Search PubMed⌕ Search

Biomedical subjects

R G Miller

Publications and source records attributed to R G Miller.

At least 109 records · Page 6Linked to original sources
PubMed1995

Antigen-presenting cells for naive transgenic gamma delta T cells. Potent activation by activated alpha beta T cells.

The function of gamma delta T cells, particularly the minor population of circulating gamma delta T cells, remains unclear. To study these lymphoid gamma delta T cells, a transgenic SCID mouse containing the KN6 gamma delta TCR whose ligand is the TL gene product, T22b, was created. KN6-SCID mice contain a monoclonal population of naive KN6+ gamma delta T cells. Using these mice, we have studied the APC required for activation of KN6+ gamma delta T cells in vitro and in vivo. Analysis of an in vitro mixed lymphocyte response identified a hierarchy of potency for stimulation: dendritic cells = T cell blasts > B cell blasts > B cells > resting T cells. In contrast, in vivo, only alpha beta T cells fully activated KN6+ gamma delta T cells as measured by an increase in the number of splenic KN6+ cells, the development of blast morphology, and the development of proliferative anergy in the responding KN6+ cells. The strong stimulatory properties of C57BL/6J T cells appeared to depend on their having been activated by KN6-SCID alloantigens. T cells from (C57BL/6J x BALB/c)F1 mice, which are tolerant of KN6-SCID alloantigens, could not fully activate KN6+ cells. However, the F1 T cells could activate KN6+ cells if they were activated in vivo by the mitogen, staphylococcal enterotoxin B. A mixture of third party activated T cells plus T22b+ non-T cells only partially activated KN6+ cells, implying that activated T22b+ T cells are acting directly as stimulatory cells. Although the Ags recognized by gamma delta T cells are generally unknown, Ag presentation by activated alpha beta T cells may be an important method of activation.

Animals↗
PubMed1995

Down-regulation of CD8 on mature antigen-reactive T cells as a mechanism of peripheral tolerance.

Previously we have shown that intravenous injection of male B6 lymphocytes containing CD8+ cells into B6 female anti-HY TCR transgenic mice results at 6 wk in the disappearance of the majority of male Ag-reactive T cells (TghighCD8+) from the periphery. Here we investigate the process in more detail. B6 female anti-HY TCR transgenic mice were intravenously injected with viable lymphocytes from male B6 normal, CD4-/-, CD8-/- or CD8-/- carrying a CD8 transgene lacking its cytoplasmic tail ("CD8 tail-less") mice. The fate of TghighCD8+ cells was followed in vivo. There was always a large (at least twofold) expansion of these cells in the periphery 4 days after encountering male Ag. Their subsequent fate differed, however, depending on whether or not the injected male lymphocytes contained normal CD8-expressing cells. If the injected male lymphocytes contained normal CD8 cells, at 6 wk there was a large drop in the number of TghighCD8+ cells associated with a rise in the number of TghighCD8- cells. If the injected male lymphocytes lacked CD8 cells or expressed only tail-less CD8, TghighCD8+ cell numbers returned to normal by 6 wk, while TghighCD8- cell numbers remained unchanged. The TghighCD8- cells, although carrying high levels of the male specific TCR, did not react to male Ag. In addition, their ability to respond in anti-CD3-induced activation, which does not require CD8 as a co-receptor, was significantly impaired. Our data suggest that down-regulation of CD8 on the Ag-reactive T cells accounts, at least partially, for the disappearance of HY-reactive T cells from the periphery. Further, some step in the process requires signaling through the cytoplasmic domain of CD8 on the injected Ag-bearing cells.

Animals↗
PubMed1995

CD28-mediated signaling in vivo prevents activation-induced apoptosis in the thymus and alters peripheral lymphocyte homeostasis.

