Biomedical subjects

J M Kaplan

Publications and source records attributed to J M Kaplan.

At least 19 recordsLinked to original sources

PubMed1997

Transient immunosuppression with deoxyspergualin improves longevity of transgene expression and ability to readminister adenoviral vector to the mouse lung.

Animal studies have suggested that the clinical usefulness of recombinant adenoviruses (Ad) as vectors for therapeutic gene delivery may be limited by their immunogenicity. Neutralizing antibodies elicited by capsid proteins reduce the efficiency of vector readministration whereas cytotoxic T lymphocytes (CTLs) directed against viral proteins and/or immunogenic transgene products expressed by transfected cells have the potential to limit persistence of expression. In this study, transient administration of the novel immunosuppressant deoxyspergualin (DSG) was found to inhibit the development of both humoral and cell-mediated immune responses against Ad vector delivered intranasally. DSG treatment of primed mice previously exposed to wild-type Ad impaired the development of antibodies in response to a secondary and even tertiary challenge with Ad vector. As a result, improved gene transfer was obtained upon subsequent administration of a beta-galactosidase (beta-Gal)-encoding Ad vector. Short-term administration of DSG also depressed the activation of CD4+ and CD8+ T lymphocytes as assessed by measurement of antigen-specific proliferation and CTL activity, respectively. The marked suppression of CTL activity against Ad vector in DSG-treated mice correlated with improved persistence of transgene expression in the lung.

Adenoviridae↗

PubMed1997

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.

Administration, Intranasal↗

PubMed1997

Safety of airway gene transfer with Ad2/CFTR2: aerosol administration in the nonhuman primate.

In this study, the safety and efficacy of aerosol delivery to non-human primates of an adenoviral vector encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) were evaluated. The technique of concurrent flow spirometry was used to determine the deposited dose of Ad2/CFTR-2, which ranged from 3 to 8 x 10(10) I.U. Transgene DNA was detected by the polymerase chain reaction (PCR) in lung tissue from all treated animals, and human CFTR mRNA was detected on days 3, 7, and 21 post-exposure. The treatment was well tolerated, with no evidence of respiratory distress. Histologic changes in the lungs from Ad2/CFTR-2-treated animals were mild and, overall, indistinguishable from animals exposed to aerosolized vehicle. One vector-treated animal demonstrated an increase in lavage lymphocyte numbers 3 days after treatment and another had an abnormal chest radiograph 14 days after treatment. A third vector-treated animal had histologic evidence of a bronchointerstitial pneumonia 7 days after aerosol treatment that resolved by day 21. This study demonstrated that Ad2/CFTR-2 can effectively be delivered to the lungs of nonhuman primates and result in minimal adverse effects.

Adenoviruses, Human↗

PubMed1997

Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung.

One potential limitation of adenovirus (Ad)-based vectors for the gene therapy of cystic fibrosis (CF) and other genetic diseases is the transience of expression observed in most in vivo systems. In this study, the influence of various factors on persistence of transgene expression in the lung was investigated. In the absence of immune pressure, such as in the nude mouse, the genomic structure of the vector was found to be predominant in determining the persistence of expression; Ad vector constructs with an E1-E3+E4ORF6+ backbone encoding beta-galactosidase (beta-Gal) or the cystic fibrosis transmembrane conductance regulator (CFTR) produced declining levels of expression while an Ad/CMV beta Gal vector with an E1-E3+E4+ backbone gave rise to sustained, long-term reporter gene expression. The ability of the latter vector to persist was in turn limited in part by the presence of cytotoxic T lymphocytes (CTLs). Adoptive transfer experiments indicated that CTLs directed against either viral proteins or the beta-Gal reporter gene product were able to reduce expression in nude C57BL/6 mice stably expressing beta-Gal from the E4+ vector. Finally, the specificity and strength of the CTL response elicited by Ad vector was found to vary considerably depending on mouse strain haplotype. These results indicate that persistence of transgene expression in a given system is determined by the interplay between several factors including genomic structure of the vector, host background, and immune response.

Adenoviridae↗

PubMed1997

Contribution of caudal brainstem to d-fenfluramine anorexia.

