in patients with pulmonary tuberculosis. There is a small population of peripheral T cells bearing the gamma delta T-cell receptor, which may be involved in the defense against invading microorganisms and tumor cells. The present study was designed to evaluate the levels of gamma delta T cells in patients with pulmonary tuberculosis, bacterial pneumonia, chronic lower respiratory tract infection, lung cancer, and normal control subjects with or without old tuberculous lesion. The results showed that only patients with tuberculosis had significantly increased proportions of peripheral blood gamma delta T cells. This study suggests that the increased proportions of gamma delta T cells in tuberculosis could be related to T-cell activation by Mycobacterium tuberculosis, although it remains to be investigated which components of mycobacteria are the major ligands for gamma delta T cells.


Secretion of granulocyte-macrophage colony-stimulating factor by human blood monocytes is stimulated by engagement of Fc gamma receptors type I by solid-phase immunoglobulins requiring high-affinity Fc-Fc gamma receptor type I interactions. Despite reports on the secretion of granulocyte-macrophage-colony-stimulating factor (GM-CSF) by murine peritoneal macrophages in response to inflammatory stimuli, the ability of human monocytes to generate this growth factor has remained doubtful. Neither endotoxin, phorbol compounds, nor inflammatory cytokines have been shown to elicit GM-CSF by these cells. Our present studies indicate that exposure of monocytes to solid-phase murine IgG2a, but not to murine IgG1 and thus cross-linkage of the 72-kDa Fc gamma RI results in transcription of the GM-CSF gene, accumulation of stable GM-CSF mRNA and finally in release of biologically active GM-CSF protein. Cross-linking of Fc gamma RI by a murine anti-Fc gamma RI monoclonal antibody and goat anti-mouse antibody failed, however, to stimulate GM-CSF release. This suggests that high affinity Fc-Fc gamma RI interactions are required for induction of expression of GM-CSF by monocytes.


Inhibition of abnormal T cell development and autoimmunity in gld mice by transgenic T cell receptor beta chain. Mice homozygous for the gld (generalized lymphoproliferative disease) mutation developed systemic autoimmune disease and severe lymphadenopathy due to an age-related accumulation in the peripheral lymphoid organs of polyclonal T cells bearing a unique phenotype (CD4-CD8-TCR alpha beta+B220+). These T cells overexpress T cell receptor (TcR) alpha beta chain RNA, proto-oncogenes c-myb and fyn, and proliferate poorly in response to TcR-mediated stimulation. The origin of these T cells is poorly understood. To study the influence of a functionally rearranged TcR beta chain on the T cell developmental abnormality of the gld mutation and autoimmunity, we have backcrossed TcR V beta 8.1-transgenic mice to C3H-gld/gld to homozygosity (transgenic gld mice). In transgenic gld mice, lymphadenopathy was markedly inhibited and the accumulation of CD4-CD8- T cells did not occur, although the remaining T cells overexpressed c-myb and proliferated poorly in response to TcR occupancy. These features indicate that the pattern of proto-oncogene expression and abnormal function persist in phenotypically normal T cells in transgenic gld mice, and that these characteristics can be dissociated from the accumulation of CD4-CD8- T cells. The hypergammaglobulinemia and anti-double-stranded DNA (anti-dsDNA) antibody production was partially improved in transgenic gld mice, supporting the critical role of T cells in abnormal B cell activation described in autoimmunity-prone mice. To investigate further the mechanisms underlying the inhibition of CD4-CD8- T cell accumulation in transgenic gld mice, the fetal ontogeny of T cells in transgenic mice was compared with that of non-transgenic mice. In transgenic thymus, development of TcR alpha beta+ cells was accelerated as detected by earlier expression of CD4, CD8 and TcR in fetal thymus. In contrast, the number of TcR gamma delta+ cells was reduced. We suggest that altered T cell development in transgenic mice directly or indirectly inhibits the accumulation of abnormal T cells in gld mice.


Selection of murine T cell receptor alpha beta and gamma delta cells in organ cultures established from 14-day embryos. The expression of minor lymphocyte stimulatory locus (Mls) determinants in combination with murine major histocompatibility complex (MHC) class II molecules, leads to the destruction of lymphocytes bearing specific V region-encoded T cell receptor (TcR) products. A much studied example is the elimination of V beta 6+ cells in IE+/Mls-1a mice, in which deletion can be detected 7-10 days after birth but is not fully operational earlier in embryonic life. Here we investigate this transitional period in development and show that selective deletion of V beta 6 occurs in vitro, approximately 1 week after organ cultures are established from 14 day embryos. These unmanipulated organ cultures receive no additional cell immigrants after day 14, suggesting that the cellular elements mediating negative selection (or their direct precursors), are already resident in the fetal thymus by day 14 of gestation. Hence, the developmental timing of the outset of rigorous negative selection of V beta 6 is not dictated by the postnatal entry of deleting elements into the thymus, but perhaps by the maturation of the pre-existing environment. Using a parallel organ-culture approach we have looked at the development of V delta 4 and V gamma 3, TcR gamma delta+ cells in a variety of mouse strains. These receptors have recently been reported to be subject of MHC and non-MHC linked selection, respectively. We find that after an initial period of expansion, the number of V gamma 3-expressing cells dramatically declines. However, this selective loss of V gamma 3 cells is not contingent on the C57BL/6 mouse strain (in contrast to a previous report). These findings are discussed in the context of current models of ontogeny and repertoire selection.