Activation of T lymphocytes can result in a functional immune response, anergy or apoptosis. Functional T cell activation requires the interaction of the TCR with Ag presented by MHC molecules on APC concurrent with appropriate interactions between cell surface accessory molecules. Interestingly, the level of CD28 expression is regulated during T cell development as well as during T cell activation and proliferation, suggesting that CD28 could play a role in determining the outcome of activation of TCR during T cell ontogeny. We identify, herein, a novel function of murine CD28 in the regulation of activation-induced apoptosis in thymocytes. In vivo, or combined in vivo and in vitro treatment with mAbs to CD28 prevents apoptosis of CD4+CD8+ thymocytes induced by Abs to the TCR complex. Prolonged administration of anti-CD28 Abs increased the number of both CD4+CD8- and CD4-CD8+ T cells in the thymus, while the number of CD4+CD8+ T cells is relatively unchanged. Furthermore, this treatment leads to a dramatic enlargement of peripheral lymphoid organs characterized primarily by the expansion of B cells. The number of CD4+CD8- T cells in the spleen of anti-CD28-treated mice is also moderately increased, while the number of CD4-CD8+ cells is relatively unchanged.

Animals↗
PubMed1995

Rapamycin enhances apoptosis and increases sensitivity to cisplatin in vitro.

Apoptosis can be regulated in a number of different systems by the actions of cytokines. Rapamycin has been shown to exert its effects on growth factor-induced cell proliferation, at least in part, by blocking the activation of the p70 S6 kinase and thus preventing the downstream signaling process, such as the activation of the members of the cdk family. To determine whether this pathway plays a role in the regulation of apoptosis, we assessed the effect of rapamycin on apoptosis induced by interleukin 2 deprivation in murine T-cell lines, by T-cell receptor ligation in a murine T-cell hybridoma, by enforced c-myc expression in murine fibroblasts, and by corticosteroids in murine T-lymphoma cell lines. Although rapamycin did not induce apoptosis on its own, rapamycin augmented apoptosis in each of the cell lines used as indicated by increased genomic DNA fragmentation, decreased cell viability, and characteristic apoptotic changes in morphology. These results suggest that a signal transduction pathway(s) inhibited by rapamycin plays an important role in the susceptibility of cells to apoptosis. Many chemotherapeutic agents kill cancer cells through the induction of apoptosis. Strikingly, rapamycin increased the ability of the alkylating agent, cisplatin, to induce apoptosis in the human promyelocytic leukemia cell line HL-60 and the human ovarian cancer cell line SKOV3. These data suggest that a signal transduction pathway, likely related to p70 S6 kinase, inhibited by rapamycin may be an important component of the pathway which prevents cell death in many cell lineages and also indicate that rapamycin has the potential to augment the efficacy of selected anticancer therapies.

Animals↗
PubMed1995

Early detection of apoptosis in defined lymphocyte populations in vivo.

Recently many methods have been developed for the detection of apoptosis. However, all of them have some limitations in determining whether specific subsets of cells are undergoing apoptosis. In this paper we describe a technique in which one simultaneously stains for cell surface markers with fluorescent monoclonal antibodies and for nuclear DNA breaks using in situ DNA nick translation detectable by fluorescence. The method has been evaluated using radiation-induced programmed cell death of lymphocytes and compared with some other techniques. It was found that the method is very specific and sensitive. It enabled us to enumerate apoptotic cells at the single cell level and simultaneously determine their subset-specific surface antigen profile both in vivo and in vitro. It is also insensitive to nicks present in replicating cells. Our data suggest that this method may be useful for the study of programmed cell death of antigen specific T cells in vivo.

Animals↗
PubMed1995

The NK1.1 antigen in NK-mediated F1 antiparent killing in vitro.

