Relation between Enhanced Antibody Responses Elicited by Adjuvant Injection and Those Elicited by Secondary Antigenic Stimulation

An attempt was made to determine if there is any common mechanism in the enhanced antibody response caused either by injection of adjuvant, such as bacterial endotoxin (LPS) and complexed polynucleotides, or by secondary antigenic stimulation. LPS inoculated in mice 4 days before injection of sheep red blood cells (SRBC) and polyA:U invalidated the adjuvant effect of polyA:U injected together with SRBC, and the hemolysin plaque-forming cell (PFC) response of such mice was similar to that of the mice which received SRBC alone. When mice primed with SRBC 24 days in advance were injected with LPS and 4 days later re-stimulated with SRBC, their PFC response to the secondary stimulation was suppressed to less than one tenth of the normal secondary PFC response. The suppressive effect of LPS on the secondary antibody response was abolished if the serum collected from mice injected with LPS was given to the primed and LPS-injected mice at the time of the secondary antigenic stimulation. From these results we discussed the possibility that some common mediator might play a role in the enhanced antibody response elicited by either adjuvant injection or secondary injection of antigen.     

Calcium-Dependent Release of Tyrosine in Brain Elicited by Stimulation of Neuropeptide Receptors

We sought to establish whether the endogenous opiate-receptor agonist Met-enkephalin (m-ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m-ENK (1-10 microM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylalanine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), taurine (Tau), and glycine (Gly) were similarly increased by 50-150% with m-ENK in slices of CS, but not Cx. The enhanced release of AAs by m-ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5-55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as gamma-aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m-ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8-51%) compared to that of GABA, Gly, Glu, and Tau (49-123%). Nonetheless, m-ENK (10 microM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Illusions of Visual Motion Elicited by Electrical Stimulation of Human MT Complex

Human cortical area MT⁺ (hMT⁺) is known to respond to visual motion stimuli, but its causal role in the conscious experience of motion remains largely unexplored. Studies in non-human primates demonstrate that altering activity in area MT can influence motion perception judgments, but animal studies are inherently limited in assessing subjective conscious experience. In the current study, we use functional magnetic resonance imaging (fMRI), intracranial electrocorticography (ECoG), and electrical brain stimulation (EBS) in three patients implanted with intracranial electrodes to address the role of area hMT⁺ in conscious visual motion perception. We show that in conscious human subjects, reproducible illusory motion can be elicited by electrical stimulation of hMT⁺. These visual motion percepts only occurred when the site of stimulation overlapped directly with the region of the brain that had increased fMRI and electrophysiological activity during moving compared to static visual stimuli in the same individual subjects. Electrical stimulation in neighboring regions failed to produce illusory motion. Our study provides evidence for the sufficient causal link between the hMT⁺ network and the human conscious experience of visual motion. It also suggests a clear spatial relationship between fMRI signal and ECoG activity in the human brain.     

Responses of the Smooth Muscle Membrane of Guinea Pig Jejunum Elicited by Field Stimulation

Field stimulation of the jejunum elicited successively an action potential of spike form, a slow excitatory depolarization, a slow inhibitory hyperpolarization, and a postinhibitory depolarization as a rebound excitation. The slow depolarization often triggered the spike. The inhibitory potential showed lower threshold than did the excitatory potential. Both the excitatory potentials were abolished by atropine and tetrodotoxin. Effective membrane resistance measured by the intracellular polarizing method was reduced during the peak of the excitatory potential, but the degree of reduction was smaller than that evoked by iontophoretic application of acetylcholine. Conditioning hyperpolarization of the muscle membrane modified the amplitude of the excitatory potential. The estimated reversal potential level for the excitatory potenialt was about 0 mv. No changes could be observed in the amplitude of the inhibitory potential when hyperpolarization was induced with intracellularly applied current. Low [K]_o and [Ca]_o blocked the generation of the excitatory potential but the amplitude of the inhibitory potential was enhanced in low [K]_o. Low [Ca]_o and high [Mg]_o had no effect on the inhibitory potential.     