Specificity of rat T cell receptor V beta chain usage in proliferative responses to staphylococcal enterotoxin B. The staphylococcal enterotoxins (SE) are potent stimulators of T cell proliferative responses in humans and in mice. In these systems, the toxins function as superantigens and stimulate T cells bearing particular V beta. Although homology between the V beta of mice and humans is limited, related V beta families may respond to certain SE in a similar fashion. In this report, we have characterized the rat T cell response to staphylococcal enterotoxin B (SEB). Rat T cells from several lymphoid organs proliferated strongly in response to both commercially available and recombinant SEB. Using a polymerase chain reaction assay, we identified the predominant V beta families stimulated by this enterotoxin. The T cell receptor V beta elements used by rat T cells were similar to but not completely identical with those used by mice. The V beta profile stimulated depended on the purity of the SEB preparation used.


Bone marrow cells of athymic nude mice express functional T cell receptor alpha chain transcripts rearranged to V delta 2, 3, 4, 5, 6 genes. From bone marrow cells (BMC) of athymic nude mice, T cell receptor (TcR) alpha chain transcripts were selectively amplified by polymerase chain reaction (PCR) using V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and C alpha-specific primers. Amplified DNA fragments were cloned, and 32 randomly selected clones from 5 PCR were sequenced. Twenty-three distinct rearrangement events were detected, of which 87% (20/23) were in-frame. All five tested V delta genes (V delta 2, 3, 4, 5, 6) rearranged in-frame to J alpha-C alpha. N-region diversity in V delta-J alpha junctions present in most clones was limited to two to five nucleotides. P-nucleotide additions in this region were also detected. The V delta 5 gene located 3' of C delta in reversed transcriptional orientation was rearranged to J alpha by inversion. The J alpha usage pattern of the sequenced clones was strongly biased towards rearrangement of the most 5' genes (located nearest to C delta) of the J alpha cluster: the most 5' J alpha (J alpha TA1) was used by 30% of all clones, and 78% of all J alpha rearranged to V delta were located in the 5' 12 kb of the 60-kb J alpha cluster. As distinct V delta/C delta and V alpha/C alpha TcR usage patterns are prevalent in peripheral T cell populations, our data suggest that these TcR usage patterns results from repertoire selections operating in alpha beta and gamma delta T cell lineages, but not from preferential V delta-C delta and V alpha-C alpha rearrangement patterns.


Ti gamma A + delta TCS1+ T cells in human thymus. Analysis of the T cell receptor. TcR1+ T cells in peripheral blood have been shown to express in an exclusive fashion either the Ti gamma A or the delta TCS1 epitope. Here, we characterize a subset of TcR1+ T cells in fresh thymus co-expressing the Ti gamma A and delta TCS1 epitopes. TcR1dim and TcR1bright clones can be distinguished. Biochemical and molecular studies on both types of clones generated from these thymocytes reveal a unique T cell receptor structure characterized by the use of a V gamma 9/C gamma 2-encoded 55-kDa gamma chain nondisulfide linked to a V delta 1-encoded delta chain.


Expression of opsin and IRBP genes in mutant RCS rats. The retinal pigment epithelium of RCS rats bearing the autosomal recessive rdy mutation fails to ingest shed rod outer segment tips. Accumulation of disk debris in the subretinal space of the maturing mutant retina causes a secondary degeneration of photoreceptor cells. Two hypotheses have been offered as possible explanations of the death of photoreceptor cells in this disorder: (1) photoreceptors are starved for amino acids, retinal, oxygen, etc; and (2) that IRBP levels and synthesis may be decreased and interfere with retinal transport and this deficiency is lethal to these cells. To test these hypotheses, we have studied the effect of this mutation on the levels of expression of opsin and IRBP genes, and gene products and on rates of synthesis at various ages in dystrophic RCS p+ rats and compared the results to those obtained with normal Long Evans rats. The mutant rats and normal controls had comparable amounts of opsin and IRBP mRNA transcripts and rates of synthesis up to post-natal day 45 (P45) but opsin transcripts were barely detectable at P60 and thereafter. IRBP mRNA levels were also very low after P62 although somewhat higher than opsin mRNA. Opsin could be detected immunochemically, albeit at lower levels, at all the ages studied up to P310, but IRBP levels fell below detection after P45. We localized opsin and IRBP in the retina by post-embedding EM immunocytochemical procedures and found that opsin is present in the remnants of rod outer segment debris, even at P390, long after detectable opsin synthesis had ceased. These data suggest that expression of opsin and IRBP genes is not influenced by the shape and state of the outer segments, and that the rdy mutation does not influence the expression of the opsin and IRBP in these retinas until the photoreceptor cells are profoundly damaged. Thus, neither hypothesis about the causes of cell death in this disorder is supported.


