Repeated immunization with plasmid DNA formulated in poly(lactide-co-glycolide) microparticles is well tolerated and stimulates durable T cell responses to the tumor-associated antigen cytochrome P450 1B1

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation. Immunization of HLA-A2/Kb transgenic mice with human CYP1B1 encoding plasmid DNA formulated in poly(lactide-co-glycolide) (PLG) microparticles elicits CD8+ T cells that respond to human CYP1B1-positive target cells. The duration of the immune response, the effect on the immune response of multiple injections, and the safety of repeated injections were studied. These results show that the PLG-encapsulated DNA therapeutic elicits durable immune responses to CYP1B1, the responses are dependent on repeat immunization, and that the formulation is well tolerated.     

Anaphylaxis following psyllium ingestion

Psyllium is a hydrophilic agent found in many bulk laxative preparations. We report the occurrence of an anaphylactic reaction in a patient after ingestion of a psyllium-containing laxative. IgE mediation of the reaction was suggested by a positive immediate skin test to psyllium, positive passive transfer skin test, lack of skin response during passive transfer with heat treated serum, and an elevated IgE (RAST) to psyllium seed.     

Effects of intrathecal substance P and a substance P antagonist on a reflex to noxious heat are independent of changes in tail skin temperature

Substance P or the substance P receptor antagonist (d-Arg¹, d-Trp^{7, 9}, Leu¹¹)-substance P (Spantide) was injected into the lumbar subarachnoid space in mice, and the ability to change the tail-flick reflex and the tail skin temperature was investigated. Tail-flick latency (the time needed to evoke the tail-flick reflex by noxious radiant heat) was reduced for 1–4 min after intrathecal administration of substance P (5 μg), but the tail skin temperature was not significantly changed. Nor was the tail skin temperature significantly changed after intrathecal injection of Spantide (5 pg), but this compound significantly increased tail-flick latencies 5–30 min after injection. Analysis of co-variance showed that the effects of substance P or Spantide on tail-flick latency were significant, whereas the influence of tail skin temperature on tail-flick latency was nonsignificant. Thus, intrathecal substance P induces a short-lasting increase in nociceptive sensitivity, and intrathecal Spantide produces an antinociceptive effect of longer duration. The results seem not to be the result of changes in tail skin temperature.     

Comparative nasal absorption of allergens in atopic and nonatopic subjects.

Based on sensitization following intranasal antigen administration, previous investigations have suggested greater absorption of allergens through the nasal mucous membranes of atopic than of nonatopic subjects. In this study mucosal absorption was assessed more directly by determining the capacity of allergens applied intranasally to elicit cutaneous Prausnitz-Küstner (P-K) reactions in nonatopic persons as compared with asymptomatic atopic subjects sensitive to other allergens. Two series of reaginic human serum dilutions were injected intracutaneously in recipients backs, and 48 hours later one series was challenged intracutaneously with test allergen. After the responses had been recorded, concentrated allergenic extract was sprayed into the nose and the second series of P-K sites observed for reactivity. Sometimes these P-K sites were rechallenged intracutaneously the following day to determine passive transfer neutralization. Two allergens were studied: bovine ribonuclease (RNase) and peanut extract. Two sera containing peanut reagins and one with RNase antibodies were each used in 10 to 11 atopic and 9 to 11 nonatopic recipients. The atopic group failed to show greater or more rapid absorption of either allergen through the nose based on the highest serum dilution reacting after nasal challenge. the speed of the reaction, the ratio of the titer by nasal challenge to the intracutaneous titer, or passive transfer neutralization. Controls showed that the results were not influenced by systemic absorption of allergen employed for intracutaneous tests. Drinking the amount of peanut extract applied intranasally did not elicit P-K reactions.     

Regionally defective colonization of the terminal bowel by the precursors of enteric neurons in lethal spotted mutant mice