Visual Evoked Cortical Potential (VECP) Elicited by Sinusoidal Gratings Controlled by Pseudo-Random Stimulation

The contributions of contrast detection mechanisms to the visual cortical evoked potential (VECP) have been investigated studying the contrast-response and spatial frequency-response functions. Previously, the use of m-sequences for stimulus control has been almost restricted to multifocal electrophysiology stimulation and, in some aspects, it substantially differs from conventional VECPs. Single stimulation with spatial contrast temporally controlled by m-sequences has not been extensively tested or compared to multifocal techniques. Our purpose was to evaluate the influence of spatial frequency and contrast of sinusoidal gratings on the VECP elicited by pseudo-random stimulation. Nine normal subjects were stimulated by achromatic sinusoidal gratings driven by pseudo random binary m-sequence at seven spatial frequencies (0.4–10 cpd) and three stimulus sizes (4°, 8°, and 16° of visual angle). At 8° subtence, six contrast levels were used (3.12–99%). The first order kernel (K1) did not provide a consistent measurable signal across spatial frequencies and contrasts that were tested–signal was very small or absent–while the second order kernel first (K2.1) and second (K2.2) slices exhibited reliable responses for the stimulus range. The main differences between results obtained with the K2.1 and K2.2 were in the contrast gain as measured in the amplitude versus contrast and amplitude versus spatial frequency functions. The results indicated that K2.1 was dominated by M-pathway, but for some stimulus condition some P-pathway contribution could be found, while the second slice reflected the P-pathway contribution. The present work extended previous findings of the visual pathways contribution to VECP elicited by pseudorandom stimulation for a wider range of spatial frequencies.     

Cannabinoids Facilitate the Swallowing Reflex Elicited by the Superior Laryngeal Nerve Stimulation in Rats

Cannabinoids have been reported to be involved in affecting various biological functions through binding with cannabinoid receptors type 1 (CB1) and 2 (CB2). The present study was designed to investigate whether swallowing, an essential component of feeding behavior, is modulated after the administration of cannabinoid. The swallowing reflex evoked by the repetitive electrical stimulation of the superior laryngeal nerve in rats was recorded before and after the administration of the cannabinoid receptor agonist, WIN 55-212-2 (WIN), with or without CB1 or CB2 antagonist. The onset latency of the first swallow and the time intervals between swallows were analyzed. The onset latency and the intervals between swallows were shorter after the intravenous administration of WIN, and the strength of effect of WIN was dose-dependent. Although the intravenous administration of CB1 antagonist prior to intravenous administration of WIN blocked the effect of WIN, the administration of CB2 antagonist did not block the effect of WIN. The microinjection of the CB1 receptor antagonist directly into the nucleus tractus solitarius (NTS) prior to intravenous administration of WIN also blocked the effect of WIN. Immunofluorescence histochemistry was conducted to assess the co-localization of CB1 receptor immunoreactivity to glutamic acid decarboxylase 67 (GAD67) or glutamate in the NTS. CB1 receptor was co-localized more with GAD67 than glutamate in the NTS. These findings suggest that cannabinoids facilitate the swallowing reflex via CB1 receptors. Cannabinoids may attenuate the tonic inhibitory effect of GABA (gamma-aminobuteric acid) neurons in the central pattern generator for swallowing.     

Lithium Reduqes the Accumulation or Inositol Polyphosphate Second Messengers Following Cholinergic Stimulation of Cerebral Cortex Slices

Abstract: The ability of lithium to interfere with the metabolism of inositol phosphates in brain may underlie its therapeutic action in manic-depressive illness. In these experiments, lithium, at therapeutic concentrations, enhanced the accumulation of [³H]inpsitol monophosphate but suppressed the accumulation of the putative second messengers [³H]inositol 1,4,5-trisphosphate ([³H]Ins(1,4,5)P₃) and f³H]inositol 1,3,4,5-tetrakisphosphate following stimulation of cerebral cortex slices with carbachol. Mass measurements of Ins(1,4,5)P₃showed similar inhibitory effects, which could be prevented by preincubation with myo -inositol. These data may reveal the mechanism by which lithium can reduce polyphosphoinositide-midiated neurotransmission in brain.     