RPE conditioned medium stimulates photoreceptor cell survival, neurite outgrowth and differentiation in vitro. In the present study we have investigated retinal pigment epithelium-photoreceptor cell interactions in vitro, and their contributions to photoreceptor cell survival and differentiation. Preparations enriched for intact photoreceptor cells from neonatal rat retina were grown in either serum-free medium supplemented with RPE-conditioned medium (RPE-CM) or in serum-free medium alone. A variety of substrate conditions were tested for the best neurite outgrowth. Cultures were monitored for 7 days by light and electron microscopy, as well as by opsin, vimentin and carbonic anhydrase-C immunocytochemistry. RPE-CM was found to stimulate both proliferation of flat cells and photoreceptor differentiation. The number of photoreceptors bearing neurites and their neurite length measurements showed significant differences between the RPE-CM group and the control group within 20 hr in culture. Elimination of contaminating flat cells by the addition of an antimitotic drug prevented photoreceptor cell morphological maturation; however, these cells survived as round cell bodies without processes for at least 10 days in the presence of RPE-CM and expressed opsin during this period. Conditioned medium from the flat-cell monolayers did not support photoreceptor differentiation or their survival. However, the presence of flat cells was a requisite to achieve any neurite outgrowth even in the presence of RPE-CM. In the absence of RPE-CM, neither photoreceptors nor flat cells survived or proliferated. Heat and trypsin treatment of the RPE-CM abolished all its growth-supporting activities which indicates its proteinaceous nature. This represents the first time in vitro that an RPE-derived factor(s) has been shown to be responsible for photoreceptor cell survival and differentiation.


Changes in T-cell regulation of responses to self antigens in women with pelvic endometriosis. To study autoimmune aspects of endometriosis. Lymphoblast transformation and hemolytic plaque formation were used to assess specific T- and B-cell activity against endometrial antigens. Military teaching hospital. Ninety-four healthy women of reproductive age undergoing diagnostic laparoscopy as part of an evaluation for infertility or chronic pelvalgia were accordingly grouped into those with normal pelvic peritoneum (20), mild endometriosis (50), and severe endometriosis (24). The study assessed the proliferative and humoral responses of lymphocytes from women with and without endometriosis to endometrial antigens and quantified the number of B-cell precursors, T-helper cells, and T-suppressor cells to these antigens. Unfractionated endometrial antigens were similarly blastogenic for lymphocytes from women with and without endometriosis. Despite equivalent numbers of B-cell precursors to these antigens, antiendometrial antibody responsiveness appears to have increased in women with mild endometriosis because of a decrease in T-suppressor cell activity and declined in women with severe endometriosis because of a further drop in T-suppressor cell activity and an increase in T-helper cell activity, as compared with women without endometriosis. Taken together, these experiments support the possibility that pelvic endometriosis may result from a break in specific T-cell tolerance rather than nonspecific polyclonal activation of responder lymphocytes.


The effect of the neurotoxin DSP4 on the development of a predisposition in the domestic chick. Under certain conditions, dark-reared chicks preferentially approach objects resembling conspecifics. In the current study we investigated the effect of the catecholaminergic neurotoxin DSP4 on this predisposition. After hatching, chicks received an intraperitoneal injection of either DSP4 or vehicle (controls). The chicks were individually placed in running wheels for 2 x 1 hr, 24 hr before testing. The chicks were maintained in darkness until given a preference test, which involved the simultaneous presentation of an illuminated red box and a stuffed fowl at 36, 48, 60, 66, or 72 hr after hatching. The approach of each chick toward the two objects was recorded over 5 min and a preference score calculated. Control chicks placed in the running wheels at 24 and 36 hr posthatch and tested 24 hr later showed a significant preference for the fowl compared to the red box. In contrast, DSP4-treated chicks did not show such a preference at corresponding ages, but did express a greater preference for the fowl when placed in the running wheels at 42 and 48 hr posthatch and tested 24 hr later. These results suggest that experience of the running wheel or of associated factors, e.g., handling, must occur during a restricted period of time to influence the emergence of the predisposition to approach the stuffed fowl. DSP4 treatment delays the onset of this period.


The basis of decreased recombination in certain outcrosses of Neurospora crassa. Crossing over in a multiply marked segment of linkage group I was conspicuously reduced in outcrosses between a marked laboratory strain and each of six unrelated wild-collected strains, compared with crosses between inbred laboratory strains. The marked chromosome segment was transferred intact from the inbred strain to one of the wild-collected strains by seven recurrent backcrosses, and conversely, the corresponding segment of the wild strain was transferred to the inbred background by backcrossing to the multiply marked laboratory strain. Recombination was then monitored in crosses from parents having the marked and unmarked chromosome segments from the same or from unrelated sources. Meiotic crossing-over in the marked segment remained low in crosses between parents that were dissimilar with respect to genetic background, but crossing over was restored to a high level when the genetic background of both parents was that of the inbred laboratory strain, regardless of the source of the marked segments. Reduced recombination in outcrosses was therefore not due to heterologies in the marked segment but must be attributed to modifiers that are unlinked or distant from the monitored region.


Age stability of human brain 5-HT terminals studied with [3H]paroxetine binding. The effect of age on serotonin uptake sites labeled with [3H]paroxetine was studied in two sets of brains. The first set included 28 subjects (19 males and 9 females) between the ages 0 and 100 years. The cortex of cingulate gyrus and the amygdala were studied. No age-related changes in binding capacity (Bmax) or binding affinity (Kd) were noted. In the second set, the frontal cortex and hypothalamus from 22 subjects (18 males and 4 females) between 16 and 75 years were studied. No age-related changes in the binding were observed. The interval between death and freezing of the tissue did not influence the binding. Regarding the [3H]paroxetine binding as an indirect marker for 5-HT terminals, the data suggest a stability of 5-HT terminals with increasing age in the human brain.


