Biomedical subjects

R G Miller

Publications and source records attributed to R G Miller.

At least 145 records · Page 8Linked to original sources

PubMed1993

Ultrastructural changes of articular cartilage chondrocytes associated with freeze-thawing.

In an attempt to justify the use of cryopreserved versus fresh articular cartilage (AC) allografts, we used transmission electron microscopy (TEM) to study the ultrastructure of fresh versus frozen-thawed AC with or without a dimethyl sulfoxide (DMSO) treatment. AC explants were cut aseptically from the femoral condyles of healthy, mature rabbits when they were killed. Half of all explants were incubated in Ham's F-12 medium, supplemented with antibiotics and with or without 7.5% DMSO, frozen to -80 degrees C, stored for 24 h, and thawed rapidly. These, and the control explants, were fixed with glutaraldehyde, paraformaldehyde, and acrolein in cacodylate buffer. Sections were stained for acid phosphatase (APase), postfixed with osmium, embedded, and examined under TEM. The typical organization of the matrix and the cells was noted in control sections. The chondrocytes contained intact nuclei, organelles, and discrete plasma membrane. Although some endoplasmic reticula and nuclear membrane appeared intact, distinct ultrastructural changes were observed in frozen-thawed samples treated with DMSO. These changes included condensation of chromatin, large lipid droplets, partly disrupted plasma membrane, and pericellular precipitation of APase-positive crystalites. In sections not treated with DMSO, the cytoplasm was extensively vacuolated and no distinct organelles could be detected in the chondrocytes. Little difference was noted between the matrix organization of fresh or frozen-thawed samples. Our results suggest that distinct ultrastructural changes occur in the chondrocytes following freeze-thawing of intact AC and that DMSO pretreatment may contribute to improvement in the cryopreservation of AC.

Acid Phosphatase↗

PubMed1993

Dissociation of [H+] from fatigue in human muscle detected by high time resolution 31P-NMR.

Previous in vivo studies of skeletal muscle fatigue have demonstrated significant relationships between the decline of muscular force and changes in muscle metabolism. However, these studies performed measurements over relatively long time intervals or during steady state exercise, thereby obscuring rapid metabolic changes occurring at the onset of exercise and recovery. To overcome these limitations, fatigue of human calf musculature during sustained isometric foot plantar flexion was quantified continuously as the decline in maximal voluntary contraction force (MVC), while concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), intracellular free hydrogen ion (H+), and monovalent phosphate (H2PO4-) were simultaneously measured at 2-second intervals by 31P nuclear magnetic resonance. The first major finding was that [H+], which has been thought to be a mediator of muscle fatigue, actually declined during the first 10 seconds of exercise when force was declining and rose immediately postexercise, when force partially recovered. Second, the correlations of [H+], [H2PO4-] and Pi with MVC during the first minute of exercise were determined to be curvilinear and not linear as previously suggested. Furthermore, using either a linear or curvilinear regression model, [H2PO4-] and Pi demonstrated a closer correlation to MVC than [H+] during the first minute of exercise. Thus, these results reveal nuances in the relationships of MVC to metabolites previously undetected by low time-resolution measurements. These findings suggest that during sustained isometric exercise, rising [H+] is not likely to be the sole mechanism of muscle fatigue and are consistent with the view that a rise of Pi or [H2PO4-] is a major causation factor in force reduction.

Adolescent↗

PubMed1993

Characterization of murine lymphokine-activated killer cell cultures separated according to cell size.

Incubation of murine splenocytes in high concentrations of the lymphokine interleukin 2 leads to the development of cytotoxic effector cells termed lymphokine-activated killer cells. Two populations of blast cells develop within these cultures, one of T origin and one of NK origin. The T blasts are CD8+, FcR gamma III- and exhibit slightly increased forward angle light scatter (FSC) and side angle light scatter (SSC). They have little or no oncolytic activity against the tumor target YAC-1. The NK blasts are CD8-, FcR gamma III+ and exhibit markedly increased FSC and SSC. They contain virtually all the lytic activity against the tumor target YAC-1. Cultures were split into multiple fractions on the basis of sedimentation velocity, which separates cells primarily on the basis of cell size. The sedimentation separation resolved both T and NK cells into resting (G0) small lymphocytes and cycling blast cells. NK blasts sedimented much more rapidly than all CD8+ cells such that most of the oncolytic activity could be separated relatively free of CD8+ cells.