Changes in Mitochondria and Intermitochondrial Junctions of Rat Cardiomyocytes upon Adrenergic Stimulation by Isoproterenol

Changes in rat cardiomyocytes and their mitochondria and intermitochondrial junctions (IMJs) upon -adrenoreceptor stimulation with isoproterenol were studied by the methods of light and electron microscopy and computer-aided morphometry. Isoproterenol injections (0.3 mg/kg for eight days) proved to induce myocardial hypertrophy, which was more pronounced in the right than in the left ventricle. In the hypertrophied cardiomyocytes of both ventricles, an adaptive response of mitochondria was observed: their ultrastructure, size, and number changed, and the number and average length of IMJs increased. A positive correlation between the degree of cell hypertrophy and the number of IMJs was revealed. The reactive properties of mitochondria, including IMJ formation, differed depending on their location in the cell (i.e., in the perinuclear, intermyofibrillar, or subsarcolemmal regions). These results suggest that the rates and intensities of adaptive compensatory processes developing in the mitochondria of cardiomyocytes exposed to -adrenoreceptor stimulation differed in the left and right ventricles.     

Overnight Fasting Regulates Inhibitory Tone to Cholinergic Neurons of the Dorsomedial Nucleus of the Hypothalamus

The dorsomedial nucleus of the hypothalamus (DMH) contributes to the regulation of overall energy homeostasis by modulating energy intake as well as energy expenditure. Despite the importance of the DMH in the control of energy balance, DMH-specific genetic markers or neuronal subtypes are poorly defined. Here we demonstrate the presence of cholinergic neurons in the DMH using genetically modified mice that express enhanced green florescent protein (eGFP) selectively in choline acetyltransferase (Chat)-neurons. Overnight food deprivation increases the activity of DMH cholinergic neurons, as shown by induction of fos protein and a significant shift in the baseline resting membrane potential. DMH cholinergic neurons receive both glutamatergic and GABAergic synaptic input, but the activation of these neurons by an overnight fast is due entirely to decreased inhibitory tone. The decreased inhibition is associated with decreased frequency and amplitude of GABAergic synaptic currents in the cholinergic DMH neurons, while glutamatergic synaptic transmission is not altered. As neither the frequency nor amplitude of miniature GABAergic or glutamatergic postsynaptic currents is affected by overnight food deprivation, the fasting-induced decrease in inhibitory tone to cholinergic neurons is dependent on superthreshold activity of GABAergic inputs. This study reveals that cholinergic neurons in the DMH readily sense the availability of nutrients and respond to overnight fasting via decreased GABAergic inhibitory tone. As such, altered synaptic as well as neuronal activity of DMH cholinergic neurons may play a critical role in the regulation of overall energy homeostasis.     

Pharmacological Mechanisms of Cortical Enhancement Induced by the Repetitive Pairing of Visual/Cholinergic Stimulation

Repetitive visual training paired with electrical activation of cholinergic projections to the primary visual cortex (V1) induces long-term enhancement of cortical processing in response to the visual training stimulus. To better determine the receptor subtypes mediating this effect the selective pharmacological blockade of V1 nicotinic (nAChR), M1 and M2 muscarinic (mAChR) or GABAergic A (GABA_AR) receptors was performed during the training session and visual evoked potentials (VEPs) were recorded before and after training. The training session consisted of the exposure of awake, adult rats to an orientation-specific 0.12 CPD grating paired with an electrical stimulation of the basal forebrain for a duration of 1 week for 10 minutes per day. Pharmacological agents were infused intracortically during this period. The post-training VEP amplitude was significantly increased compared to the pre-training values for the trained spatial frequency and to adjacent spatial frequencies up to 0.3 CPD, suggesting a long-term increase of V1 sensitivity. This increase was totally blocked by the nAChR antagonist as well as by an M2 mAChR subtype and GABA_AR antagonist. Moreover, administration of the M2 mAChR antagonist also significantly decreased the amplitude of the control VEPs, suggesting a suppressive effect on cortical responsiveness. However, the M1 mAChR antagonist blocked the increase of the VEP amplitude only for the high spatial frequency (0.3 CPD), suggesting that M1 role was limited to the spread of the enhancement effect to a higher spatial frequency. More generally, all the drugs used did block the VEP increase at 0.3 CPD. Further, use of each of the aforementioned receptor antagonists blocked training-induced changes in gamma and beta band oscillations. These findings demonstrate that visual training coupled with cholinergic stimulation improved perceptual sensitivity by enhancing cortical responsiveness in V1. This enhancement is mainly mediated by nAChRs, M2 mAChRs and GABA_ARs. The M1 mAChR subtype appears to be involved in spreading the enhancement of V1 cortical responsiveness to adjacent neurons.     