Effect of oral clodronate on bone pain. A controlled study in patients with metastic prostatic cancer. Although osteosclerotic metastases are characteristic of prostatic carcinoma, bone resorption is also accelerated. Since clodronate inhibits bone resorption and relieves bone pain, we have given it to patients with painful bone disease from prostatic cancer after failure of hormonal therapy. All patients received estramustine phosphate orally. Simultaneously they were randomly allocated to clodronate (36) and placebo (39) groups. Clodronate was given by mouth. The dose was 3.2 g for the first month, thereafter 1.6 g. Pain relief was more distinct in the clodronate group where one third of patients were totally free of bone pain. The use of analgesics stopped in 38% of patients on clodronate and in 18% on placebo which effect probably belongs to estramustine phosphate. Serum calcium concentration decreased more markedly in the clodronate group. Clodronate dose of 3.2 g seemed to be more potent than that of 1.6 g. Side effects were uncommon and occurred equally in both groups. No significant differences were seen in median survival or survival rates between the groups.


Psychosocial adaptation of fathers of children with autism, Down syndrome, and normal development. Fathers have been largely neglected in previous research of families of autistic children. We compared fathers of 20 autistic, 20 Down syndrome, and 20 developmentally normal children on several measures of psychosocial adaptation. Groups were matched on child's adaptive behavior age equivalent, gender, birth order, family size, and SES. The three groups differed significantly on measures of intrapersonal and family functioning but not on social-ecological variables. Fathers of children with autism or Down syndrome reported more frequent use of wish-fulfilling fantasy and information seeking as coping strategies as well as more financial impact and disruption of family activities than did fathers of developmentally normal children. There were few significant differences between fathers of children with autism and those of children with Down syndrome. These results suggest that fathers adapt relatively well to the demands associated with raising a child with a developmental disability.


In vivo and in vitro characterization of the secA gene product of Bacillus subtilis. The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.


Mutational analysis of the glycine-rich region of the c subunit of the Escherichia coli F0F1 ATPase. Eight strains carrying amino acid substitutions within the c subunit of the F0F1 ATPase of Escherichia coli have been constructed by using site-directed mutagenesis. Three strains carrying the substitutions Gly-23----Leu, Ala-24----Leu, and Gly-38----Leu, which reside in or near the highly conserved glycine-rich region of the c subunit, are unable to carry out oxidative phosphorylation. Membranes prepared from these strains possess basal levels of ATPase activity. In contrast, strains carrying the substitutions Ile-30----Phe, Gly-33----Leu, Gly-58----Leu, and Lys-34----Val and the Lys-34----Val, Glu-37----Gln double substitution were found to possess a coupled phenotype similar to that of the wild type.


Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme. The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacillus casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound lipoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the dae gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the dae gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% of the total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid.


Regulation of katF and katE in Escherichia coli K-12 by weak acids. Chromosomal transcriptional and translational lacZ fusions to the katE (structural gene for the HPII hydroperoxidase) and katF (putative sigma factor required for katE expression) genes of Escherichia coli were isolated, and the regulation of these fusions was used to identify factors that control the expression of these two important antioxidant factors. While katE was found to be regulated primarily at the level of transcription (since induction patterns were similar for both transcriptional and translational fusions), katF expression was a function of both transcriptional and translational signals. The katE gene was induced 57-fold as cells entered the stationary phase, while katF was induced 23-fold. katF induction was coincident with katE induction and occurred at the onset of the stationary growth phase. Expression of both katE and katF could be induced by resuspending uninduced exponential-phase cells in spent culture supernatant recovered from stationary-phase cells. The component of stationary-phase culture supernatant responsible for induction of the katF regulon appeared to be acetate, since expression of both katE and katF fusions was induced when exponential-phase cells were exposed to this weak acid. Other weak acids, including propionate and benzoate, were also found to be effective inducers of expression of both katF and katE. Induction of katE and katF fusions was unaffected in merodiploid strains containing both mutant and wild-type alleles, indicating that expression of both genes is independent of the wild-type gene product. Examination of catalase zymograms prepared from cells exposed to various levels of acetate revealed that both HPI and HPII catalases are induced by this weak acid, suggesting that there is a common link in the regulation of these two enzymes.


Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene. The vitamin B12 (B12)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product. It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism. Using Escherichia coli strains lysogenized with a lambda phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion. Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression. In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein. When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect. In a metJ mutant, B12 represses metE-lacZ expression less than twofold. When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored. The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-B12 holoenzyme.


Inhibition of heterotopic osteogenesis in rats by a new bioerodible system for local delivery of indomethacin. A study was done to evaluate the effect of a system for the local delivery of indomethacin on demineralized bone-induced formation of heterotopic bone in the abdominal muscles of rats. Two separate investigations were conducted on a total of forty-eight Wistar rats. In both series, two types of implants were used: polyorthoester and demineralized bone (Group A, the control group) and polyorthoester with 5 per cent indomethacin and demineralized bone (Group B, the experimental group). In the first series, host-tissue responses and osteoinduction were evaluated histologically at two, three, and four weeks after the implantation. In the second series, the formation of bone was quantified on the basis of uptake of 85Sr at four weeks after the implantation. The polyorthoester system for the local delivery of indomethacin significantly inhibited demineralized bone-induced heterotopic formation of bone, as demonstrated by light microscopy and by uptake of 85Sr. The polyorthoester, with or without the drug, caused little tissue reaction and was resorbed almost completely at four weeks.