NK cells in lethally irradiated F1(A x B) hybrid mice can reject parental A or B strain bone marrow cells, a phenomenon called "hybrid resistance." The recognition mechanism used by the NK cells remains unknown. Our laboratory has previously described an in vitro model for hybrid resistance, and we have used it here to test whether the NK surface marker, NK1.1, is involved in such recognition. We found that 1) an anti-NK1.1 mAb (PK136) inhibited F1 lymphokine-activated killer (LAK) antiparent lysis if the LAK expressed NK1.1. Other mAb, even a mAb such as 2B4 that recognizes the same LAK as PK136, did not produce inhibition. 2) The F(ab')2 fragment of PK136 also inhibited lysis. 3) F1 LAK generated from athymic nude mice were as effective antiparent killers as LAK from normal mice and were equally inhibitable by anti-NK1.1 mAb, strengthening the conclusion that killing is mediated by NK cells and not T cells. 4) As previously shown by others, addition of anti-NK1.1 mAb to a mixture of NK1.1+ LAK cells and NK-resistant FcR+ cells allowed lysis of the FcR+ cells via "redirected lysis," in which the anti-NK1.1 mAb binds to NK1.1 on the NK cells and FcR on the target cell. The ability of anti-NK1.1 mAb to inhibit direct lysis and enhance redirected lysis is most consistent with NK1.1 being a receptor involved in NK activation.

Animals↗
PubMed1995

Allo-skin graft rejection, tumor rejection and natural killer activity in mice lacking p56lck.

Mice lacking the p56lck molecule (lck -/-) have a profound block in the maturation of thymocytes and a greatly reduced number of peripheral mature T cells. To analyze further the functions of the T cells developed in lck -/- mice in vivo, we evaluated the ability of lck -/- mice to reject allo-skin grafts and methylcholanthrene (MCA)-induced syngeneic fibrosarcoma, and also examined the biological activity of lck -/- natural killer (NK) cells. Mice lacking p56lck failed to reject skin grafts from either MHC-disparate or minor-histocompatibility-different donors, even after they had been primed with donor spleen cells. They also failed to reject the MCA-induced immunogenic syngeneic fibrosarcoma, MC57X. NK activity in mice lacking p56lck was normal, and there were no differences in the NK cell activation induced by poly(I).poly(C) stimulation or interleukin-2 stimulation (lymphokine-activated killer induction) between mice lacking p56lck and their immunocompetent heterozygous littermates. NK cells lacking p56lck mediated a normal antibody-dependent cell-mediated cytotoxicity (ADCC) response. The results of this study indicate that the loss of p56lck severely impairs the effectors of the immune system which mediate the rejection of allo-skin grafts and syngeneic tumors. The normal NK activity in lck -/- mice suggests that p56lck is not required for the development and activation of NK cells.

Animals↗
PubMed1995

Factors which influence alterations of phosphates and pH in exercising human skeletal muscle: measurement error, reproducibility, and effects of fasting, carbohydrate loading, and metabolic acidosis.

Previous studies have shown considerable variability in the metabolic response of human skeletal muscle during a standardized exercise protocol. The goal of these studies was to investigate the factors responsible for the broad range of metabolic changes produced by fatiguing exercise. Experiments were performed to quantitate the measurement error of 31P nuclear magnetic resonance spectroscopy of human muscle, the reproducibility of changes within a single subject, and the effects of fasting, carbohydrate loading, and metabolic acidosis. The results show that none of these factors appear to be responsible for the wide variation between subjects. However, the effects of training and genetic factors were not investigated and are likely to be responsible for the substantial variability between subjects.

Acidosis↗
PubMed1995

Evidence of an abnormal intramuscular component of fatigue in multiple sclerosis.

The goals of this study were to investigate muscle fatigue in patients with multiple sclerosis (MS), and to determine the relationships between muscle fatigue, clinical status, and perceived fatigue. The fatigability of the anterior tibial muscle was quantitated in patients and controls during 9 min of intermittent stimulation (used to eliminate central sources of muscle fatigue). During exercise, the decline in tetanic force, phosphocreatine, and intracellular pH was greater in patients than in controls. The compound muscle action potential amplitude did not decrease during exercise, indicating that there was no failure of neuromuscular transmission during fatigue. Thus, the excessive fatigue in MS developed from sources beyond the muscle membrane. Following exercise, the recovery of tetanic force was delayed in patients (a pattern that suggests abnormal excitation-contraction coupling), whereas the recovery of metabolites was complete in both groups. Muscular fatigue was correlated with clinical disability but not with perceived fatigue. These results suggests that fatigue in MS has both central (perception, upper motor neuron dysfunction) and peripheral (impaired metabolism and excitation-contraction coupling) components.