Cytoarchitecture of the Ferret Suprasylvian Gyrus Correlated with Areas Containing Multiunit Responses Elicited by Stimulation of the Face

The cytoarchitecture was studied in a segment of the ferret suprasylvian gyrus containing at least two and possibly four somesthetic representations of the face that were observed in the primary somatosensory cortex. These representations were restricted to the crown of the gyrus and were surrounded by somesthetically unresponsive cortex that extended down both sides to the base of adjacent sulci. Numerous cytoarchitectonic subdivisions were found on a qualitative basis, and were confirmed quantitatively by cluster analyses and relevant statistical tests of 10 prominent features from layers III, IV, and V. Four distinct cytoarchitectonic subdivisions, each with a well-developed and homogeneous granular layer IV, were found distributed from anterior to posterior along the crown of the gyrus at sites corresponding to the locations of the four facial representations. The surrounding unresponsive cortex had a fragmented cytoarchitecture, especially along the medial bank and base of the coronal sulcus. This unresponsive cortex separated the facial representations from the body representations, which were located on the adjacent posterior cruciate gyrus. Most of the unresponsive subdivisions had a heterogeneous or agranular layer IV and fairly well-developed sublamination in layer III, which may be indicative of extensive corticocortical connections. One set of unresponsive subdivisions had comparable cytoarchitectures that directly bordered the facial representations. Another set of unresponsive subdivisions with comparable architectures occupied most of the lateral bank of the gyrus. The implications of multiple representations and cytoarchitectonic fragmentation of the ferret primary somatosensory cortex are discussed in relation to the organization of the primary somatosensory cortex in other species.     

Role of Egr-1 in Cholinergic Stimulation of Phenylethanolamine N-Methyltransferase Promoter

Abstract: The effects of the cholinergic agonist carbachol on phenylethanolamine N -methyltransferase promoter activity and Egr-1 mRNA expression in PC12-derived RS1 cells were examined to investigate the potential involvement of Egr-1 in the neural regulation of phenylethanolamine N -methyltransferase gene expression. Carbachol stimulated luciferase expression in cells transfected with a rat phenylethanolamine N -methyltransferase promoter-luciferase reporter gene construct and also elevated Egr-1 mRNA levels in untransfected cells. Maximum induction of Egr-1 mRNA by carbachol was rapid (0.5 h), whereas by comparison, peak luciferase activity was delayed (6 h). In addition, carbachol stimulation of both luciferase and Egr-1 mRNA expression could be completely inhibited by atropine but not hexamethonium. Furthermore, bethanechol but not nicotine could mimic the effects of carbachol, indicating that carbachol activation was medicated through muscarinic cholinergic receptors. Finally, carbachol failed to stimulate luciferase expression in cells transfected with a mutant construct, in which the Egr-1 binding element in the phenylethanolamine N -methyltransferase promoter was mutated. These results suggest that carbachol activates the phenylethanolamine N -methyltransferase promoter through stimulation of Egr-1 expression, and are consistent with the potential involvement of Egr-1 in the cholinergic activation of the phenylethanolamine N -methyltransferase gene.     

Stimulation of ethylene or ethane production by malformin

Malformin stimulated ethylene production of Phaseolus vulgarisL. seedlings and explants. However, when malformin was vacuum-infiltratedinto apical bud sections, the production of ethylene was inhibited,ethane production was stimulated and the sections became softand pliable; in pure oxygen, ethylene production was- stimulatedand the sections remained firm. Prolonged stimulation of ethaneproduction by malformin-treated sections required oxygen. Indoleaceticacid (IAA) had no effect on the stimulation of ethane productionby malformin-infiltrated tissues; malformin and IAA stimulatedethylene production synergistically at the same time that malformininducedethane production had increased markedly. ¹This work was supported by grant GB-7158 from the NationalScience Foundation. ²Journal Paper No. 3560 of the Purdue Agricultural ExperimentStation. (Received July 23, 1969; )     