Animals↗

PubMed1993

Auger electron spectroscopy of dentin: elemental quantification and the effects of electron and ion bombardment.

Auger electron spectroscopy was used in this investigation to quantify the elemental composition of various dentin surfaces. The surfaces included the "smeared layer," produced by abrasion, as well as surfaces prepared by fracturing in vacuum and in air. Quantification was aided by spectra obtained from standard samples of the two main components of dentin, hydroxyapatite, and collagen. Experimental conditions were found whereby the samples could be analyzed without conductive coatings. Ion sputter etch rates were measured for dentin, hydroxyapatite, and collagen. Under the conditions used here, it was observed that collagen etched at a rate approximately five times greater than hydroxyapatite. Auger spectra of the sputtered surfaces indicated significant ion beam-induced sample changes in collagen and dentin; no significant changes were found with hydroxyapatite. The results of this investigation demonstrate that Auger electron spectroscopy can be used to characterize dentinal surfaces and that ion sputtering can cause changes in the surface composition of dentin.

Collagen↗

PubMed1993

The correlation of prolonged survival of maternal skin grafts with the presence of naturally transferred maternal T cells.

Hypoimmune responses towards noninherited maternal MHC antigens (NIMA) have been observed on both the humoral and cellular level in humans. The mechanism involved is not known but is thought to involve transfer of material from mother to child. Here we use a mouse model to test this hypothesis. Mice exposed to NIMA either in utero and/or via breast milk showed enhanced survival of maternal skin grafts and also had maternal lymphocytes in their lymph nodes. Survival of the maternal skin graft correlated with the number of maternal T cells present and was not correlated with the number of maternal B cells. We propose that the transferred maternal T cells selectively inactivate host T cells capable of recognizing them.

Animals↗

PubMed1993

Slow force recovery after long-duration exercise: metabolic and activation factors in muscle fatigue.

To investigate the roles of metabolic and nonmetabolic factors in human muscle fatigue, two relatively brief nonexhausting exercise protocols that produced similar levels of moderate fatigue were used: short-duration exercise (SDE; 2-min sustained maximal voluntary contraction) and long-duration exercise (LDE; 15- to 20-min intermittent exercise). After exercise and during recovery, multiple potential mechanisms of fatigue were studied from measurements of voluntary, twitch, and tetanic forces; intracellular metabolites (using 31P-nuclear magnetic resonance spectroscopy); and electromyographic signals. The major findings were as follows. 1) After SDE, fatigue closely correlated with increased [Pi]. Both force and [Pi] recovered within approximately 5 min after exercise. 2) After LDE, force recovered slowly, with significant fatigue beyond 15 min after exercise; however, recovery of [Pi] was not slowed. 3) Electromyographic signals were little affected by either protocol. These findings suggest that multiple mechanisms contribute to moderate fatigue. Fatigue from SDE may arise primarily from metabolic mechanisms, whereas fatigue from LDE involves an additional slowly recovering nonmetabolic mechanism that may arise from impaired activation, beyond the cell membrane, at the level of excitation contraction-coupling.

Adult↗

PubMed1993

Phases of metabolism during progressive exercise to fatigue in human skeletal muscle.

The purpose of this study was to assess noninvasively both the "oxidative potential" and the sequence of metabolic events that occur during the transition from rest to fatigue in an isometrically exercising muscle. 31P-magnetic resonance spectroscopy was used to obtain continuous measures of intracellular phosphocreatine (PCr), inorganic phosphate (Pi), and proton concentration ([H+]) in the tibialis anterior muscle of eight healthy human volunteers during a progressive isometric exercise protocol. The exercise protocol consisted of 2-min stages, with a duty cycle consisting of 4 s of contraction and 6 s of relaxation, beginning at 10% of the force from an initial maximal voluntary contraction (MVC) and increasing by 10% to a final load of 80% MVC. An MVC was performed at the beginning of each stage; a decrease in MVC indicated fatigue. The initial linear slope of the relationship between force and Pi/PCr was used as an index of oxidative potential for the muscle. This initial slope ranged from 85 to 167% MVC/(Pi/PCr), indicating substantial variability of oxidative potential in these subjects. The changes in Pi/PCr and [H+] over time were best described with a bilinear fit of the individual data. The inflection point for each fit was defined as the point at which the slopes intersected. The Pi/PCr inflection point occurred at a similar value of Pi/PCr in all subjects [0.47 +/- 0.04 (SE)]. The Pi/PCr inflection point occurred significantly earlier (7.8 +/- 0.7 min) than the [H+] inflection point (9.3 +/- 0.4 min, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Central basis of muscle fatigue in chronic fatigue syndrome.