Exclusion of close linkage of bipolar disorder to dopamine D1 and D2 receptor gene markers. A potential role of dopamine in bipolar disorder has been suggested by several strands of evidence, namely the ability of dopaminergic agonists to induce mania and the effects of lithium, carbamazepine and the antipsychotics on central dopamine receptors and/or turnover. We therefore aimed to determine if bipolar disorder in two large bipolar pedigrees was linked to the recently cloned dopamine D1 (DRD1) and D2 (DRD2) receptors. (These have been mapped to chromosomal regions 5q35.1 and 11q22.3-q23, respectively). Linkage of bipolar disorder and recurrent depression to DRD1 and DRD2 was tested using a series of genetic models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective status (ranging from bipolar I alone to all affective illnesses). Close linkage to these markers was strongly excluded using each model and definition. The findings for DRD1 also persisted when a wide range of rates of 'sporadic' (non-genetic) presentations of illness were incorporated in the analysis, but the DRD2 results did not remain statistically significant at high sporadic rates. The exclusion of linkage to DRD2 is consistent with other recent reports.


Facet theoretic analysis of the Hamilton-D scale. The items of the HAM-D(17-item version) were analyzed by a nonmetric (ordinal) multi-dimensional scaling procedure (Smallest Space Analysis, SSA-I) and the structure of the test items characterized within the framework of Guttman's facet theory. Two systematic components (facets) were discerned: 'centrality' and 'aspect'. Properties of the facets as well as their relations were assessed and examined empirically by analyzing the inter-relations among different items. The spatial configurations obtained by the scaling procedure were found only partially to fit the expectations derived from the facet-theory model. The facet 'centrality' was found to have a strong overriding influence over the 'aspect' facet. The results suggest the value of a new combination and selection of items reflecting different facets of depression consistently over time.


[Laparoscopic hysterectomy. Results in 44 cases]. Laparoscopic hysterectomy is a recent procedure. We present our preliminary results about 44 patients. In 77.3% of cases (34 patients) the operation was carried out completely by laparoscopy and 10 patients (22.7%) required conversion of the laparoscopy to a standard laparotomy. The indications for laparotomy were: hemostasis difficulties (6 cases); bladder injury (1 case); inability to expose the uterine pedicles and or the ureter (3 cases). Three post-operative complications occurred: one small bowel occlusion which was explored by laparoscopy, and two infection treated by antibiotics only. These preliminary results enable us: to affirm that laparoscopic hysterectomy is feasibility without an important risk of per and or post-operative complications. to specify the four situations in which laparoscopic surgery is particularly advantageous for hysterectomy: absence of genital prolapse; when uni or bilateral adnexectomy is required; previous past-history of abdomino-pelvic surgery, salpingitis, endometriosis ...; neoplastic pathologies (lymphadenectomy); to propose a laparoscopic hysterectomy classification.


Ondansetron, a 5-HT3 receptor antagonist, partially attenuates the effects of amphetamine: a pilot study in healthy volunteers. Preclinical studies suggest that 5-HT3 antagonists modulate dopamine-mediated responses in the limbic system and may therefore have a therapeutic role in psychiatry. We have examined the effect of ondansetron, a specific 5-HT3 antagonist, on the psychological and psychomotor changes induced by amphetamine in human volunteers. Nine healthy males took part in this double-blind placebo-controlled balanced-crossover study. Each subject received one of three treatments in a randomised manner: (a) placebo/placebo; (b) placebo/amphetamine (15 mg); (c) ondansetron (4 mg)/amphetamine (15 mg). Subjects were assessed for self-ratings of hunger, mood, energy, alertness, restlessness, irritability, and asked to rate the abnormality of their overall subjective state. In addition, systolic blood pressure, and performance on psychomotor tests were repeatedly assessed. Although amphetamine did not cause any significant changes in self-rating of mood, energy, alterness, restlessness or irritability, it induced a significant increase in self-ratings for overall subjective state, and a significant decrease in self-ratings of hunger. Amphetamine also caused an increase in systolic blood pressure and a decrease in the mean time taken to complete the psychomotor tests. Pretreatment with ondansetron attenuated the effects of amphetamine on hunger and subjective state, but not on blood pressure or psychomotor performance tests. These findings suggest that in humans 5-HT3 receptor antagonists may partially modify the subjective effects of amphetamine, and are in keeping with results from animal studies that 5-HT3 receptor antagonists might affect neurotransmission within mesolimbic brain regions. However, it was not possible to exclude a pharmacokinetic interaction to explain the effects of ondansetron.


