Biomedical subjects
Publications and source records attributed to D A Thomas.
CD8+ cytotoxic lymphocytes, natural killer cells and lymphokine-activated killer cells depend primarily on the perforin/granzyme system to kill their targets, while CD4+ T cells utilize Fas and other mechanisms to induce cell death. The molecular mechanisms used by these pathways to induce target cell apoptosis may converge on common death substrates.
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In mice deficient in either lymphotoxin alpha (LT-alpha) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-alpha and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-alpha-deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-alpha-expressing cells required to establish organized FDC are derived from BM. The role of LT-alpha in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)-deficient BM cells into LT-alpha-deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-alpha-deficient recipient. Thus, expression of LT-alpha in the BM-derived cells, but not in the non-BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I-deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non-BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I-deficient BM was able to restore FDC organization and GC formation in LT-alpha-deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-alpha-expressing BM-derived cells and by TNFR-I-expressing non-BM-derived cells.
Patients with risk factors for VTE and developing signs or symptoms remotely consistent with DVT or PE should undergo formal testing. Ultrasound and IPG have good sensitivity and specificity in symptomatic DVT. V/Q lung scans should be obtained in all patients with suspected PE or proven DVT. A prior clinical assessment of probability, based on risk factors, history, physical examination, chest film, and arterial blood gas analysis, can aid in the interpretation of V/Q scans. Normal scans exclude PE. High-probability scans confirm PE if the clinical probability is at least intermediate. Nondiagnostic scans are common, but diagnosis in such cases can be aided by noninvasive leg studies. Heparin therapy should be started when there is suspicion of VTE. To avoid recurrence, a therapeutic aPTT of 1.5 to 2.5 times the control rate should be achieved as soon as possible after the diagnosis of VTE is confirmed. Thrombolytics are reserved for hemodynamically compromised patients. Warfarin should be administered to achieve an INR of 2.0 to 3.0 and should be continued for at least 3 months in patients with low risk of recurrence and probably for at least 6 months in all other patients.
This study tested the ability of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), to attenuate behavioral hyperalgesia in a rat model of neuropathic pain [Bennett, G.J. and Xie, Y.-K., Pain, 33 (1988) 87-107]. A mononeuropathy was produced by chronic constriction injury (CCI) of the sciatic nerve. Thermal hyperalgesia was assessed by a reduction of paw withdrawal latency to a noxious heat source. Following CCI, there was significant hyperalgesia in groups of rats treated with D-NAME (n = 7), an inactive isomer of L-NAME, saline (n = 7) or systemic L-NAME (n = 10). In contrast, when L-NAME was applied directly and continuously to the site of CCI (5.0 micrograms/microliter per h for up to 2 weeks) via an osmotic pump implanted at the time of the injury, no significant thermal hyperalgesia was observed (n = 8). The results suggest the involvement of nitric oxide in the development and maintenance of thermal hyperalgesia in a rat model of neuropathy. The blockade of nitric oxide production at the site of injury may provide a new approach for treatment of neuropathic pain.
This study examined the sites in the central nervous system at which subcutaneously-administered R(+)-baclofen hydrochloride (baclofen), the most active isomer of this prototypic gamma-aminobutyric acid (GABA)B receptor agonist, acts to produce antinociception in the rat. To determine whether baclofen acts in the spinal cord, either saline or the GABAB receptor antagonist CGP 35348 was injected intrathecally in rats pretreated 24 min earlier with 1 or 3 mg/kg s.c. baclofen. Intrathecal (i.t.) injection of 3 or 10 micrograms of CGP 35348 antagonized the increase in tail-flick and hot-plate latency produced by either dose of baclofen. To determine whether baclofen acts at sites in the ventromedial medulla (VMM), either saline or CGP 35348 was microinjected in the nucleus raphe magnus or nucleus reticularis gigantocellularis pars alpha of rats pretreated 24 min earlier with 1 or 3 mg/kg s.c. baclofen. Microinjection of 0.5 or 3 micrograms of CGP 35348 at sites in the VMM produced at best only a very modest attenuation of the antinociceptive effects of baclofen. These data suggest that systemically-administered baclofen acts at sites in both the spinal cord and the VMM, but that its antinociceptive effects are likely to be mediated to a greater extent by a spinal, rather than medullary site of action. However, a definitive comparison of the relative contribution of GABAB receptors in these two regions is precluded by differences in the diffusion and concentrations of the antagonist in the spinal cord and brainstem. Finally, microinjection of 0.5 or 3.0 micrograms of CGP 35348 in the nucleus raphe magnus or nucleus reticularis gigantocellularis pars alpha of saline-pretreated rats did not alter tail-flick or hot-plate latency. This finding suggests that, unlike GABAA receptors, GABAB receptors do not mediate the tonic GABAergic input to neurons in these nuclei.
