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Biomedical subjects

J F Miller

Publications and source records attributed to J F Miller.

At least 145 records · Page 8Linked to original sources
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↗
PubMed1991

Peripheral deletion of self-reactive B cells.

B LYMPHOCYTES are key participants in the immune response because of their specificity, their ability to take up and present antigens to T cells, and their capacity to differentiate into antibody-secreting cells. To limit reactivity to self antigens, autospecific B cells can be functionally inactivated or deleted. Developing B cells that react with membrane antigens expressed in the bone marrow are deleted from the peripheral lymphocyte pool. It is important to ascertain the fate of B cells that recognize membrane autoantigens expressed exclusively on peripheral tissues because B cells in the peripheral lymphoid organs are phenotypically and functionally distinct from bone-marrow B cells. Here we show that in immunoglobulin-transgenic mice, B cells specific for major histocompatibility complex class I antigen can be deleted if they encounter membrane-bound antigen at a post-bone-marrow stage of development. This deletion may be necessary to prevent organ-specific autoimmunity.

Animals↗
PubMed1991

Dysmyelination in transgenic mice resulting from expression of class I histocompatibility molecules in oligodendrocytes.

Major histocompatibility complex (MHC) molecules are not normally expressed in the central nervous system (CNS). However, aberrant expression has been observed in multiple sclerosis lesions and could contribute to the destruction of myelin or the myelinating cells known as oligodendrocytes. The mechanism of cell damage associated with aberrant MHC molecule expression is unclear: for example, overexpression of class I and class II MHC molecules in pancreatic beta cells in transgenic mice leads to nonimmune destruction of the cells and insulin-dependent diabetes mellitus. We have generated transgenic mice that express class I H-2Kb MHC molecules, under the control of the myelin basic protein promoter, specifically in oligodendrocytes. Homozygous transgenic mice have a shivering phenotype, develop tonic seizures and die at 15-22 days. This phenotype, which we term 'wonky', is due to hypomyelination in the CNS, and not to involvement of the immune system. The primary defect appears to be a shortage of myelinating oligodendrocytes resulting from overexpression of the class I MHC molecules.

Animals↗
PubMed1991

Overexpression of beta 2-microglobulin in transgenic mouse islet beta cells results in defective insulin secretion.

Overexpression of heavy chains of the class I major histocompatibility complex in islet beta cells of transgenic mice is known to induce nonimmune diabetes. We have now overexpressed the secretory protein beta 2-microglobulin in beta cells. Transgenic mice of one lineage had normal islets. Mice of another lineage did not become overtly diabetic but showed significant depletion of beta-cell insulin. When mice were made homozygous for the transgene locus, they developed diabetes. Introduction of the beta 2-microglobulin chain into class I heavy chain transgenic mice resulted in a significant improvement in their islet morphology and insulin content, and the female mice remained normoglycemic. These results suggest that different transgene molecules overexpressed in beta cells can cause islet dysfunction, though not necessarily overt diabetes, and that this effect is mediated by the level of transgene expression. Evidence is provided to show that beta-cell disruption by transgene overexpression occurs at the level of protein and involves a defect in insulin secretion.

Animals↗
PubMed1991

Iodometric measurement of lipid hydroperoxides in human plasma.

Many assay techniques have been used to measure lipid hydroperoxides in plasma, including absorbance of conjugated dienes and reactivity with thiobarbituric acid. Because these measurements are not specific for lipid hydroperoxides, we modified an exisiting iodometric method to correct for interfering phenomena and to provide a more specific measurement of the lipid hydroperoxide content of plasma. To ensure reproducible extraction of hydroperoxides from the many possible forms in plasma, the plasma was treated to hydrolyze enzymatically cholesterol ester, triglycerides, and phospholipids, and the nonesterified fatty acid peroxides were then extracted with ethyl acetate. Extracted lipids were reacted with potassium iodide in acetic acid and methylene chloride, and the resulting triiodide ion (I3-) was measured spectrophotometrically. Correction for nonoxidizing chromophores was made after back-titration of the triiodide ion to iodide with sodium thiosulfate and other non-peroxide oxidants were estimated by their resistance to reduction with glutathione peroxidase. Recovery of added hydroperoxide standards provided routine validations of the procedure's efficiency. The method indicated that insignificant amounts of hydroperoxide may be in the less polar lipids, but the total amount of lipid hydroperoxide esterfied in the plasma lipids of apparently healthy humans may be as much as 4.0 +/- 1.7 microM.

Esterases↗
PubMed1991

Use of a voltage-responsive timer to quantitate hydroperoxides by the cyclooxygenase activation assay.