We studied whether muscle fatigue, metabolism, or activation are abnormal in the chronic fatigue syndrome (CFS). Subjects performed both an intermittent submaximal and a sustained maximal voluntary isometric exercise protocol of the tibialis anterior muscle. The extent of fatigue, metabolic response, and changes in both M-wave amplitude and twitch tension during exercise were similar in patients and controls. The response to systemic exercise was also normal in the patients. However, voluntary activation of the tibialis was significantly lower in the patients during maximal sustained exercise. The results indicate that patients with CFS have (1) normal fatigability and metabolism at both the intracellular and systemic levels, (2) normal muscle membrane function and excitation-contraction coupling, and (3) an inability to fully activate skeletal muscle during intense, sustained exercise. This failure of activation was well in excess of that found in controls, suggesting an important central component of muscle fatigue in CFS.

Adult↗

PubMed1993

Cyclosporine increases muscular force generation in Duchenne muscular dystrophy.

We investigated the effect of cyclosporine (CsA) on force generation in 15 boys with Duchenne muscular dystrophy (DMD) by obtaining monthly measures of tetanic force and maximum voluntary contraction (MVC) of both anterior tibial muscles. During 4 months of a natural history phase, both tetanic force and MVC declined significantly. During 8 weeks of CsA treatment (5 mg/kg/day), significantly increased tetanic force (25.8 +/- 6.6%) and MVC (13.6 +/- 4.0%) occurred within 2 weeks. The maximum mean increase during treatment was 35.2 +/- 5.9% (tetanic force) and 19.0 +/- 4.6% (MVC). Side effects from CsA, gastrointestinal and flu-like symptoms, were transient and self-limiting. Thus, as previously reported with prednisone, CsA increases muscular force generation in the anterior tibial muscles of DMD patients.

Action Potentials↗

PubMed1993

The fatigue of rapid repetitive movements.

Normal subjects (n = 7) performed rapid voluntary isometric contractions of the adductor pollicis and the tibialis anterior. Within the first minute of this low-intensity exercise, the speed of tension development slowed, accompanied by a prolongation in EMG burst duration. In contrast, electrically evoked contractions either became more rapid (twitch) or did not change (tetanus), suggesting a fatigue of central origin. 31P NMR measurements of high-energy phosphates showed that the relationship between the fall of maximum force and changes of phosphates and pH was similar to that produced by other high-intensity fatiguing exercise protocols. Thus, rapid movements produce fatigue through two major mechanisms. First, there is slowing of the speed of tension development which appears secondary to central fatigue. Second, the decline of muscle force is primarily attributable to changes in muscle pH or inorganic phosphate, which usually occurs only after high-intensity (but non-rapid) exercise.

Adenosine Triphosphate↗

PubMed1993

The natural history of amyotrophic lateral sclerosis.

Using 42 strength and functional assessments recorded monthly, the natural history of amyotrophic lateral sclerosis (ALS) is described in 167 patients (98 men, 67 women) followed in five medical centers in the western United States. The mean age at onset was 57.4 years, and symptoms were present for 2.64 years before study entry. Although there was a highly variable rate of decline within the group of patients, there were no differences in rate of decline by age or gender. Older patients and women were weaker on entry. Forty-eight patients died during the study. The median survival was 4.0 years for the study cohort but 2.1 years for newly diagnosed cases. Decline in pulmonary function most closely correlated with death. Our results emphasize the importance of considering clinical variability in planning clinical trials. One possible strategy is to identify and stratify patients by rate of decline in pulmonary function since prospectively identifying homogeneous subgroups allows investigators to substantially reduce sample size in therapeutic trials.

