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J F Miller

Publications and source records attributed to J F Miller.

At least 19 recordsLinked to original sources
PubMed1992

Autoimmune diabetes as a consequence of locally produced interleukin-2.

During cell differentiation in the thymus, self-reactive T cells can be generated. The majority of these seem to be deleted after intrathymic encounter with the relevant autoantigen. As all self antigens are unlikely to be present in the thymus, some autoreactive T cells may escape censorship. Here we study the fate of these cells using transgenic mice expressing the class I molecule H-2Kb (Kb) in the insulin-producing beta-cells of the pancreas. These mice were crossed with mice transgenic for genes encoding a Kb-specific T-cell antigen receptor (TCR) which could be detected using a clonotype-specific monoclonal antibody. Although T cells expressing the highest level of transgenic TCR were deleted intrathymically in double-transgenic mice, Kb-specific T cells were detected in the periphery. These cells caused the rejection of Kb-expressing skin grafts, but ignored islet Kb antigens even after priming. But when double-transgenic mice were crossed with transgenic mice expressing the lymphokine interleukin-2 in the pancreatic beta-cells, there was a rapid onset of diabetes. These results indicate that autoreactive T cells that ignore self antigens may cause autoimmune diabetes when provided with exogenous 'help' in the form of interleukin-2.

Animals↗
PubMed1992

The Croonian Lecture, 1992. The key role of the thymus in the body's defence strategies.

For centuries the thymus has remained a mysterious organ with largely unknown functions. The first demonstration of its crucial role in the development of the immune system was reported in 1961, when it was found that mice thymectomized at birth had poorly developed lymphoid tissues, impaired immune reactivities, and an inordinate susceptibility to develop infections. Although thymus lymphocytes were for a long time deemed immunoincompetent, it was shown in 1967 that they could respond to antigen by proliferating to give rise to a progeny of cells which did not secrete antibody (T cells), but which had a remarkable ability to induce bone marrow cells (B cells) to become antibody formers. This was the first unequivocal demonstration of a major division of labour among mammalian lymphocytes. Tremendous progress in our understanding of the function of the thymus and of the T cells derived from it followed. Distinct T cell subsets were characterized and shown to have an essential role in initiating and regulating a variety of immune responses. The ontogenetic events which occurred during their differentiation were mapped, and this allowed studies of the selection of the T cell repertoire. The major histocompatibility complex and associated peptides were shown to govern T cell selection and antigen activation, and the antigen-specific T cell receptor and the genes which code for it were characterized. Future studies should allow some insight into how to activate T cells more effectively for vaccination purposes, and how to switch them off to prevent autoimmune reactions and to induce tolerance to transplanted tissues.

Animals↗
PubMed1992

HIV-1 and HIV-2 reverse transcriptases: a comparative study of sensitivity to inhibition by selected natural products.

One hundred and fifty six pure natural products, which had previously been tested against HIV-1 reverse transcriptase, were evaluated for HIV-2 reverse transcriptase inhibitory activity. Compounds that lacked effect in the HIV-1 reverse transcriptase system were found also to be inactive against HIV-2 reverse transcriptase. However, compounds belonging to the benzophenanthridine and protoberberine classes of alkaloids, certain flavonoids, the iridoid, fulvoplumierin, and the ansamycin antibiotic, daunomycin, exhibited similar potencies in both enzyme systems. In contrast, HIV-2 reverse transcriptase was observed to be four-fold more sensitive toward the inhibitory effects of the ipecac alkaloids, O-methylpsychotrine sulfate heptahydrate and psychotrine dihydrogen oxalate. Such differences in susceptibilities to inhibitors may indicate subtle dissimilarities in enzyme structure and function.

Alkaloids↗
PubMed1992

Tolerance induction by thymic medullary epithelium.

To study the role of thymic medullary epithelium in tolerance induction, the third and fourth branchial clefts of embryos from E mu-Kb transgenic mice, which express the major histocompatibility complex class I antigen H-2Kb exclusively on medullary thymic epithelium, were grafted to athymic nude mice. The grafts differentiated into tissue that morphologically resembled normal thymus. These grafts expressed the H-2Kb antigen appropriately and gave rise to a functional T cell repertoire. In vivo tolerance to H-2Kb disparate skin grafts was invariably found in mice expressing H-2Kb in the medulla or in both medulla and cortex of C57BL/6 branchial cleft-grafted controls. In marked contrast, in vitro cytotoxicity assays demonstrated reactivity toward H-2Kb in the presence of interleukin 2, and limiting-dilution analyses showed similar frequencies of cytolytic T cell precursors reactive to H-2Kb and to third-party stimulators. Medullary epithelium can, therefore, induce split tolerance, in which in vivo tolerance is accompanied by strong in vitro responses in the presence of interleukin 2.