The importance of lipid hydroperoxides in the interpretations of physiological and pathological processes requires a valid and reliable microanalytical procedure. However, quantitation of the small amounts of lipid hydroperoxides in biological samples is often attempted with methods that have low sensitivity, low validity, and/or low reliability. The highly sensitive and specific enzymatic assay based upon cyclooxygenase activation has many complex kinetic features that have caused it to be cumbersome and unreliable. Analysis of the cyclooxygenase kinetics showed that a fixed percentage (20%) of full reaction accurately substituted for the measurements of the time required to reach optimal velocity. Consequently, a voltage-responsive timer can report endpoints that are dependent upon picomole amounts of lipid hydroperoxide. Additional features of the timer are described which make the hydroperoxide assay simpler, faster, more reliable, and easier to perform than the previous method while still retaining its sensitivity and specificity.

Animals↗
PubMed1991

Regulation of MHC molecules on MBP positive oligodendrocytes in mice by IFN-gamma and TNF-alpha.

The expression of class I and class II histocompatibility antigens by myelin basic protein (MBP)-positive oligodendrocytes, in response to exogenous cytokines, has been investigated in vitro. It has been found that interferon-gamma (IFN-gamma), although capable of class I induction, does not induce class II on oligodendrocytes. Furthermore, tumour necrosis factor-alpha (TNF-alpha), which was shown to induce class I MHC on other neural cells, failed to induce class I on oligodendrocytes. A combination of IFN-gamma and TNF-alpha also failed to facilitate the expression of class II antigens on oligodendrocytes, nor did it amplify the expression of class I seen with IFN-gamma alone. Thus it appears that MBP+ murine oligodendrocytes are refractory to class II induction, and express class I in response to IFN-gamma but not TNF-alpha. The differential regulation and class of MHC expression may have implications in terms of the initiation and targeting of immune responses directed toward the oligodendrocyte.

Brain↗
PubMed1991

Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes.

Each of the two mutants isolated from a fliC (= hag, flagellin-deficient) Escherichia coli strain made motile by a plasmid carrying the fliC gene of Salmonella muenchen by selection for motility in the presence of anti-d (Salmonella flagellar antigen) serum had both lost and gained one or more subfactors of the wild-type antigen. In one mutant codon 246 was GAC (alanine) instead of GCC (asparagine); the other had a deletion of 105 base pairs, explicable by a 10bp direct repeat, starting at bases 782 and 887. The in vitro removal of a 48bp EcoRV(631)/EcoRV(679) fragment produced plasmid pLS408, which was found to lack a subfactor of wild-type antigen d but able to confer motility on flagellin-negative Salmonella sp. (and used for insertion of epitope-specifying oligonucleotides at its EcoRV site). Immunoblotting with absorbed and unabsorbed sera from rabbits immunized with E. coli with wild-type or mutated antigen d showed that the fusion proteins specified by lambda gt11 with the N-terminal part of gene lacZ joined to a restriction fragment coding for residues 145-391 of flagellin gave the same pattern of parent-specific and mutant-specific reactions as the flagellate bacteria. Four out of five similarly selected mutants had the same 105 bp deletion as the first-isolated mutant; the fifth had a 72 bp deletion made possible by a 7-base pair direct repeat, starting at positions 649 and 721. All these changes in serological character without loss of function affected segment IV, specifying residues 182 to 308 of the total of 505, where there is little homology between different flagellar-antigen alleles.

Antigens, Bacterial↗
PubMed1991

A transgenic approach to the study of peripheral T-cell tolerance.

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 and other investigators have used transgenic technology to direct expression of a known "nonself" gene to a given extrathymic tissue. No lymphocytic infiltration was ever seen in transgene-expressing tissues, even if the mice were given normal syngeneic (nontransgenic) spleen cells intravenously or were stimulated with H-2Kb spleen cells. Infiltration did, however, occur in irradiated transgenic recipients of H-2Kb immune spleen cells. In MET-Kb mice, this infiltrate diminished with time, raising the possibility that peripheral tolerance may even have been induced in immune cells. H-2Kb-bearing skin was accepted in young RIP-Kb mice but rejected in older mice, which had lost more than 75% of their beta cells as a result of the overexpression of H-2Kb. This loss of tolerance thus occurred when the concentration of the tolerogen, H-2Kb, fell below some critical threshold. Following in vitro stimulation, spleen cells from young RIP-Kb mice could not kill H-2Kb-bearing targets, but could respond to third party targets. Thymus cells, on the other hand, could be stimulated to kill both targets, clearly indicating that tolerance was not imposed intrathymically. Spleen cells from older RIP-Kb mice (those that had lost most of their beta cells) killed both targets, which is in agreement with the in vivo data. Reactivity to H-2Kb was restored to young spleen cells by providing them with IL-2. Two hypotheses were proposed to account for the above findings: tolerance results either from the deletion or functional silencing of high-affinity effector cells or of regulatory, IL-2-producing helper T cells. As it is difficult to distinguish between these, we have produced a second series of transgenic mice (F3+) with rearranged TCR genes encoding an anti-H-2Kb TCR and derived "double-transgenic" (F3+RIP+) offspring by mating these mice with RIP-Kb mice. The transgenic TCR utilized the V beta 11 segment which can be detected by a monoclonal antibody. There were in the thymus very few CD4+ and very few CD4+8+ cells in both F3+ and F3+RIP+ mice and, in the double-transgenic mice, there was no evidence of deletion of CD8+V beta 11+ cells in the periphery although they showed tolerance to H-2Kb-bearing skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Clonal deletion of autospecific B lymphocytes.