Of the central 5-HT substrates that may mediate the anorexic actions of systemically administered d-fenfluramine (d-FEN), those in the forebrain have received the most attention. As a counterpoint to this forebrain focus, we evaluated the contribution of caudal brainstem substrates to the anorexic action of d-FEN. Two experimental protocols were employed. In one we compared the feeding response (intra-oral intake of 12.5% glucose) of intact and chronic supracollicular decerebrate (CD) rats to systemic administration of d-FEN. In the other, d-FEN was administered via fourth intracerebroventricular (ICV) injection to determine whether a dose-related suppression of intra-oral intake could be obtained. A dose-dependent suppression of intra-oral intake was obtained in the CD rat treated with d-FEN (0-8 mg/kg, delivered IP 20 min before testing). The threshold dose was two to three times higher in CD rats than in their intact controls, but the dynamic range of the dose-response curves of the two groups were overlapping with similar slopes of decline and with comparable maximal intake suppression. Fourth ICV administration of d-FEN in the intact rat yielded a dose-related suppression of intra-oral intake. Intake was also suppressed by fourth ICV d-FEN (30 mg) when rats drank 12.5% glucose solution from a spout. The reduced intra-oral intake following fourth ICV d-FEN treatment was partially attenuated by the systemic administration of the serotonin antagonist metergoline (0.4 mg/kg; IP). The CD results demonstrate the sufficiency of caudal brainstem receptors in mediating intake suppressive responses to systemic d-FEN. The fourth ICV results suggest further that 5-HT receptors in the caudal brainstem play a significant role in normal meal size control in the neurologically intact rat.

Animals↗

PubMed1997

Pulmonary dysfunction and reduced exercise capacity in patients with myelomeningocele.

OBJECTIVE: To evaluate pulmonary function and exercise capacity in children with myelomeningocele. STUDY DESIGN: Prospective evaluation in a randomly selected cohort of 12 subjects (10 to 17 years of age) with myelomeningocele and 12 control subjects matched for age, sex, and arm span. METHODS: Spirometry, lung volumes, maximum respiratory pressures, maximum oxygen expenditure during arm ergometry, and anaerobic threshold were measured. RESULTS: Mean total lung capacity and fractional lung volumes were significantly lower in case subjects than control subjects. Eleven subjects (92%) had a reduced forced vital capacity; seven (58%) had restrictive disease as evidenced by reductions in total lung capacity with normal or increased forced expiratory volume in 1 second/forced vital capacity ratio. Nine subjects (75%) had respiratory muscle weakness as evidenced by reduced maximum respiratory pressures or a low maximum voluntary ventilation. Exercise capacity was reduced as evidenced by a lower maximum oxygen consumption at peak exercise (13.8 +/- 4.8 vs 21.3 +/- 7.5 ml/min per kilogram of body weight; p < 0.02) and a lower anaerobic threshold (12.4 +/- 5.1 vs 17.3 +/- 4.2 ml/min per kilogram; p < 0.01) than the control group. Though the majority of subjects with myelomeningocele had a significant degree of restrictive disease, respiratory muscle weakness, or both, only one subject had pulmonary symptoms during exercise. CONCLUSIONS: Though most subjects with myelomeningocele had a significant degree of restrictive lung disease, respiratory muscle weakness, or both, exercise capacity was mostly limited by arm weakness. Skeletal muscle weakness may mask the symptoms of an underlying pulmonary abnormality, which may not be evident unless a pathologic cause of increased ventilation is present. Pulmonary function testing is suggested to screen for these abnormalities.

Adolescent↗

PubMed1997

Effects of central oxytocin administration on intraoral intake of glucose in deprived and nondeprived rats.

We evaluated the effects of lateral intracerebroventricular administration of oxytocin (OT) and/or a selective oxytocin-receptor antagonist (OTX), 1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-OT, on ingestion of intraorally delivered 12.5% glucose in rats that were either nondeprived or deprived of food for 20 h. In deprived rats, OT delivered 30 min before an initial intake test yielded a dose-related reduction of intraoral glucose intake. The highest dose tested, 20 nmol, reduced intraoral glucose intake by 45%. The effect was short-lived, however. Intraoral intake for a second test, initiated 60 min after the termination of the first, increased as a function of OT dose so that total session intake was unaffected by OT treatment. The suppression of intraoral intake by 20 nmol OT was reversed by pretreatment (45 min before testing) with OTX. In nondeprived rats, by contrast, OT yielded no effect on first-test, second-test, or total session intakes. Significant increases in first-test and total session intakes were obtained when OTX (20 nmol) was administered alone both in deprived (32% increase in first-test intake) and nondeprived (31% increase) rats. In general, the results obtained are consistent with the suggestion that OT contributes to the control of meal size and, in particular, to the process of satiation, which is the aspect of ingestive control highlighted by the specialized intake test used in the present study.