Animals↗
PubMed1992

Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor.

We have constructed a large library of random peptides fused to the C terminus of the lac repressor. The DNA binding activity of the repressor protein physically links the peptides to the plasmid encoding them by binding to lac operator sequences on the plasmid. This linkage allows efficient enrichment for specific peptide ligands in the random population of peptides by affinity purification of the peptide-repressor-plasmid complexes with an immobilized receptor. After transformation of Escherichia coli with recovered plasmids, the library can be amplified for additional rounds of affinity enrichment or specific plasmids can be sequenced to determine the primary structure of the peptides. We used a monoclonal antibody specific for the peptide dynorphin B as a model receptor to screen a random dodecamer library. After only two rounds of enrichment, the majority of the plasmids in the selected population encoded fusion peptides that bound specifically to the antibody. These peptides contain a consensus sequence similar to a segment of dynorphin B (RQFKVV). This technique should be useful to find peptide ligands for a variety of biological receptors.

Amino Acid Sequence↗
PubMed1992

Inflammation but not autoimmunity occurs in transgenic mice expressing constitutive levels of interleukin-2 in islet beta cells.

Transgenic mice expressing murine interleukin (IL)-2 constitutively in islet beta cells were generated (RIP-IL-2 mice). They died at an early age, when higher levels of IL-2 were produced, because of a predominant macrophage inflammatory response that destroyed the exocrine pancreas. Animals with lower levels of IL-2 survived and had islets that became increasingly infiltrated with lymphocytes over time. However, in spite of the presence of impressive peri- and intra-islet infiltrates, autoimmunity to islet antigens was not seen. Autoimmunity was also not induced to extrathymic H-2Kb molecules known to induce tolerance by a peripheral mechanism when the RIP-IL-2 mice were mated to other mice expressing H-2Kb in islet beta cells (RIP-Kb mice). Apparently, IL-2 can act only on activated T cells and is unable to reverse tolerance in T cells that have been made unresponsive through inappropriate presentation of antigen.

Animals↗
PubMed1992

Differences between steady-state and transient post-synaptic potentials elicited by stimulation of the sural nerve.

In cat medial gastrocnemius motoneurons, single stimuli to the cutaneous sural nerve evoke a post-synaptic potential with a mixture of depolarization and hyperpolarization, depolarization being dominant in type F cells and hyperpolarization in type S cells. This pattern is consistent with previous reports showing that activation of the sural nerve can sometimes reverse the normal order of motor unit recruitment by inhibiting S motor units while simultaneously exciting F motor units. However, during repetitive stimulation for 1-2 s, we found that the hyperpolarizing component of the sural input to medial gastrocnemius motoneurons was not persistent, but instead gave way to depolarization after the first 30 ms. The net steady-state response after 0.5-1.0 s of stimulation was depolarization in all cells, regardless of motor unit type. This suggests that tonic sural input may be incapable of producing prolonged recruitment reversals.

Animals↗
PubMed1992

Post-thymic tolerance to self antigens.

There is now convincing evidence for the imposition of self-tolerance by means of the clonal deletion of self-reactive T cells operating within the thymus. Since not all self components may be encountered there, the question must be asked whether tolerance can occur post-thymically. To test this, we have used transgenic technology to direct expression of a known 'non-self' gene, H-2Kb, to the insulin producing beta cells of the pancreas of mice. H-2Kb-bearing skin, but not skin from other mouse strains, failed to be rejected by the 'RIP-Kb' transgenic mice indicating specific tolerance. Following in vitro stimulation, their spleen cells could not kill H-2Kb-bearing targets, but could respond to third party targets. Their reactivity to H-2Kb was restored by providing them with IL-2. Two hypotheses could account for the above: tolerance results either from the deletion or functional silencing of high affinity effector cytotoxic cells or of regulatory, IL-2-producing helper T cells. Since it is difficult to distinguish between these, we have produced a second series of transgenic mice with rearranged T cell receptor (TCR) genes encoding an anti-H-2Kb TCR, and obtained 'double transgenic' offspring by mating these mice with RIP-Kb mice. The TCR utilized the V beta 11 segment which can be detected by a monoclonal antibody. Although the double transgenic mice were tolerant of H-2Kb, there was no evidence of deletion of anti-H-2Kb T cells. It seems, therefore, that a non-deletional mechanism operates to induce post-thymic tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A transgenic window on peripheral T cell tolerance.