Using mice transgenic for functional, rearranged immunoglobulin heavy and light chain genes, it can be demonstrated that B lymphocytes reactive with cell surface-bound class I MHC antigen can be controlled by clonal elimination. Even low-affinity cell-bound ligands can induce deletion. Deletion can occur in the pre-B to B cell transitional stage or after the B cells exist the bone marrow, depending on where the cells first encounter autoantigen. IgD appears to play no role in protecting cells from deletion. It is argued that defects in B-cell tolerance alone may be sufficient to lead to systemic autoimmunity.

Animals↗
PubMed1991

Inherent beta-cell dysfunction induced by transgenic expression of allogeneic major histocompatibility complex class I antigen in islet cells.

Insulin-dependent diabetes mellitus (IDDM) is generally believed to be an autoimmune disease resulting from T-cell dysfunction that produces beta-cell damage, but it is conceivable that some forms of IDDM are not immunologically mediated. The effect of the expression of a foreign transgenic MHC class I antigen (H-2Kb), restricted to pancreatic islet beta-cells, was tested in vitro and in nude (athymic) mice to determine whether beta-cell dysfunction was due to non-immune mechanisms. The models used clearly excluded immune involvement in beta-cell damage. Fetal pancreas from transgenic and littermate control mice was maintained in organ culture for up to 18 days and insulin secretion into the medium assessed. For the initial 3-4 days in vitro, fetal control and transgenic pancreas secreted similar amounts of insulin, but thereafter insulin secretion by the transgenic tissue decreased in comparison with the controls. When the cultured pancreas was transplanted into nude mice, the transgenic issue produced smaller grafts than the control pancreas, but there was wide variation in graft size. Expression of H-2Kb antigens in beta-cells of nude transgenic mice also resulted in early-onset diabetes. The insulin content in the pancreas of young H-2Kb transgenic euthymic mice, (previously shown not to have insulitis), was reduced but glucagon content was normal. The reduction in in vivo insulin production was similar chronologically to the reduced insulin production by transgenic islets in vitro. These data confirm the non-immune loss of beta-cell function in MHC-transgenic mice and they may be a model for atypical Type I diabetes.

Animals↗
PubMed1991

Developing and maintaining hope in families of the critically ill.

Because of the interdependent, reciprocal dynamics of families and patients, responses of one affect the other. Hope is an important state of being and has been noted to be an example of such a response. Developing and maintaining family members' hope has a contagious effect on the patient. A model of nursing strategies to inspire hope in families of the critically ill with possible outcomes is presented.

Adaptation, Psychological↗
PubMed1991

Physiological and perceptual responses to cycle and treadmill exercise during pregnancy.

We compared physiological and perceptual responses to non-weight bearing (cycle) and weight bearing (treadmill) exercise in 16 sedentary women throughout a normal term pregnancy. Subjects were recruited late in the first trimester (less than 13 wk gestation) and were studied at 4 wk intervals throughout pregnancy and 4 wk postpartum (PP). Exercise consisted of four 5 min protocols; two were performed on the cycle (C1 = 50 W; C2 = 75 W) and two on the treadmill (T1 = 66 m.min-1, 2.5% grade; T2 = 66 m.min-1, 12% grade). Measured variables included oxygen consumption (VO2), heart rate (HR), minute ventilation (VE), and ratings of perceived exertion (RPE, 10-point Borg scale). Absolute VO2 (ml.min-1) during cycle exercise increased (P less than 0.001) by 25 wk gestation, while relative VO2 (ml.kg-1.min-1) during treadmill walking was unchanged through late pregnancy and PP. This suggests that cycle exercise is not a true non-weight bearing exercise within a given group of women throughout gestation. Subjects' VO2max values were estimated at each test interval and found to increase (P less than 0.001) by 25-28 wk gestation. Heart rate and RPE responses to exercise remained constant throughout gestation and decreased (P less than 0.01) PP. Although they did not participate in a regular exercise program, it appears that our subjects experienced a mild aerobic training effect during late pregnancy.

Adult↗