Animals↗

PubMed1997

EGL-36 Shaw channels regulate C. elegans egg-laying muscle activity.

The C. elegans egl-36 gene encodes a Shaw-type potassium channel that regulates egg-laying behavior. Gain of function [egl-36(gf)] and dominant negative [egl-36(dn)] mutations in egl-36 cause reciprocal defects in egg laying. An egl-36::gfp reporter is expressed in the egg-laying muscles and in a few other tissues. Expression of an egl-36(gf) cDNA in the egg-laying muscles causes behavioral defects similar to those observed in egl-36(gf) mutants. Gain of function EGL-36 subunits form channels that are active at more negative potentials than wild-type channels. The egl-36(gf) alleles correspond to missense mutations in an amino terminal subunit assembly domain (E138K) and in the S6 transmembrane domain (P435S), neither of which were previously implicated in the voltage dependence of channel activation. Altogether, these results suggest that EGL-36 channels regulate the excitability of the egg-laying muscles.

Animals↗

PubMed1997

Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse airway.

Adenoviral vectors have been used successfully to transfer the human CFTR cDNA to respiratory epithelium in animal models and to CF patients in vivo. However, studies done primarily in mice, indicate that present vector systems have limitations. Among other things, transgene expression in the lung is transient and the production of neutralizing antibodies against adenovirus correlates with a reduced ability to readminister a vector of the same serotype. Here we demonstrate that in mice, a transient blockade of costimulation between activated T cells and B cells/antigen presenting cells using a monoclonal antibody (MR1) against murine CD40 ligand inhibits the development of neutralizing antibodies to adenoviral (Ad) vector. MR1 also decreased the cellular immune response to Ad vector and allowed an increase in persistence of transgene expression. Furthermore, when administered with a second dose of Ad vector to mice preimmunized against vector, MR1 was able to interfere with the development of a secondary antibody response and allowed for high levels of transgene expression upon a third administration of vector to the airway.

Adenoviridae↗

PubMed1997

Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype.

Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population.

Adenovirus E1A Proteins↗

PubMed1997

Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo.

The recent findings that prolonged expression of certain adenovirus (Ad) vector-encoded proteins, including human alpha1-antitrypsin (huAAT), mouse erythropoietin (EPO), and human factor IX, can be achieved in animals that do not mount an immune response to the reporter protein were obtained with mouse strains which have been shown to be capable of mounting a cellular immune response against Ad vector antigens. This suggests either that Ad vectors expressing nonimmunogenic transgenes fail to elicit a cellular immune response or that an Ad-specific cellular immune response does develop but is ineffective against cells expressing nonimmunogenic transgenes. Here we demonstrate that an Ad vector expressing huAAT administered by intravenous injection does stimulate an Ad-specific cellular immune response but that this response fails to abolish vector-directed gene expression in vivo. Moreover, expression of huAAT remained stable in animals stimulated by concurrent and multiple administrations of different Ad vectors or viruses. We also demonstrate prolonged expression of huAAT in CD1 mice transgenic for the huAAT gene, indicating that long-term expression is not restricted to C57BL/6 mice. These results demonstrate that under some circumstances, an Ad vector can direct prolonged expression of a nonimmunogenic transgene despite the presence of a robust Ad-specific cellular immune response.

Adenoviridae↗

PubMed1997

Intake suppression after hepatic portal glucose infusion: all-or-none effect and its temporal threshold.