In order to gain insight into the process of colonization of the bowel by the neural crest-derived precursors of enteric neurons, the development of the enteric nervous system was examined in lethal spotted mutant mice, a strain in which a segment of bowel is congenitally aganglionic. In addition, nerve fibers within the ganglionic and aganglionic zones of the gut of adult mutant mice were investigated with respect to their content of acetylcholinesterase, immunoreactive substance P, vasoactive intestinal polypeptide and serotonin, and their ability to take up [³Hserotonin. In both the fetal gut of developing mutant mice and in the mature bowel of adult animals abnormalities were limited to the terminal 2 mm of colon. The enteric nervous system in the proximal alimentary tract was indistinguishable from that of control animals for all of the parameters examined. In the terminal bowel, the normal plexiform pattern of the innervation and ganglion cell bodies were replaced by a coarse reticulum of nerve fibers that stained for acetylcholineserase and were continuous with extrinsic nerves running between the colon and the pelvic plexus. These coarse nerve bundles contained greatly reduced numbers of fibers that displayed substance P- and vasoactive intestinal polypeptide-like immunoreactivity, but a serotonergic innervation was totally missing from the aganglionic bowel. During development, acetylcholineserase and uptake of [³Hserotonin appeared in neural elements in the foregut of mutant mice on the 12th day of embryonic life (E12), about the same time these markers appeared in the forgut in normal mice. By day E14, neurons expressing one or the other marker were recognizable as far distally as about 2 mm from the anus. The appearance of neurons in segments of gut grown for 2 weeks as expiants in culture was used as an assay for the presence of neuronal progenitor cells in the segments of fetal bowel at the time of explantation. Both acetyl- cholinesterase activity and uptake of [³Hserotonin developed in neuronsin vitro in expiants of proximal bowel between days E10 and E17. At all times, however, the terminal 2mm of mutant but not normal fetal gut gave rise to aneuronal cultures. In some mutant mice rare, small, ectopically-situated pelvic ganglia were found just outside aganglionic segments of fetal colon. Uptake of [³Hserotonin, normally a marker for intrinsic enteric neurites, was found in these ganglia.The experiments suppport the hypothesis that the terminal 2 mm of the gut in lethal spotted mutant mice is intrinsically abnormal and thus cannot be colonized by the precursors of enteric neurons. The defect seems to be specific in that both cells and processes of intrinsic enteric neurons, including all serotonergic and most peptidergic neurites, seem to be excluded from the abnormal region while extrinsic nerve fibers, including sympathetic and sensory axons, are able to enter the aganglionic zones. Since examination of neural progenitor cells has failed to reveal a significant proximo-distal displacement of these cells through the enteric tube during development of the murine bowel, a defect in the migration of precursor cells down the alimentary tract to the terminal gut seems unlikely to be substantially involved in the pathogenesis of aganglionosis. This conclusion is supported by the normal enteric nervous system in proximal regions of the mutant gut and the presence of enteric type neurons outside of, but at the same level as the aganglionic region.     

Substance P and somatostatin metabolism in sympathetic and special sensory ganglia in vitro

Mechanisms regulating the content of the putative peptide transmitters, substance P and somatostatin, were examined in several neuronal populations in culture. Substance P levels increased more than 25-fold within 48 h in sympathetic neurons in the explanted rat superior cervical ganglion, and remained elevated for 4 weeks. Identity of the peptide was authenticated by combined high pressure liquid chromatography-radioimmunoassay. Veratridine prevented the increase of substance P in vitro, and tetrodotoxin blocked the veratridine effect, suggesting that sodium ion influx and membrane depolarization prevent peptide elevation. Veratridine (or potassium)-induced membrane depolarization released substance P into the culture medium through a calcium-dependent process. Consequently, at least some veratridine effects are attributable to release and subsequent depletion of ganglion peptide. However, the inhibitory effects of veratridine were far greater than could be accounted for by the quantity of peptide released, suggesting a separate influence on net synthesis (synthesis less catabolism) of substance P. Viewed in conjunction with previous in vivo studies, our observations suggest that trans-synaptic impulses, through the mediation of postsynaptic sodium flux, release substance P from sympathetic neurons and also regulate intracellular peptide metabolism. To determine whether the processes regulating substance P in sympathetic neurons reflect generalized mechanisms, a different peptide, somatostatin, was examined in sympathetic neurons; moreover, substance P was examined in a different neuronal population, special sensory neurons in the nodose ganglion. Substance P levels increased significantly in both sympathetic and sensory neurons after explantation, and somatostatin levels increased in sympathetic neurons. In each instance, the increase was dependent upon the presence of the calcium ions. Moreover, these increases were all prevented by veratridine, in a tetrodotoxin-sensitive manner. Our observations suggest that common regulatory mechanisms govern peptide transmitter metabolism in diverse neuronal populations.     

Correspondence between the dopamine islands and striosomes of the mammalian striatum

During the development of the mammalian striatum, the early-forming dopamine innervation is broken up into macroscopic patches called "dopamine islands". These express high tyrosine hydroxylase-like immunoreactivity and are also rich in acetylcholinesterase activity. The mature striatum has prominent macroscopic compartments called "striosomes" that were first characterized by their low acetylcholinesterase activity and since have been related to heterogeneities in striatal input-output organizations. This report describes two sets of experiments designed to determine the relationship between the dopamine islands and the striosomes. The distributions of striatal tyrosine hydroxylase-like immunoreactivity and acetylcholinesterase activity were first compared in a series of kittens and young cats ranging in age from 1-228 postnatal days. During this time, the pattern of tyrosine hydroxylase-like immunoreactivity changed from islandic (patchy) to diffuse, and the pattern of acetylcholinesterase staining changed from one of acetylcholinesterase-rich patches to one of acetylcholinesterase-poor striosomes. The dopamine islands were in register with the acetylcholinesterase-poor patches at early developmental stages and at later stages the islands matched striosomes. These observations establish a correspondence between the dopamine islands and striosomes and demonstrate that the acetylcholinesterase-rich patches of the immature caudate nucleus become the acetylcholinesterase-poor striosomes of the adult. In a second set of experiments, cat fetuses were exposed to [3H]thymidine at embryonic days 22-29 in order to label the clustered subpopulations of striatal neurons known from previous experiments to lie in striosomes [Graybiel and Hickey (1982) Proc. natn. Acad. Sci. U.S.A. 79, 198-202]. The [3H]thymidine-labeled brains were examined at late fetal (embryonic days 50-52), early postnatal (days 1-21) and later postnatal (days 62-199) ages. The clusters of [3H]thymidine-labeled neurons were aligned with tyrosine hydroxylase-rich, acetylcholinesterase-rich patches early in development, and with acetylcholinesterase-poor striosomes at later stages. There were marked dorsoventral differences in the intensity of tyrosine hydroxylase-like immunoreactivity in the dopamine islands and this was confirmed in neonatal rats. A "dorsal islandic system" was defined as having crisp, highly immunoreactive islands; ventrally, regions of low and medium tyrosine hydroxylase-like immunoreactivity formed a mosaic.(ABSTRACT TRUNCATED AT 400 WORDS)     