Evidence for extrathymic development of TNK cells. NK1+ CD3+ cells responsible for acute marrow graft rejection are present in thymus-deficient mice. The predominant mechanism responsible for acute specific rejection of allogeneic and parental bone marrow by irradiated mice is due to a cell (TNK) that expresses the NK cell surface markers NK1 and ASGM1 as well as TCR. Here we analyze the question as to whether TNK cells require a functional thymus for their development. Using adoptive cell transfer assays, evidence is presented that, as is the case in normal mice, NK1+ CD3+ effector cells are responsible for rejection in thymus-deficient nude mice and that the specificity of rejection is indistinguishable from that of normal mice. To reveal the presence of TNK cells in the spleen of nude mice, double staining for NK1 and CD3 followed by FACS analysis was done. It is shown that NK1+ CD3+ cells are present in the spleens of nude but not euthymic mice, suggesting that the lack of a functional thymus stimulates either Ag expression or the number of TNK cells. In support of this finding, the treatment of irradiated marrow reconstituted mice with cyclosporin A leads to the appearance of TNK cells in the spleen. The relative efficiency of spleen cells from nude and cyclosporin A-treated mice to transfer resistance in adoptive cell transfers was assessed and found to be higher than that of normal spleen, consistent with the higher frequency of these cells in thymus-defective mice. The fate of NK1+ CD3+ cells subsequent to stimulation with an allogeneic marrow graft indicates that these cells proliferate in nude mice without gaining cytolytic activity. In euthymic mice, however, NK1+ CD3+ cells appear transiently but disappear in favor of CD4+ and CD8+ cells that proliferate in response to an allogeneic marrow graft. The CD8+ cells express cytolytic activity with specificity similar to that of the acute rejection mechanism, consistent with the suggestion that TNK cells differentiate into CD8+ killer cells. The reason why TNK cells in nude mice fail to differentiate into CD8+ CTL is explained by the lack of Th cells.


Developmentally regulated expression of the beta 4 integrin on immature mouse thymocytes. Integrins are a superfamily of alpha beta heterodimers, most of which serve as cell surface receptors for extracellular matrix proteins. In this report, we demonstrate that the recently described alpha 6 beta 4 integrin, previously thought to be limited to epithelial cells and Schwann cells, is expressed on immature mouse thymocytes. The presence of alpha 6 beta 4 is controlled by regulation of beta 4 expression, because alpha 6 was expressed by virtually all cells examined, paired with the beta 1 integrin chain to form VLA-6. During fetal ontogeny, beta 4 was highly expressed by 35% of day-13 thymocytes, 75% of day-14 to -15 thymocytes, then rapidly declined to low levels by birth. In neonates and adults, beta 4 expression was highest on CD4- CD8- CD3- and TCR(+)-gamma delta subsets. Correlation of IL-2R, CD44 and beta 4 on CD4- CD8- thymocytes revealed maximal levels on the intermediate CD44- IL-2R+ subset. Most CD4- CD8+ TCR- thymocytes and a significant fraction of CD4+ CD8+ thymocytes were beta 4lo, whereas the most mature J11d- single positive thymocytes were beta-4. Overall, down-regulation of beta 4 was associated with up-regulation of CD4, CD8, and CD3 in the thymus. alpha 6 beta 4 was undetectable on fetal liver or bone marrow cells, lymphocytes from lymph node, spleen, or blood, and mitogen-activated splenic T cells cultured up to 10 wk with IL-2. The data suggest that alpha 6 beta 4 is up-regulated after pro-T cells enter the thymus and may have a thymus-specific function for T cells. The developmentally regulated pattern of expression and the prominence of alpha 6 beta 4 on day-13 to -16 fetal and adult CD4- CD8- CD3- thymocytes further suggest this unusual integrin may play a role in early T cell development, including stages before acquisition of the TCR.


The N-terminus and C-terminus of IFN-gamma are binding domains for cloned soluble IFN-gamma receptor. The mechanism of binding of murine IFN-gamma to its receptor has not been determined. We have studied this mechanism by examining the binding of overlapping synthetic peptides of IFN-gamma to cloned soluble murine IFN-gamma R. IFN-gamma (1-39) and IFN-gamma (95-133) were able to compete with [125I]IFN-gamma for binding to cloned soluble receptor. Peptides corresponding to the inner region of IFN-gamma--IFN-gamma (36-60), IFN-gamma (54-91), and IFN-gamma (78-107)--showed a markedly reduced ability to compete with [125I]IFN-gamma for receptor binding relative to the N-terminal and C-terminal peptides. In direct binding studies, the binding of [125I]-IFN-gamma (1-39) to soluble receptor could only be competed by IFN-gamma (1-39) and IFN-gamma and not by any of the other peptides including IFN-gamma (95-133). This suggests that the N- and C-termini of IFN-gamma bind to different regions of the receptor. These data in conjunction with previous structure/function studies and x-ray crystallographic data have allowed us to formulate a "velcro-key" model of IFN-gamma binding to receptor that involves both the N- and C-terminal domains. The N-terminus binds in the classical "lock-and-key" manner characterized by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct from the N-terminus likely through the polycationic region, which is conserved across species barriers. Binding of this type would exhibit high affinity and low specificity similar to a piece of velcro. This interaction becomes specific when the C-terminus is in the context of the whole IFN-gamma molecule and may act to increase the affinity of receptor binding and/or facilitate signal transduction.