The toxicity of cadmium with regard to the vegetative reproduction of duckweed, Lemna gibba, grown in sterile culture, was determined. The EC50 was found to be 800 ppb. Duckweed grown in 2.24 ppm cadmium (supplied as cadmium nitrate) for 7 days accumulated 98.5% of the available cadmium from the growth medium. Plants that had been grown for 7 days in 2.24 ppm cadmium and control plants were fed to red swamp crayfish, Procambarus clarkii, for 14 days. The concentrations of cadmium were measured in hepatopancreata and muscles of crayfish on Day 0 and in crayfish fed duckweed grown in cadmium for 14 days. Accumulation of this metal in hepatopancreata increased 26-fold, i.e., 176.80 ppb on Day 0 to 4657.56 ppb on Day 14, and in muscles almost 7-fold, i.e., 6.75 ppb on Day 0 to 46.28 ppb on Day 14. Crayfish fed cadmium-containing duckweed demonstrated inhibition (55% after 14 days of feeding) of acetylcholinesterase activity in their central nervous tissue compared to crayfish fed cadmium-free duckweed. The ovarian index and total lipids content in the ovaries of crayfish fed cadmium-containing duckweed demonstrated significant increases on Day 14.
The dynamics of appetitive-aversive interaction theory were assayed in an experiment where excitors and inhibitors from the same and different incentive classes were compounded. Excitatory and inhibitory incentive properties acquired by the discriminative stimuli resulted from the reinforcement differences between components of four-component multiple training schedules, with the influence of competing peripheral responses factored into the design. Rats' barpressing was maintained in tone and in click by food or free-operant shock avoidance; extinction was programmed in click-plus-light and in the simultaneous absence of tone, click, and light. In Test 1, an excitor that occasioned operant responding and an inhibitor that occasioned response cessation were compounded. Here, compounding an inhibitor with an excitor from the same incentive class reduced responding significantly more than did compounding an inhibitor with an excitor from the other incentive class. In Test 2, the compounds consisted of elements that individually occasioned operant responding. Here, compounding stimuli that were excitors from different incentive classes appeared to create reciprocal inhibition that counteracted the additive effects produced when these stimuli were excitors from the same incentive class. Predictions from appetitive-aversive interaction theory and Weiss' two-factor model of stimulus control were confirmed within this experimental design, where all conditioning was a by-product of the behavioural contingencies programmed on the operant baselines.
The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5-transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF-alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.