The effects of hepatic portal infusions of isotonic glucose on glucose intake (3.2%) were evaluated with use of the intraoral intake test, which, unlike traditional tests, permits delivery of portal infusions in explicit temporal relationship to intake onset in nondeprived rats. Continuous or discontinuous portal infusions (0.1 ml/min) of isotonic glucose or saline were initiated 0, 30, 60, or 120 min before meal onset. Jugular infusions of isotonic saline or glucose and portal infusions of isotonic saline were without effect. For all effective portal glucose infusions, intake was suppressed by approximately 30% of baseline values. Because the duration (and quantity) of effective portal glucose infusions varied by a factor of 10, we conclude that intraoral intake suppression under these conditions is all or none in nature. Whether an intake suppression was obtained depended more on when the infusion was delivered than on how much was infused. Thus 1.5 ml of isotonic glucose infused between 60 and 45 min before the intake test was effective, whereas 3.0 ml infused for the 30 min before intake was without effect. These results suggest that the liver participates in the control of future intake but not in the termination of an ongoing meal. The temporal requirement for intake suppression should be considered in analyses of the metabolic, hormonal, and/or neural mechanisms that underlie the liver's contribution to intake control.

Animals↗

PubMed1997

Reward comparison in chronic decerebrate rats.

The simultaneous contrast paradigm was used to evaluate responsiveness to a low (0.05 M) and a high (0.5 M) concentration of sucrose under two conditions in intact and chronic decerebrate rats. In one condition the low concentration was presented on one day and the high concentration on another. In the other condition presentation of the two concentration was alternated within the same daily session. In each case there was a total of 40 trials/day during which the stimulus was delivered intraorally for 2 s at a rate of 1.5 ml/min with a 30-s intertrial interval. The results showed that the intact rats always licked more for the high than for the low concentration of sucrose but that the magnitude of the effect was larger when given the opportunity to compare the two concentrations within the same daily session. The decerebrate rats produced a similar pattern, but the concentration effect was evident only when the stimuli were alternated within the same daily session. These data stand as the first evidence that the isolated caudal brain stem is adequate for the expression of a behavior that depends on comparison processes involving short-term memory.

Animals↗

PubMed1997

Effect of oral versus gastric delivery on gastric emptying of corn oil emulsions.

Several studies have shown that fluids delivered to the stomach tend to empty more rapidly than when ingested by mouth. To better characterize the "delivery route effect" for corn oil, rats received intragastric or intraoral infusions matched for concentration and for the rate and duration of stimulus delivery. We showed, first, that more than twice as much oil emptied by the end of 12-min intragastric versus intraoral infusions but that the emptying curves remained roughly parallel for 1 h after infusion offset. Remaining experiments therefore focused on stimulus parameters of relevance to emptying control during stomach fill. Emptying during intragastric infusions approximately doubled with doublings of oil concentration (25-50%), infusion duration (6-12 and 12-24 min), and infusion rate (0.5-1.0 ml/min). Emptying during intraoral infusions, by contrast, was entirely unaffected by these manipulations. Unlike oil emptying, glucose emptying did not vary as a function of delivery route. The nutrient specificity of the delivery route effect cannot be explained in terms of energy density, as the effect was obtained for oil but not for glucose when their energy densities were equated (50% glucose, 25% corn oil). In discussion, we suggest that the oral influence on corn oil emptying during stomach fill is a gating factor that enables the expression of inhibition derived from postgastric nutrient stimulation.

Administration, Oral↗

PubMed1997

Gastric branch vagotomy and gastric emptying during and after intragastric infusion of glucose.

The effect of gastric branch vagotomy (GVX) on the gastric emptying of glucose was evaluated during two phases of emptying control: as the stomach fills and in the postload period. GVX and control rats received a series of intragastric glucose infusions (1.0 ml/min) through indwelling gastric fistulas. In experiment 1, gastric samples were withdrawn either immediately after the offset of 9- or 18-min infusions of 12.5% glucose or at various times up to 36 min postinfusion. In experiment 2, samples were withdrawn either immediately or 30 min after termination of 12-min infusions of 12.5 or 25% glucose. After gastric fill, glucose solute emptying rate was stable over time not influenced by concentration doubling, and, surprisingly, not affected by GVX. During gastric fill, solute emptying rate doubled with concentration in both GVX and control rats. For each concentration, however, glucose emptied during fill at almost twice the rate in GVX compared with control rats. This accelerated emptying of glucose during fill in GVX rats is consistent with a gastric vagal contribution to inhibitory mechanisms (e.g., receptive relaxation) that operate as the stomach fills under normal conditions. The absence of a GVX effect on emptying after fill suggests either that gastric branch vagal efferents play little role in feedback inhibitory control of glucose emptying under normal conditions or that other systems compensate for the function previously served by vagal gastric branch efferents. Further work is required to address the possible role of the gastric vagus in feedback control of gastric emptying when nutritive fluids other than glucose are delivered.