Adult↗

PubMed1993

Antigen-specific human T-cell colony formation in a liquid culture system.

We have developed a limiting dilution microculture T-cell cloning technique to generate antigen-specific T-cell clones. Cultures contained peripheral blood T-cells, irradiated autologous mononuclear cells (feeders), human AB serum and recombinant IL-2. Colonies were enumerated at 14 days. Under these conditions the cloning efficiency expressed as the frequency of proliferating cells in response to PHA, ranged from 1/1.07 to 1/3.35. In response to tetanus toxoid, the frequency of proliferating cells ranged from 1/415 to 1/5655. Average colony size ranged from 0.7 x 10(5) to 6.2 x 10(5) cells with a mean T-cell recovery of 2.5 x 10(5) cells per microculture. Restimulation of 14-day tetanus toxoid T-cell colonies revealed marked proliferation to PHA and tetanus toxoid but only background responses to PPD. These studies document the development of a rapid, antigen-specific liquid culture T-cell cloning system which is likely to detect T-cell clones that are representative of the original T-cell population. The clones are of sufficient size to be useful in studies of antigen cross-reactivity.

Cells, Cultured↗

PubMed1992

Effect of class I MHC binding peptides on recognition by natural killer cells.

NK cells can recognize and destroy a broad range of cells, including many tumor cells and virally infected cells, yet spare most normal cells. Identification of the target structure recognized by these cells has proved elusive. An attractive hypothesis is that, unlike B cells and T cells that recognize a specific foreign marker, NK cells respond to the absence of a "self" marker. Class I MHC molecules have been implicated as the self markers whose absence can trigger lysis. We show here that normal cells are lysed on incubation with IL-2-activated NK cells if peptides that can bind to the class I MHC molecules of the normal cells are also included in the assay, and speculate that this binding is somehow removing a self marker that normally protects a cell from lysis. NK cells were derived from splenocytes of young (5 to 8 wk old) athymic nude BALB/c (H-2d) or nude C57Bl/6 (H-2b) mice incubated with 1000 U/ml rIL-2, and target cells were derived from splenocytes of normal BALB/c or C57Bl/6 mice incubated with Con A. Peptides were from xenogeneic, viral, self, and mutated self protein sequences and included sequences specific for Kd, Kb, Db, and Ld. All peptides increased lysability of those targets to which they could bind.

Amino Acid Sequence↗

PubMed1992

Correlation between lymphocyte-induced donor-specific tolerance and donor cell recirculation.

Intravenous infusion of mice with major histocompatibility complex (MHC) incompatible lymphocytes can inhibit the response of recipient T cells capable of recognizing the injected cells, and can enhance survival of grafts sharing MHC with the injected cells. However, neither T cell inactivation nor graft survival enhancement is always achieved. This is particularly true for donor cells that are fully allogeneic (as compared to semiallogeneic) to the recipient. We show here that both donor-specific induced response reduction and graft survival enhancement are directly correlated with the ability of the injected lymphoid cells to persist in the recirculating lymphocyte pool of the host. Whether donor cells persist correlates inversely with the level of natural killer cell (NK) activity in the host. Fully allogeneic cells can only persist in hosts with low NK activity and can then induce response reduction. Both persistence and response reduction are abrogated by injection of the host with poly-I:C, a treatment that boosts host NK activity. The same treatment also destroys the ability of semiallogeneic injected cells to persist, to induce response reduction, and to enhance skin graft survival.

Animals↗

PubMed1992

Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen.