The effect of heparin, aspirin, and recombinat tissue-type plasminogen activator (rt-PA) on TP-9201 pharmacokinetics and pharmacodynamics was investigated in beagles. Animals received TP-9201, an Arginine-Glycine-Aspartic acid (RGD)-containing synthetic peptide glycoprotein (gp)IIbIIIa antagonist as a bolus of 0.31 mg/kg, followed by a 4-h infusion of 0.5 mg/kg/h. rt-PA was administered as a modification of the weight-adjusted standard regimen. Heparin was administered as a bolus followed by an infusion producing a 1.5- to 2-fold increase in the activated prothromboplastin time (aPTT) above baseline values. Aspirin was administered orally, approximately 24 and 2 h before TP-9201. TP-9201 had a plasma clearance of 9.9 +/- 2 ml/min/kg and a volume of distribution that was larger than plasma volume. Administration of heparin and aspirin with TP-9201 did not affect the clearance of TP-9201, whereas rt-PA resulted in a faster clearance (p = 0.05). Whether the faster clearance is physiologic or a result of rt-PA interference in the TP-9201 assay is unclear. TP-9201 completely inhibited ADP-mediated platelet aggregation. After discontinuation of TP-9201, recovery of platelet aggregation had a half life (t1/2) of 2-3 h and was complete < or = 24 h. Coadministration of heparin did not interfere with TP-9201 pharmacodynamics, whereas aspirin and rt-PA slowed the recovery of platelet aggregation. The template bleeding time profile for the TP-9201-treated animals was similar to that of the aspirin-treated animals.
We wished to investigate the effects of sleep deprivation on sleep, arousal propensity, respiratory events and peripheral chemoresponses in healthy infants, since these effects might be relevant to mechanisms concerned with some cases of sudden infant death syndrome. Paired observations were made overnight during natural sleep and following sleep deprivation, in a randomized fashion, in 15 healthy infants aged 78 (7) days (mean (SD)). Polysomnograms were recorded and sleep was scored using Anders' criteria. Respiratory events were categorized into central, mixed and obstructive apnoeas. Peripheral chemoresponses were measured during quiet sleep from the respiratory response to two-breath alternations in fractional inspiratory oxygen (F1, O2) (0.42 and 0.00). Arousal propensity was determined from awakening and arousal thresholds to graded photic and auditory stimuli during quiet sleep, and from spontaneous awakenings and limb movements. Compared with natural sleep, following sleep deprivation infants maintained a greater proportion of quiet sleep (39 vs 44%). There was no measurable change in arousal propensity. During quiet sleep, obstructed breathing events tended to be more common after sleep deprivation (0.1 vs 0 events.h-1) and the expiratory time during baseline breathing increased significantly (1.27 vs 1.58 s) although the decrease in respiratory rate was not significant (32 vs 30 breaths.min-1). Peripheral chemoresponses altered significantly, alternations in tidal volume/inspiratory time (VT/tI) as a measure of inspiratory drive increased after sleep deprivation (9 vs 21%). In conclusion, following short-term sleep deprivation in infancy, respiratory control alters, peripheral chemoresponsiveness increases in magnitude and the timing of baseline breathing alters, without any detectable alteration in arousal propensity. This state may be associated with an increased vulnerability to obstructive respiratory events.
The aim of this study was to develop a minimally invasive and reliable method for measuring peripheral chemoresponsiveness to oxygen in infants, and to establish baseline data from normal infants at 12 weeks of age. Two-breath alternations in fractional inspired oxygen (FI,O2), switching between 0.42 to 0.00 were given for 2 min periods via a face mask (held close to the face but without contact) to 18 healthy infants during quiet sleep. End-tidal oxygen concentrations alternated between 21 and 11%. Instantaneous minute ventilation (V'E) and its components tidal volume (VT), respiratory frequency (fR) inspiratory and expiratory times (tI and tE), inspiratory flow (VT/tT), and inspiratory duty cycle (tI/ttot) were measured by respiratory inductance plethysmography. Two-breath alternations in each of the ventilatory components were matched with the corresponding alternating end-tidal oxygen record and compared with contiguous pre- and post-test data obtained in control periods of air breathing. Alternations in all ventilatory components except fR changed significantly during FI,O2 alternations; VT 26%, tE-8%, VT/tI 18%, tI/ttot 11% and V'E 28% of baseline values. Within and between infant variances are reported for the individual components of ventilation. Differences among infants were best detected by alternations in V'E; within infant variance 76, between infant variance 171. We conclude that the test described is a safe, reliable and relatively easily applied method of measuring peripheral chemoresponsiveness, which is suitable for clinical application in infancy.