There is convincing evidence for the imposition of self-tolerance by means of the clonal deletion of self-reactive T cells within the thymus. Since not all self components may be encountered there, the question must be asked whether tolerance can occur post-thymically. To test this, we have used transgenic technology to direct expression of a 'non-self' gene, H-2Kb, to the insulin-producing beta cells of the pancreas in mice. These 'RIP-Kb' transgenic mice were specifically tolerant of H-2Kb-bearing skin grafts. To test the fate of potentially reactive H-2Kb-specific T cells in these tolerant mice, the RIP-Kb mice were mated to a second series of transgenic mice with rearranged T cell receptor (TCR) genes encoding an H-2Kb-specific TCR to produce 'double transgenic' offspring. The TCR was detectable by a clonotype-specific antibody. Although there was some evidence of intrathymic deletion of those T cells that expressed the highest density of the H-2Kb-specific TCR, lower density cells were present in the periphery. These may have been either indifferent to the H-2Kb molecule expressed on the beta cells or functionally silenced by it. Further experiments are planned to determine which of these two situations exists.

Animals↗
PubMed1992

Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene.

The products of the bvgAS locus coordinately regulate expression of the Bordetella pertussis virulence regulon in response to environmental signals. Transcription of bvgAS-activated genes is nearly eliminated by several modulating conditions, including the presence of sulfate anion or nicotinic acid and growth at low temperature. We have isolated spontaneous mutations that result in the constitutive synthesis of multiple bvg-regulated loci. Several of these mutations have been analyzed and were found to result from single-nucleotide substitutions within bvgS, in a region encoding a 161-amino-acid segment which links the transmembrane sequence with cytoplasmic domains that appear to be involved in signaling events. The effect of signal transduction mutations in Escherichia coli was determined by measuring the expression of an fhaB-lacZYA transcriptional fusion, and that in B. pertussis was determined by measuring expression of both fhaB-cat and ptxA3201-cat fusions. The constitutive mutations have little effect on fhaB-cat or fhaB-lacZYA expression in the absence of modulating signals but result in a nearly complete insensitivity to MgSO4, nicotinic acid, or growth at low temperature. Furthermore, insertion and deletion mutations in bvgS sequences encoding the periplasmic domain eliminate activity of the wild-type product, whereas constitutive mutants remain active. In B. pertussis cultures grown in Stainer-Scholte broth, expression of ptxA3201-cat differed from that of fhaB-cat in several respects. In combination with a wild-type bvgS allele, ptxA3201-cat expression required the addition of heptakis-(2,6-O-dimethyl)-beta-cyclodextrin, and this requirement was eliminated by the presence of the constitutive mutations.

Alleles↗
PubMed1992

The bvgAS locus negatively controls motility and synthesis of flagella in Bordetella bronchiseptica.

The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.

Amino Acid Sequence↗
PubMed1992

Peripheral T cell tolerance.

The most efficient way to ensure self-tolerance in the T-cell repertoire is by intrathymic deletion of self-reactive clones. Antigens not present intrathymically may, however, influence the peripheral T-cell pool in various ways. The may of course activate T cells, provided that these have the correct specificity and affinity and that the antigens are presented in sufficient amounts on professional antigen-presenting cells. They may be ignored by T cells if some of these conditions are not met. In some forms, the antigen may be toleragenic for mature T cells. If the antigens persist in an immunogenic form, unresponsiveness may eventually be imposed as the end result of a powerful immune response. Extrathymic self-antigenic components are generally encountered early in development, and the way in which these influence peripheral T lymphocytes has been studied by transgenic technology. They may be ignored by T cells if they are sequestered from the immune system, or if they are present in low amounts or on nonprofessional antigen-presenting cells which lack the appropriate accessory molecules or signals needed to activate the relevant T-cell subset. On the other hand, some of these self-antigens readily induce anergy in peripheral T cells, which may or may not involve downregulation of antigen receptors and coreceptors. Tolerance in the T-cell repertoire is therefore achieved not only by intrathymic deletion of self-reactive clones but also by several postthymic mechanisms.

Animals↗
PubMed1992

Treating hopelessness: nursing strategies from six countries.

Strategies to treat hopelessness were described by 188 critical care nurses from Belgium, Canada, Colombia, England, France, and the United States. Data were collected by an open-ended questionnaire distributed by site coordinators to 50 randomly selected critical care nurses in one or more hospitals in each of the six countries. Data were analyzed using content analysis and data reduction techniques. Prior to collapsing data into hope-inspiring categories, discrete words or phrases to treat hopelessness were enumerated for each country. Interrater consensus was reached on each category label. Hope-inspiring strategies included use of interpersonal self (listening, presence, empathy, directing the interaction away from hopelessness themes); involvement and caring for the family; sharing success stories; use of progress reports; empowerment; and referral to experts (psychologists, clinical nurse specialists). Differences were evident among the countries. Providing physical comfort was emphasized by Belgium, Colombia, and Canada but not mentioned by other countries. Nurses from France used strategies labeled "positive provocation" to keep patients self-sufficient, autonomous, and able to move beyond dwelling on hopelessness.