It has been well established that T cell tolerance to self Ag occurs primarily via clonal deletion of immature thymocytes in the thymus. Evidence also exists that there are additional mechanisms operative on mature T cells for establishing and maintaining tolerance in the periphery. To follow the fate of mature Ag-specific T cells in vivo, we used female transgenic mice, which contain a large population of male H-Y Ag-specific T cells that can be identified by immunostaining with mAb directed against CD8 and the transgenic TCR. H-Y Ag was introduced into these mice by injecting Ag-bearing male lymphocytes using conditions known to induce CTL precursor response reduction. The number of Ag-reactive CD8+ transgenic T cells in the periphery started to decrease after 2 days of in vivo exposure to male Ag. Decline was maximum (up to 80% of total) by 7 days, and stayed at this level for at least 6 wk. CD4+ cells and those CD8+ cells that did not carry the transgenic TCR were not affected. Most or all of the remaining Ag-reactive CD8+ cells in the periphery were fully responsive when stimulated by male Ag in vitro. Maturation of transgenic T cells in the thymus of injected mice remained the same as that of control animals. Our data provide direct evidence that mature Ag-reactive CD8+ cells are susceptible to clonal deletion in the periphery when exposed to the Ag in vivo. These findings suggest the presence of two types of APC in the periphery: stimulatory APC (e.g., macrophages and dendritic cells) required for initiating an active immune response; and functionally deleting APC (or veto cells) capable of deleting mature T lymphocytes that recognize Ag presented on their surface. Functionally deleting APC that present self Ag to peripheral T cells may provide a fail-safe mechanism against autoreactive cells that escaped deletion during differentiation in the thymus.

Animals↗

PubMed1992

Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation.

Gene delivery by transplantation of normal myoblasts has been proposed as a treatment of the primary defect, lack of the muscle protein dystrophin, that causes Duchenne muscular dystrophy (DMD), a lethal human muscle degenerative disorder. To test this possibility, we transplanted normal myoblasts from a father or an unaffected sibling into the muscle of eight boys with DMD, and assessed their production of dystrophin. Three patients with deletions in the dystrophin gene expressed normal dystrophin transcripts in muscle biopsy specimens taken from the transplant site one month after myoblast injection. Using the polymerase chain reaction we established that the dystrophin in these biopsies derived from donor myoblast DNA. These results show that transplanted myoblasts persist and produce dystrophin in muscle fibres of DMD patients.

Adult↗

PubMed1992

Hybrid resistance in vitro. Possible role of both class I MHC and self peptides in determining the level of target cell sensitivity.

NK cells of F1 hybrids frequently exhibit an enhanced capacity to reject semisyngeneic parental bone marrow grafts, a phenomenon known as hybrid resistance. Attempts to define the mechanism whereby this occurs have been hampered by factors inherent in the use of an in vivo model, including the host immune response, microenvironment, immune cell trafficking patterns, and host irradiation. We show here that IL-2-activated NK cells (lymphokine-activated killer cells) can be used to establish an in vitro model that appears to mimic hybrid resistance. These effectors lyse parental lymphoblast targets in a pattern consistent with that observed in vivo, bear NK not T cell surface markers, and recognize target structures mapping to the MHC. By using this model, we then demonstrate that sensitivity to lysis of syngeneic F1 lymphoblasts can be augmented by exposing the targets to conditions that have been reported to permit the exchange of endogenous class I-bound self peptides for exogenous foreign sequences. These data suggest that both MHC class I molecules and the "self" peptides they bind contribute to determining the susceptibility of a target to hybrid resistance, and the data are discussed in the context of a model of NK recognition in which the sensitivity of a target to NK lysis is mediated by a complex of the class I MHC molecule and the self peptides it samples from the intracellular peptide pool.

Animals↗

PubMed1992

Role of plasmapheresis in acute disseminated (postinfectious) encephalomyelitis.

Acute disseminated encephalomyelitis (ADEM) is a demyelinating central nervous system disease that is associated with high morbidity and mortality. Although the recognition of ADEM may be facilitated by newer imaging techniques, the optimal treatment of this disease remains uncertain. We describe 4 patients with severe ADEM who responded to treatment that included intensive plasmapheresis. Two of the patients were in coma at the time that plasmapheresis was instituted, and all 4 patients made an excellent recovery. Immunologic studies revealed increased serum IgA levels, increased circulating immune complex levels as measured by the Raji cell assay, and decreased numbers of T and B cells prior to treatment of ADEM. These abnormalities improved following plasma exchange. Plasmapheresis appears to be effective in reversing the neuropathologic process in ADEM. The role of this treatment modality in ADEM requires further evaluation in controlled clinical trials.

Acute Disease↗