Modulation of Cortical Oscillations by Low-Frequency Direct Cortical Stimulation Is State-Dependent

Cortical oscillations play a fundamental role in organizing large-scale functional brain networks. Noninvasive brain stimulation with temporally patterned waveforms such as repetitive transcranial magnetic stimulation (rTMS) and transcranial alternating current stimulation (tACS) have been proposed to modulate these oscillations. Thus, these stimulation modalities represent promising new approaches for the treatment of psychiatric illnesses in which these oscillations are impaired. However, the mechanism by which periodic brain stimulation alters endogenous oscillation dynamics is debated and appears to depend on brain state. Here, we demonstrate with a static model and a neural oscillator model that recurrent excitation in the thalamo-cortical circuit, together with recruitment of cortico-cortical connections, can explain the enhancement of oscillations by brain stimulation as a function of brain state. We then performed concurrent invasive recording and stimulation of the human cortical surface to elucidate the response of cortical oscillations to periodic stimulation and support the findings from the computational models. We found that (1) stimulation enhanced the targeted oscillation power, (2) this enhancement outlasted stimulation, and (3) the effect of stimulation depended on behavioral state. Together, our results show successful target engagement of oscillations by periodic brain stimulation and highlight the role of nonlinear interaction between endogenous network oscillations and stimulation. These mechanistic insights will contribute to the design of adaptive, more targeted stimulation paradigms.     

Stimulation of vesicular-arbuscular mycorrhiza by fungicides or rhizosphere bacteria

Stimulation of vesicular-arbuscular (VA) mycorrhizal fungi may secure the early establishment of symbiosis and benefit the host plant at an earlier stage of development. The application of Bacillus mycoides resulted in particular in the acceleration of early VA mycorrhiza formation. An increase in vigour of the symbiosis could be measured later in terms of increased sporulation of the mycorrhizal fungi after shoot removal. Natural sporulation during later mycorrhizal development was affected by combination of bacteria and just one mycorrhizal isolate. The stimulation of mycorrhizal development was shown to be non-specific with regard to host plant and the isolate of the VAM fungus. However, the effect could not be achieved in all combinations of soil types and host plants. Application of the systemic fungicides triadimefon and pyrazophos promoted VAM formation. Combinations of fungicide and bacterial treatments were not synergistic.     

Whole-Cell Mechanosensitive Currents in Rat Ventricular Myocytes Activated by Direct Stimulation

Mechanosensitive channels may have a significant role in the development of cardiac arrhythmia following infarction, but the data on mechanical responses at the cellular level are limited. Mechanosensitivity is a ubiquitous property of cells, and although the structure of bacteriological mechanosensitive ion channels is becoming known by cloning, the structure and force transduction pathway in eukaryotes remains elusive. Isolated adult rat ventricular myocytes were voltage clamped and stimulated with a mechanical probe. The probe was set in sinusoidal motion (either in, or normal to, the plane of the cell membrane), and then slowly lowered onto the cell. The sinusoidal frequency was held constant at 1 Hz but the stimulation amplitude was increased and the probe gradually lowered until a mechanically sensitive whole cell current was seen, which usually followed several minutes of stimulation. The whole cell mechanosensitive current in rat cells had two components: (i) a brief large inward current spike current; (ii) a more sustained smaller inward current. The presence of the initial sharp inward current suggests that some structure within the cell either relaxes or is broken, exposing the mechanosensitive element(s) to stress. Metabolic changes induced by continued stress prior to the mechanosensitive response may weaken the elements that break producing the spike, or simple stress-induced fracture of the cytoskeleton itself may occur.     

Muscarinic Cholinergic Stimulation of Exogenous Phosphatidylinositol Hydrolysis Is Regulated by Guanine Nucleotides in Rabbit Brain Cortical Membranes