Lymphocyte development of adherence and motility in extracellular matrix during IL-2 stimulation. To extravasate into normal and neoplastic tissue, lymphocytes must migrate through the subendothelial basement membrane and underlying interstitium, structures rich in extracellular matrix (ECM). We have performed a time-course study of the development of motility in ECM by murine lymphocytes during in vitro exposure to high titers of IL-2 (1000 Cetus units/ml). This protocol generates immunotherapeutic lymphocyte populations expressing lymphokine-activated killer activity. Spontaneous motility was measured in three-dimensional gels of type I (interstitial) collagen or Matrigel, a model basement membrane. A newly developed assay permitted not only the measurement of distance traveled by the leading cell front, but also the separation of lymphocytes on the basis of three types of behavior. The motile fraction consisted of lymphocytes that penetrated beneath the ECM gel surface during an 18-h migration period. There were also two nonmotile fractions: the nonadherent fraction, which failed to bind to the gel surface; and the adherent fraction, which bound but did not penetrate during the assay period. During a 3- to 5-day exposure to high titer IL-2, both adherence and motility increased significantly. In type I collagen, cells of the NK lineage developed greater surface adherence and less motility than cells of the T lineage. The surface-adherent fraction expressed higher lymphokine-activated killer and NK activity than did the nonadherent or motile fractions. Under prolonged IL-2 stimulation (7 to 12 days), there was a decline in the percentage of cells exhibiting motility in both types of ECM, and an increase in the percentage of surface-adherent cells. The findings indicate that the behavior of an IL-2-stimulated lymphocyte population in ECM is profoundly influenced by the duration of IL-2 exposure. Furthermore, lack of lymphocyte motility may reflect two different behaviors, nonadherence and adherence without motility. The nonadherent and surface-adherent populations may differ in phenotypic distribution and function. The motility system described in this report will be useful in separating and studying the mechanisms that produce lymphocyte adherence and motility, and in understanding the in vivo implications of these behaviors.


Effects of similarity and repetition on memory: registration without learning? We investigated judgments of the frequency of test items (Y) that were highly similar to studied items (X) to test a prediction made by several memory models: that the judged frequency of Y should be proportional to the judged frequency of X. Whether stimuli were pictures or words, judged frequency of Y was bimodally distributed with 1 mode at zero, suggesting that frequency judgments involve a 2-stage process in which a zero judgment is made if there is a mismatch between retrieved information and the test item. Nonzero judgements, taken by themselves, were consistent with the prediction of proportionality. In 2 experiments, the percentage of zero judgments made to Y increased with repetition of X, but in 2 others the percentage did not change beyond frequency = 1. The percentage of "new" judgments in recognition memory followed this same pattern. Because the judged frequency of X increased even as X-Y discrimination showed no improvement, we characterize the result as "registration without learning."


Subjective memorability and the mirror effect. The mirror effect refers to the common finding that hit and false alarm rates on a recognition test are inversely related. The present research investigated the generality of the mirror effect (to rare words) and tested whether the effect might be grounded in accurate estimates of word memorability. The first 2 experiments showed that although high- and low-frequency words exhibit a mirror effect, rare words do not. Furthermore, contrary to expectations, Ss consistently (and mistakenly) predicted that memorability was directly correlated with frequency of usage. These findings weight against the idea that the mirror effect arises because of a S's ability to reject low-frequency lures on the grounds that such words would have been remembered had they appeared previously. Instead, the rejection of lures from different frequency categories may be determined by their semantic or phonemic overlap with list targets, and an analysis along these lines may help to explain why rare words constitute an exception to the otherwise ubiquitous mirror effect.


On the relationship between recall and recognition memory. The relationship between recall and recognition has been a central topic for the study of memory. A test of alternative views about recall and recognition was arranged by studying amnesic patients. In amnesia, damage has occurred to a brain system important for declarative (conscious) memory, but skill learning, priming, and other forms of nonconscious memory are intact. Recall and recognition were found to be proportionately impaired in amnesic patients, and confidence ratings for the recognition judgments were commensurate with the level of impaired performance. The results are contrary to views that either recognition memory or associated confidence judgments are ordinarily supported significantly by nonconscious memory. The results favor the view that recall and recognition are related functions of declarative memory and equivalently dependent on the brain system damaged in amnesia.


Influence of contextual features on the activation of ambiguous word meanings. Three studies examined whether initial meaning activation is sensitive to context. Experiment 1 demonstrated that contextually appropriate targets were activated more than inappropriate targets. Experiment 2 evaluated activation across intervals of 0, 300, and 600 ms. Constraining sentences activated contextually appropriate meanings over inappropriate meanings. This was maintained across the intervals for highly salient targets. Less-salient targets, although initially activated, were no longer activated 300 ms following the homograph. Experiment 3 converged on context-sensitive activation following a 50-ms exposure of the sentence-final homograph. Conclusions are (a) initial meaning activation can be sensitive to context, (b) when a homograph is instantiated, it is congruent with a broad scope of targets, and (c) less-salient targets receive less activation over the time course.


Children's and adults' reading of nonwords: effects of regularity and consistency. The procedures used by novice readers to assemble pronunciations for nonwords were investigated. Children in Grades 1-3 read aloud consonant-vowel-consonant and longer monosyllabic nonwords. By the end of Grade 1, children displayed a good grasp of grapheme-phoneme (G-P) correspondences (e.g., ai, ow). Grade 2 and 3 readers increasingly used larger orthographic correspondences termed rimes (e.g., -ook, -ild). However, G-P correspondences determined most responses. Adults likewise used G-P rules when reading aloud nonwords and were more accurate at applying the rules. The strong reliance of Grade 1 and 2 readers on G-P rules was also demonstrated by their superior oral reading of regular words along with a tendency to regularize exception words (e.g., reading bull to rhyme with dull).