Nerve growth factor (NGF) reverses some effects of axotomy and prevents toxic neuropathy in adult rodents. We tested the effect of NGF on behavioral hyperalgesia resulting from a chronic constriction injury (CCI) of the sciatic nerve in the rat [5]. CCI rats exhibit thermal hyperalgesia as demonstrated by a reduction of paw withdrawal latency to a noxious thermal stimulus applied to the paw on the side of injury. The mechanical sensitivity of the ipsilateral hindpaw, assessed with von Frey filaments, was also significantly increased. There were no significant changes in nociceptive thresholds on the contralateral side. When NGF was infused directly on the ligated nerve via an osmotic pump (0.5 microgram/microliter/h for 7 days) immediately after the ligation, thermal hyperalgesia was abolished from postoperative days 5 up to at least two weeks. The CCI-induced decrease in mechanical threshold was also abolished by NGF. However, NGF had only a minor effect on the abnormally long response duration, a second measure of mechanical sensitivity, to the mechanical stimulus. Delayed infusion of NGF four days after the ligation failed to block hyperalgesia. Infusion of NGF on the sciatic nerve of rats that had no CCI had no significant effect on paw withdrawal latency. Infusion of anti-NGF antiserum did not enhance hyperalgesia in CCI rats. These results suggest that alterations in neurotrophic factor(s) contribute to the development of behavioral hyperalgesia in an animal model of neuropathy and that NGF may have therapeutic value in the treatment of neuropathic pain in humans.
It has been proposed that opioids act at the level of the medulla to produce facial pruritus. Supporting this hypothesis, microinjection of mu-opioid receptor agonists into the medullary dorsal horn (MDH; trigeminal subnucleus caudalis) of monkeys produces facial scratching behavior. The present study sought to establish a rodent model of opioid-induced facial pruritus. To this end, morphine (0.1, 0.3 or 1.0 micrograms/0.2 microliter) or saline (0.2 microliter) was unilaterally microinjected into the MDH of male Sprague-Dawley rats. Behavior for the 20 min preceding and the 80 min after this microinjection was videotaped. Morphine produced dose-dependent increases in facial scratching behavior ipsilateral to the microinjections with the peak effect at 30-40 min after microinjection. Facial scratching continued for the entire 80 min post-microinjection test period. Morphine also produced a lesser degree of facial scratching contralateral to the microinjections. Increases in facial scratching ipsilateral to the microinjection of 0.3 microgram morphine into the MDH were attenuated by 0.4 mg/kg s.c. naloxone. These findings support the hypothesis that the MDH is a critical site of action of opioid agonists in producing facial pruritus.
L-Canavanine (CAV) is a potent L-arginine antagonist, produced by legumes such as the jack bean, Canavalia ensiformis. CAV is cytotoxic to MIA PaCa-2 human pancreatic cancer cells. We sought to determine whether CAV's efficacy as an anticancer agent might be increased in combination with 5-fluorouracil (5-FU), a pyrimidine antimetabolite with activity against solid tumors. Using optimal conditions for the expression of CAV's cytotoxicity against MIA PaCa-2 cells, CAV was more cytotoxic to the cells than 5-FU. The combination of both drugs at a fixed molar ratio of 1:1 exhibited synergistic effects in the cells as determined by combination index analysis. The combination of 5-FU:CAV was tested at a ratio of 5:1 and exhibited antagonism at lower effect levels, additivity at 50% effect levels and slight synergism at higher effect levels. A 10:1 combination of both drugs (5-FU:CAV) exhibited antagonistic effects at all levels. When the drugs were combined at a molar ratio of 20:1, increased antagonism was observed. When CAV (1.0 or 2.0 g/kg daily) and/or 5-FU (35 mg/kg daily) was administered to colonic tumor-bearing rats for five consecutive days, the antitumor activity of the drug combination was significantly greater than the combined effects of either drug alone. However, the body weight loss experienced by CAV-treated rats was increased in those rats exposed to a combination of both drugs. These