Adult↗
PubMed1995

Fatigue brought on by malfunction of the central and peripheral nervous systems.

Increased fatigability necessarily occurs in every patient with muscle weakness, regardless of whether the latter is due to a central or peripheral neurological disorder. The tendency for disuse to increase fatigability, as a secondary phenomenon, must also be considered; disuse affects both motoneuron recruitment and the biochemical and physiological properties of the muscle fibers. In recent studies impaired recruitment has been observed in postpolio patients, while patients with multiple sclerosis or spinal cord injury have shown, in addition, altered neuromuscular function. Findings are also presented in ALS and the chronic fatigue syndrome. In general, the most dramatic increases in fatigability take place in disorders of the peripheral nervous system and almost any cell component can be incriminated. There is a need to study fatigability systematically in neurology and rehabilitation.

Central Nervous System Diseases↗
PubMed1995

Decreased cysteine and proline synthesis in parenterally fed, premature infants.

Little is known about the amino acid (AA) biosynthetic capacity and requirements of premature infants. This study assessed the synthesis of seven biochemically nonessential AA from a universal precursor, glucose, in stable, parenterally fed, premature neonates. Seven infants (six boys, one girl) were studied at a mean age of 6.3 +/- 0.6 (SEM) days; mean gestational age was 29.7 +/- 1.3 (SEM) weeks, and mean birth weight was 1,222.8 +/- 176.5 (SEM) grams. All infants were parenterally fed a mixture of 7.5% to 12.5% dextrose and 2.2% Trophamine, with or without lipid. Mean caloric intake was 93 +/- 8.4 (SEM) kcal/kg/d, and total AA intake was standardized at 2.86 g/kg/d AA, plus supplemental cysteine (30 mg/g AA/d). Each infant received a 4-hour continuous, unprimed intravenous infusion of a stable isotope tracer of D(-)[U13C] glucose (200 mg/kg). Blood samples were obtained before and at the end of the infusion. Conversion of the glucose tracer into seven biochemically nonessential AA (cysteine [Cys], proline [Pro], aspartate [Asp], serine [Ser], glutamate [Glu], alanine [Ala], and glycine [Gly]) was assessed by measuring their isotopic enrichment in plasma, using gas chromatography/mass spectrometry (GC/MS), and expressed as mole percent excess (MPE) (mean +/- SEM). The isotopic enrichment of plasma glucose was also measured using GC/MS. Free plasma AA concentrations (mean +/- SD) were measured using an automated amino acid analyzer. Mean MPE for M + 1, M + 2 and M + 3 Cys, and for M + 1 and M + 3 Pro were not significantly different from 0; M + 2 Pro barely achieved statistical significance (P = .048).(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1995

A new stable isotope tracer technique to assess human neonatal amino acid synthesis.

The amino acid (AA) synthetic ability and requirements of human infants are undefined. A stable isotope tracer technique was employed in neonates to assess conversion of uniformly labeled 13C glucose into biochemically nonessential AA (NEAA). Ten neonates (5 males, 5 females) were studied at a mean age of 7 +/- 2.0 (SEM) days. The mean gestational age was 35.5 +/- 1.1 weeks, and the mean weight at time of study was 2,191 +/- 181 g. Six infants were fed enterally, and four received only intravenous 10% dextrose (D10W). Blood samples were obtained before, and 30, 60, and 120 minutes after an orogastric bolus of D-[U-13C]glucose (100 mg/kg). The conversion of glucose carbon into seven NEAA was assessed by measuring their isotopic enrichments in plasma, using gas chromatography/mass spectrometry (GC/MS), and was expressed as mole percent excess (MPE), with detectable MPE defined as > or = 0.2. The isotopic enrichment of plasma glucose also was measured using GC/MS. Free plasma AA concentrations were assayed using an automated AA analyzer and expressed in micromoles per liter. The mean glucose enrichment was 9.33 +/- 1.8 MPE (range, 5.82 to 13.48). Detectable 13C-labeling of the NEAA was observed as follows: Glu in 100% of infants; Gly, 100%; Ala, 90%; Ser, 80%; Asp, 70%; Cys, 60%; and Pro, 60%. Detectable Pro enrichment was observed in none of three premature infants on D10W. Free plasma Cys concentration was markedly lower than normal (19.8 v 86 mumol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1995

Muscular fatigue in Duchenne muscular dystrophy.