Nonword naming: further exploration of the pseudohomophone effect in terms of orthographic neighborhood size, graphemic changes, spelling-sound consistency, and reader accuracy. Five experiments examined nonword pronunciation. As reported by McCann and Besner (1987), accurate, regular pronunciations increased as the number of orthographic neighbors (N) increased. Adults read pseudohomophones (nonwords that sound like a word) more accurately than other nonwords only when the nonwords were low n, shared the consonants with the words on which they were based, and overall accuracy was lower. Children showed a pseudohomophone advantage even when N was high. Adults pronounced nonwords comprised of inconsistent endings (with existing regular and irregular pronunciations) in an irregular fashion when this resulted in a word; this applied to relatively high-N items.


Reaction time measures of spelling: testing a two-strategy model of skilled spelling. Two strategies have been suggested to be important in skilled English spelling: a lexical strategy, which relies on word-specific graphemic information, and a rule strategy, which relies on phoneme-to-grapheme correspondences. Limitations in the usefulness of spelling accuracy as a dependent variable make it difficult to test claims about how the strategies might work together. Two experimental paradigms that measure both spelling accuracy and time are suggested to be useful. Results produced with the paradigms were consistent with the claims that skilled spellers make use of both strategies and that the lexical strategy is more useful than the rule strategy. Working memory may be important in combining products of both strategies. Results were inconsistent with the claim that the rule strategy is invoked only when the lexical strategy fails to produce a complete spelling.


Comprehension strategies in the development of a mental model. It is generally assumed that the comprehension strategy used in the development of a mental model for narrative texts focuses on information that is relevant to the protagonist. Experiments 1a and 1b confirmed that readers remain sensitive to the location of the protagonist even when strategies based on text-base level representations predict this information should not be active. Experiments 2 and 3 tested the stronger claim that readers adopt the perspective of the protagonist. Ss did not notice information that was contradictory from the perspective of the protagonist unless explicitly instructed to adopt that perspective. It was concluded that, at the level of the mental model, readers focus on information relevant to the protagonist but that they do not adopt the perspective of the protagonist unless characteristics of the text induce such a strategy.


Conscious knowledge and changes in performance in sequence learning: evidence against dissociation. Two experiments examined the relation between explicit knowledge and motor performance on the serial reaction time task developed by Nissen and Bullemer (1987). Tests of free recall and recognition of sequence components revealed that reliable explicit knowledge was acquired after an amount of practice that was hardly sufficient to improve mean motor performance. In addition, reaction time improvement was limited to the ending trials of the 3- and 4-trial sequence components that Ss recalled or recognized. These results were replicated in Experiment 3, in which Ss were trained under attentional distraction in the task developed by Cohen, Ivry, and Keele (1990). Overall, these findings undermine the most direct experimental support for the widespread view that conscious knowledge and performance in sequence-learning tasks tap 2 independent knowledge bases in normal Ss.


Persistence of stimulus-response compatibility effects with extended practice. The influence of extended practice on stimulus-response (S-R) compatibility effects was investigated. Three experiments, each extending over a period of 8 sessions, were conducted. The nature and degree of compatibility was manipulated across experiments. In all experiments, a persistent effect of S-R compatibility on reaction times was observed. Thus, the lower bound for reaction times appears to be less for compatible assignments than for incompatible assignments. This persistence of S-R compatibility indicates that the initial codings used to perform a novel task continue to exert an influence on later performance.


Differential effects of voluntary expectancies on reaction times and event-related potentials: evidence for automatic and controlled expectancies. Expectancy has been used to explain the effects of stimulus sequences both on reaction times (RTs) and on the P300 component of the human event-related potential. However, there are conflicting views about the control obtainable over these underlying expectancies. We compared the effects of voluntary expectancies for stimulus changes or repetitions in random tone series on RTs and the P300. Ss responded according to either stimulus identity (Experiment 1) or stimulus sequence (Experiment 2). In both experiments RTs were strongly affected by event expectedness. P300 amplitude, on the other hand, was affected (as a trend) only in Experiment 2. The results suggest that there are at least 2 types of "expectancy", one that is largely automatic and inflexible, reflected in P300 amplitude, and a second, controlled process that is reflected mainly in RT. The latter type of expectancy appears to affect processing stages beyond stimulus evaluation and classification.


Reasoning with conditionals containing negated constituents. Three experiments investigated matching bias in conditional reasoning tasks. Matching bias occurs when Ss ignore negations and match named items. Experiment 1 used an abstract and a thematic version of Evans's (1972) construction task. Results showed that matching may be due to an interaction between task demands and constructing contrast classes when interpreting negations. Experiment 2, which used Wason's (1968) selection task, introduced a manipulation to ease contrast-class construction. Confirmation plus falsification dominated over matching. Experiment 3 introduced two other manipulations to aid contrast-class construction with abstract material. Confirmation was facilitated, matching was suppressed, and falsification remained unchanged. These results suggest that matching occurs only when insufficient or ambiguous information prevents the intended interpretation of negations.


Effects of probability mode on preference reversal. Six analysts estimated verbally and numerically the chances that specific events will occur. Sixty decision makers used each type of estimate to make binary-choice decisions and to bid for lotteries based on the events. The usual reversal of preference between choice and bidding procedures was found in the numerical condition, but the frequency of preference reversals was significantly reduced in the verbal condition. This reduction occurred because risk aversion was reduced in choice when verbal estimates were given, whereas bidding was unaffected by presentation mode. The pattern of results was predicted by and supports the hypothesis that the relative importance given to the dimensions of a prospect depends on the form in which the information is displayed.