Americas↗
PubMed1992

Maternal respiration and blood gases during aerobic exercise performed at moderate altitude.

We studied whether maternal acid-base status during aerobic exercise performed at moderate altitude is affected by pregnancy. Seven primiparus women were tested at 37 wk gestation and 12 wk postpartum. Subjects were studied at rest, and during two cycle (50 W, 75 W) and two treadmill (67 m.min-1; 2.5% grade, 67 m.min-1; 12% grade) protocols. Exercise bouts lasted 6 min with a 10-min rest between sessions. Minute (VE) and alveolar (VA) ventilation, tidal volume (VT), and ventilatory equivalent for carbon dioxide (VE/VCO2) were significantly (P less than 0.01) greater when exercise was performed during pregnancy. Physiological dead space (VD) was not affected by pregnancy status and did not differ between rest and exercise. Decreases (P less than 0.01) in arterial pH during exercise averaged 0.04 units in both pregnancy and postpartum. Despite similar change in maternal pH, carbon dioxide tension (PaCO2) remained unchanged during exercise at 37 wk gestation but decreased at 12 wk postpartum. Decreases in arterial bicarbonate [HCO3-] associated with exercise were smaller during pregnancy. Our findings indicate that pregnancy did not compromise maternal acid-base status during aerobic exercise.

Acid-Base Equilibrium↗
PubMed1992

A food frequency questionnaire for adolescents: defining eating patterns.

A self-administered food frequency questionnaire (FFQ) was developed to indicate weekly consumption of 64 foods. Reliability, validity, and usefulness of the tool to define a protein eating pattern were determined. Adolescents (N = 1,108) completed the FFQ during the Bogalusa Heart Study. Two-hour and 2-week reliability measures demonstrated consistency of intake of specific foods. Frequency of foods obtained from seven consecutive 24-hour recalls was compared with frequency obtained from the FFQ. A mean 50% agreement for both frequency and quantity of food intake was observed. Geometric means showed differences in mean number of protein foods by age of adolescent but the only significant difference was for beef intake of 15-year-olds. White children reported more servings of beef, cheese, and vegetables with meat than did black children. Black children reported more servings of eggs, luncheon meat, pork, poultry, and total protein than did white children. Boys reported a greater frequency of total protein foods, specifically, eggs, milk, and poultry, than did girls. Significant correlations were noted between low-density-lipoprotein cholesterol and intake of eggs and luncheon meat. We were able to quantitate the reliability and validity of the FFQ and to use it to explore the association of specific eating patterns with cardiovascular disease risk.

Adolescent↗
PubMed1992

Does interleukin-2 abrogate peripheral immunologic self-tolerance in vivo?

Transgenic mice that constitutively expressed murine IL-2 in islet beta cells (RIP-IL-2 mice) had pancreatitis from birth which resolved into a peri- and intra-islet infiltrate in adult animals. In spite of the impressive infiltration, these mice did not develop autoimmunity to islet antigens. Neither was autoimmunity found to extrathymic H-2Kb molecules known to induce tolerance by a peripheral mechanism, when IL-2 and H-2Kb were coexpressed in the beta cells. Apparently, IL-2 can only act on activated T cells and is unable to reverse tolerance in T cells that have been made unresponsive through inappropriate antigen presentation in our system.

Animals↗
PubMed1991

A nondeletional mechanism of peripheral tolerance in T-cell receptor transgenic mice.

To investigate tolerance to extrathymic self molecules, we produced two groups of transgenic mice: one expressed the major histocompatibility complex molecule H-2Kb in pancreatic beta cells, and the other expressed rearranged T-cell receptor genes encoding an anti-H-2Kb receptor. The transgenic T-cell receptor genes were shown to confer the correct specificity and to be expressed appropriately. T cells bearing this receptor were activated by H-2Kb in vitro and in vivo, and they underwent negative selection in mice expressing H-2Kb in the thymus. To determine the fate and function of these anti-H-2Kb T cells in mice expressing H-2Kb exclusively in the periphery, the two groups of transgenic mice were mated to produce double transgenic offspring. In these, transgene-expressing T cells were present in both thymus and periphery. Persisting T cells had not down-regulated either their antigen-specific receptors or their CD8 molecules. Despite the persistence of large numbers of potentially reactive T cells, the mice were tolerant of H-2Kb in that they could not reject H-2Kb-bearing skin grafts, although they did reject third-party grafts. The results show that peripheral T-cell tolerance, unlike that imposed in the thymus, does not involve deletion of T cells. The existence of T cells bearing receptors specific for self components raises the possibility that aberrant activation of such cells may lead to the development of autoimmune disease.

Animals↗