Rabbit brain cortical membranes, which have been extracted with 2 M KCl, hydrolyze exogenously added [3H]phosphatidylinositol [( 3H]PI) in a guanine nucleotide- and carbachol-dependent manner. Both oxotremorine-M and carbachol are full agonists with EC50 values of 8 and 73 microM, respectively. Pirenzepine and atropine inhibit carbachol-stimulated [3H]PI hydrolysis. The hydrolysis-resistant guanine nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is the most potent in supporting carbachol-stimulated hydrolysis of PI. There is no effect of carbachol in the absence of guanine nucleotides or in the presence of 100 microM adenosine 5'-O-(3-thiotriphosphate), adenosine-5'-(beta, gamma-imido)triphosphate, or sodium pyrophosphate. Guanylyl-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] in the presence of carbachol also stimulates PI hydrolysis although much less than that seen with GTP gamma S. GDP and Gpp(NH)p are potent antagonists of the GTP gamma S-dependent carbachol response. Optimal stimulation by carbachol and GTP gamma S was observed at 0.3-1 microM free Ca2+ and 6 mM MgCl2. Limited trypsinization resulted in loss of receptor-regulated PI breakdown and a slight decrease in basal activity. These results demonstrate that phospholipase C hydrolysis of exogenous PI by rabbit cortical membranes may be stimulated by carbachol in a guanine nucleotide-dependent manner.     

Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization

A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors.Recombinant adenovirus (rAd)-based vectors are currently under investigation in a variety of gene therapy and T-cell-based vaccine clinical trials. There are more than 370 such ongoing clinical trials for broad applications, including infectious diseases and cancer therapy (http://www.wiley.co.uk/genetherapy/clinical/). Based on supportive data from nonhuman primate studies, rAd-based vectors have been developed and tested in human clinical trials to deliver human immunodeficiency virus (HIV-1) gene products that stimulate HIV-specific immune responses. Preexisting immunity to Ad serotype 5 (Ad5), from which most vectors are derived, is common in humans. Though neutralizing antibodies to Ad5 may reduce the immunogenicity of Ad5-based vectors in animal models (16), their effect on immunity in subjects with previous Ad5 infection is poorly understood. In the STEP trial, which tested a Merck rAd5 vaccine encoding HIV-1 Gag, Pol, and Nef, vaccination failed to show protection, either by lowering viral load or by decreasing acquisition of infection (3, 9, 12, 21). Furthermore, the possibility was raised that subjects with preexisting neutralizing antibodies from natural Ad5 infection may have carried an increased risk of HIV infection after vaccination. Thus, understanding the nature and immune effects of Ad5 seropositivity in humans is important to the development of vaccines against AIDS and other diseases.Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis in humans, and exposure before adolescence is common in human populations (19). Such exposure stimulates both innate and adaptive immune responses that generate neutralizing antibodies and virus-specific T-cell responses (6). These antibodies can also synergize with each other to achieve maximum viral neutralization (7, 22). The capsid protein specificity of Ad5 neutralizing antibodies has been reported for humans following administration of rAd5 gene therapy vectors for advanced liver or lung cancer (7, 10). However, results were presented solely for antibodies induced by administration of rAd5. One report has assessed Ad5 neutralizing antibodies with a healthy human population that was Ad5 seropositive from natural exposure to the virus (18). The median titer of the population was presented, but the frequency of protein-specific neutralizing antibody has not been defined for humans.Here we describe the first report of the natural frequency and effect on immunization of neutralizing antibodies specific for different Ad capsid proteins in human subjects. We address the fundamental mechanisms of how humans generate neutralizing antibodies to a common cold virus that is in widespread use as a vector for gene therapy and vaccines. Such mechanisms may also be applicable to other nonenveloped viruses, including adeno-associated viruses and other viruses containing multiple envelope surface proteins, like influenza. To analyze the contribution of anti-capsid antibodies to neutralization by different human serum samples, wild-type and chimeric vectors were utilized. For example, a rAd type 5 (rAd5) vector with a fiber derived from Ad35 fiber (rAd5 F35) can be used to analyze the anti-Ad5 capsid response independent of fiber. Conversely, a rAd35 vector with a fiber transposed from Ad5 can determine the specificity of neutralization mediated by the Ad5 fiber. Using these vectors, we have analyzed human serum samples from two HIV vaccine clinical trials, VRC 006 and HVTN 204, in which a single-dose rAd5 vaccine alone and a three-dose DNA prime/single dose rAd5 boost vaccine encoding HIV-1 Env A,B, and C; Gag; and Pol, respectively, were administered. Thus, we sought to characterize the specificity of rAd5 neutralizing antibodies in Ad5-immune subjects and to determine their effect on immune responses elicited by vaccination.