Perceptual and Motor Space Representation: An Event-Related Potential Study

The purpose of this experiment was to study the brain potentials generated during spatial tasks related to the “schema corporel” (a mental map of sensory-motor relationships). Seven right-handed subjects performed a choice reaction-time task (Experiment 1), in which the spatial position of a visual stimulus (right or left of a fixation point) was varied independently of the spatial position of the response (right or left hand). The subjects also made self-paced extensions and flexions of the right and left index fingers (Experiment 2). Experiments 1 and 2 were performed with the hands both crossed and uncrossed. Spatio-temporal maps showed that the P300 component elicited by the choice RT situation in Experiment 1 was largest ipsilateral to the hand involved in the response, whether or not the hands were crossed. The later part of the pre-movement potentials during Experiment 2 and the motor potential were significantly larger contralateral to the moving hand under all conditions. Thus this pattern of lateralization can be attributed to the superimposition of a bilateral P300 wave on the asymmetrical motor potential. This suggests that distinct neuronal populations are involved in the generation of these two components. P300 latency and RT reflected the spatial conflict: both were longer when the stimulus and response were on opposite sides than when they were on the same side, even when the hands were crossed. However, the average P300 latency was not increased when the hands were crossed, whereas the average RT was substantially increased. Since the additional time required for programming the movement in the crossed hand situation had no effect on P300 generation, we infer that the P300 does not index this motor programming. However, P300 does reflect the stimulus-response spatial matching, since its latency was delayed by spatial conflict.     

The beta1 adrenergic effects of antibodies against the C-terminal end of the ribosomal P2beta protein of Trypanosoma cruzi associate with a specific pattern of epitope recognition

BALB/c mice immunized with recombinant Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) develop a strong and specific antibody response against its 13 residue-long C-terminal epitope (peptide R13: EEEDDDMGFGLFD) that has a concomitant beta1-adrenergic stimulating activity. However, other animals that undergo similar immunizations seem tolerant to this epitope. To evaluate further the antibody response against the ribosomal P proteins, 25 BALB/c and 25 Swiss mice were immunized with TcP2beta. From the 50 animals, 31 developed a positive anti-R13 response, whereas 19 were non-responsive. From the 31 anti-R13 positive mice, 25 had anti-R13 antibodies that recognized the discontinuous motif ExDDxGF, and their presence correlated with the recording of supraventricular tachycardia. The other six had anti-R13 antibodies but with a normal electrocardiographic recording. These anti-R13 antibodies recognized the motif DDxGF shared by mammals and T. cruzi and proved to be a true anti-P autoantibody because they were similar to those elicited in Swiss, but not in BALB/c mice, by immunization with the C-terminal portion of the mouse ribosomal P protein. Our results show that the recognition of the glutamic acid in position 3 of peptide R13 defines the ability of anti-R13 antibodies to react with the motif AESDE of the second extracellular loop of the beta1-adrenergic receptor, setting the molecular basis for their pathogenic beta1 adrenoceptor stimulating activity.     

Changes in the P100 latency of the visual evoked potential and the saccadic reaction time during isometric contraction of the shoulder girdle elevators

We investigated changes in the P100 latency of the visual evoked potential (VEP) and the saccadic reaction time (SRT) in relation to the degree of activity of the shoulder girdle elevators. Muscle force was set in 10% increments from 0% to 50% of the maximal voluntary contraction (MVC). The VEP was derived from a midline occipital electrode with reference electrodes on the ears when the right retina was stimulated through the eyelid by light emitting diodes while the eyes were closed. The P100 latency of the VEP was defined as the time from the stimulus onset to the main positive peak. The SRT was defined as the latency until the beginning of eye movement toward the lateral target, which was moved at random time-intervals. P100 latency was shortened until 30% of the MVC, and which it lengthened. The SRT changed in a pattern similar to that observed for the P100 latency. The ratio of the shortening in P100 latency relative to that of the SRT was approximately 20%. All data is presented as the mean value, plus the standard deviation. We believe that the information processing time in the neural pathway from the retina to the visual cortex was shortened up to a certain muscle force of the shoulder girdle elevators, and then this processing time lengthened. These findings indicate that shortening of information processing time in the neural pathway beyond the visual cortex is included in the shortening of the SRT.