Animals↗

PubMed1996

Relationships between antibodies against human soluble complement receptor 1 (hsCR1) from various species.

The relationships between antibodies against human soluble complement receptor 1 (hsCR1) were studied in rodents, dogs, nonhuman primates, and humans. An antibody response occurred in all species except humans. The anti-hsCR1 antibodies from the various species were characterized to determine if they recognize similar epitopes on the hsCR1 molecule. Dog and monkey sera, positve for hsCR1 binding, were used as blocking antibodies against mouse anti-hsCR1 monoclonal antibodies as well as mouse and rat anti-hsCR1-positive sera. Human sera (blood group antisera: anti-Knops, anti-McCoy, anti-Knops/McCoy, anti-Swain-Langley) and serum from one burn patient (who became seropositive despite ever receiving treatment with hsCR1) were also used to test blocking of mouse, rat, dog, and monkey anti-hsCR1. Characterization of anti-hsCR1 antibodies from different species demonstrated that hsCR1 causes divergent antibody responses among animals. While mouse, rat, and dog antibodies cross inhibit binding by approximately 50%, monkey antibodies recognize primarily different epitopes of the hsCR1 molecule. Moreover, human antibodies binding hsCR1 are completely different from the animal antibodies, including monkey. This study indicates that although hsCR1 is immunogenic in animals, there is a difference in response between species, particularly between nonprimates and primates, and finally, that this antibody response is not predictive for humans.

Animals↗

PubMed1996

Sensory signaling in Caenorhabditis elegans.

The simple anatomy, behavior, and genetics of the nematode Caenorhabditis elegans make it an attractive organism for studying sensory circuits and their functions in vivo. Recent advances in our understanding of C. elegans sensory signaling stem from work on topographic maps, chemosensory receptors, modality coding, and the integration of antagonistic sensory inputs.

Animals↗

PubMed1996

A new taste reactivity analysis of the integration of taste and physiological state information.

We used conjoint manipulation of taste and physiological state to address the theoretical issue of signal integration. The interaction between taste (glucose concentration) and state (food deprivation) was evaluated using the taste reactivity method in which oral motor responses elicited by direct intraoral infusion are measured. The time frame of the typical taste reactivity paradigm, where observation is limited to the infusion period, was expanded to include the postinfusion interval. In each test session, rats received a series of trials consisting of 15-s intraoral infusions and 45-s postinfusion observation intervals. Two experiments were run in which glucose concentration was varied and rats were run nondeprived and after 24 h food deprivation. In experiment 1, glucose concentrations (0, 3.2, 6.25, 12.5, and 25%) were randomly presented during each test session. In experiment 2, individual glucose concentrations (0, 6.25, or 25%) were presented during separate sessions. For both, a deprivation condition was flanked by nondeprived (baseline) sessions. Concentration-response functions were comparable in both experiments. In each experiment, the shape of the concentration-response function was dramatically different during and after infusions. During infusions, there were no increases in glucose-elicited rhythmic oral responses beyond a very dilute concentration. After infusions, the concentration-response functions appeared linear across the concentration range. In both experiments, deprivation elevated responding only in the after-infusion periods. In experiment 1, the concentration-response function was uniformly elevated (on average, 27%) by deprivation, which if taken at face value would suggest an additive combination of taste and state feedback signals. In experiment 2, however, deprivation increased responding (approximately 30%) for 6.25%, but not for 0 or 25%, suggesting a stimulus specificity of the taste-state integration. Clearly then, the taste-state profiles differed as a function of experimental design. In the GENERAL DISCUSSION, we suggest that the uniform elevation of responding to all glucose concentrations, and to water, seen in experiment 1, may be an artifact of the random presentation of all stimuli during individual sessions. Experiment 2, in which stimuli were presented in a between-sessions design, may provide a truer reflection of the underlying integrative process.

Animals↗