studies using different tumors provide in vitro and in vivo evidence that combination therapy offers a viable means of improving CAV's intrinsic efficacy while decreasing the concentration of 5-FU required to produce the same cytotoxic effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Neurons of the nucleus raphe magnus (NRM) and adjacent nucleus reticularis gigantocellularis pars alpha (NGCp alpha) receive a tonic inhibitory input from gamma-aminobutyric acid (GABA)-ergic neurons that is mediated by GABAA receptors. However, comparatively little is known about the role of GABAB receptors in these nuclei. The present study examined the effects on nociceptive threshold of microinjection of a wide dose range (0.1-150 ng) of the GABAB receptor agonist, baclofen hydrochloride (BAC), in the NRM, NGCp alpha or the nucleus reticularis giganto-cellularis (NGC). Microinjection of low doses of R(+)-BAC (0.1-1.0 ng) in the NRM or the NGCp alpha, but not the NGC increased response latencies in the tail flick test. As the dose of BAC was increased to 30.0 to 50.0 ng, response latencies in the tail flick test diminished to control values. Microinjection of the highest dose, 150 ng, in either the NRM, NGCp alpha or NGC significantly decreased response latencies in the tail flick test. Response latencies in the hot plate test were not increased by microinjection of 0.1 to 5.0 ng of R(+)BAC in the NRM, NGCp alpha or NGC. Although hot plate latency was increased after microinjection of 30.0 to 50.0 ng of R(+)-BAC in these nuclei, the effect was confounded by the occurrence of motor dysfunction. The effects of BAC were stereospecific as nociceptive threshold and motor function were not altered by microinjection of either 0.5 ng or 150 ng of the less active stereoisomer, S(-)-BAC. The biphasic effect of R(+)-BAC suggests the existence of multiple mechanisms by which GABAB receptors modulate the activity of neurons in the ventromedial medulla. It is proposed that the antinociception produced by low doses of R(+)-BAC results from disinhibition (activation) of neurons in the NRM and NGCp alpha as a consequence of the presynaptic inhibition of inhibitory GABAergic and/or noradrenergic inputs. The decrement in antinociceptive effect and hyperalgesia produced by higher doses of R(+)-BAC may result from a presynaptic inhibition of excitatory inputs to the NRM and NGCp alpha, postsynaptic hyperpolarization of neurons in these nuclei or the unmasking of a descending facilitatory pathway originating from the NRM, NGCp alpha and NGC.
We administered capsaicin or vehicle in 2-day-old rat pups, and for over 6 months examined the rats for damaged skin and for the behaviors of scratching, gnawing and biting their skin. By 35 days of age, all rats in the capsaicin group (n = 10) had damaged skin (i.e., lesions, hair loss and red skin) on the rostral half of their bodies. Skin damage remained prevalent over 6 months, whereas vehicle-treated rats (n = 8) had virtually no skin damage. Gnawing and biting behaviors were rarely observed, however, rats in the capsaicin group frequently scratched themselves. There was a significant positive correlation between the frequency at which rats scratched themselves and the total area of skin damage. Morphine (3.0 mg/kg, i.p.) greatly increased scratching behavior in only the capsaicin-treated rats and naloxone (0.5 mg/kg, i.p.) significantly reduced scratching in these rats. Thus, neonatal capsaicin, in its destruction of the majority of primary afferent C-fibers, is capable of inducing opioid-sensitive scratching behavior.
The medullary dorsal horn is a site of action of opiates in producing facial scratching. Extracts of plasma (0.4 microliter) from 4 patients with the pruritus of cholestasis induced facial scratching when microinjected into the medullary dorsal horn of monkeys. This extract-induced scratching could be abolished or prevented by administering the opioid receptor antagonist, naloxone. Neither saline nor an extract of plasma from a nonpruritic cholestatic patient induced scratching when similarly administered. We infer that plasma of patients with the pruritus of cholestasis contains a factor which induces pruritus by a central opioid receptor-mediated mechanism.