We used a 4-minute sustained maximum voluntary contraction to investigate fatigability of the anterior tibial muscle in eight healthy boys and 11 boys with Duchenne muscular dystrophy (DMD) (ages 5 to 10 years). Before exercise, the force generation of dystrophic muscle and the compound muscle action potential amplitude were lower and half-relaxation time of the tetanus was longer in patients than in controls. During exercise, the decline in tetanic force and potentiation in twitch tension were similar in both groups. However, during exercise, there was less decline in maximum voluntary contraction and less added force in DMD patients, suggesting that there was less central fatigue in patients than in controls. Thus, patients with DMD and controls have similar intramuscular fatigability and excitation-contraction coupling, and central activation in patients is functioning as well as or better than in healthy controls.

Adolescent↗
PubMed1995

Physiology of fatigue in amyotrophic lateral sclerosis.

We investigated the mechanisms of muscle fatigue in ALS. In the muscles of ALS patients and healthy control subjects, we examined (1) fatigue using measurements of muscle force, (2) energy metabolism using phosphorus-31 magnetic resonance spectroscopy, and (3) activation using neurophysiologic measures and MRI. During 25 minutes of intermittent isometric exercise of the tibialis anterior muscle, both maximum voluntary and tetanic force declined more in patients than in controls, indicating greater fatigability in ALS. There was a similar decline of voluntary and tetanic force, suggesting that much of the fatigue was not central. Evoked compound muscle action potential amplitudes were preserved during exercise in both groups, indicating no failure of neuromuscular transmission; this result suggests that the source of fatigue was not at the neuromuscular junction or within the muscle membrane. In spite of greater fatigability, changes during exercise in energy metabolites and proton signal intensity tended to be less in ALS patients compared with controls, suggesting impaired muscular activation. We conclude that the greater muscle fatigue in ALS patients results from activation impairment, due in part to alterations distal to the muscle membrane.

Action Potentials↗
PubMed1995

Linkage of Miyoshi myopathy (distal autosomal recessive muscular dystrophy) locus to chromosome 2p12-14.

Miyoshi myopathy (MM) is a young-adult-onset, autosomal recessive distal muscular dystrophy initially affecting the plantar flexors. We analyzed 12 MM families, five with consanguineous marriage, for chromosomal linkage using polymorphic microsatellite DNA markers to map the MM gene. A significant lod score was obtained with the 2p12-14 locus D2S291 (Zmax = 15.3 at theta = 0). Two additional 2p12-14 markers, D2S286 (Z = 10.7 at theta = 0) and D2S292 (Z = 7.2 at theta = 0.05), also gave significant lod scores. These markers will be useful for diagnosis of symptomatic and presymptomatic patients, prenatal and carrier diagnosis of family members carrying MM, and ultimately identification of a gene responsible for MM.

Adolescent↗
PubMed1994

Specific T cell receptor gene rearrangements at the site of muscle degeneration in Duchenne muscular dystrophy.

Mononuclear cells infiltrate degenerating muscles of Duchenne muscular dystrophy (DMD) patients. Using a quantitative PCR, we first characterized the T cells infiltrating muscle biopsies from six DMD patients. High levels of TCR V beta 2 transcripts were observed in DMD muscle tissue. TCR V beta 2 transcripts from seven DMD patients and five controls were sequenced, and the VDJ junctional region analyzed in 166 clones. One specific amino acid motif, RVSG, was found in the third complementary determining region (CDR3) of TCR V beta 2 chains in samples from five DMD patients, but not in controls. A specific immune reaction at the site of tissue degeneration may play an important role in the pathogenesis of DMD.

Amino Acid Sequence↗