Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

BACKGROUND: Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. METHODS: Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. RESULTS: Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. CONCLUSIONS: LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

Investigation of electro-vascular phase-amplitude coupling during an auditory task.

Multimodal neuroimaging using electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS) provides complementary views of cortical processes, including those related to auditory processing. However, current multimodal approaches often overlook potential insights that can be gained from nonlinear interactions between electrical and hemodynamic signals. Here, we explore electro-vascular phase-amplitude coupling (PAC) between low-frequency hemodynamic and high-frequency electrical oscillations during an auditory task. We further apply a temporally embedded canonical correlation analysis (tCCA)-general linear model (GLM)-based correction approach to reduce the possible effect of systemic physiology on fNIRS recordings. Before correction, we observed significant PAC between fNIRS and broadband EEG in the frontal region (p ≪ 0.05), ß (p ≪ 0.05) and γ (p = 0.010) in the left temporal/temporoparietal (left auditory; LA) region, and γ (p = 0.032) in the right temporal/temporoparietal (right auditory; RA) region across the entire dataset. Significant differences in PAC across conditions (task versus silence) were observed in LA (p = 0.023) and RA (p = 0.049) γ sub-bands and in lower frequency (5-20 Hz) frontal activity (p = 0.005). After correction, significant fNIRS-γ-band PAC was observed in the frontal (p = 0.021) and LA (p = 0.025) regions, while fNIRS-α (p = 0.003) and fNIRS-ß (p = 0.041) PAC were observed in RA. Decreased frontal γ-band (p = 0.008) and increased ß-band (p ≪ 0.05) PAC were observed during the task. These outcomes represent the first characterization of electro-vascular PAC between fNIRS and EEG signals during an auditory task, providing insights into electro-vascular coupling in auditory processing.

Individual-specific characterization of event-related hemodynamic responses during an auditory task: An exploratory study.

Functional near-infrared spectroscopy (fNIRS) has been established as an informative modality for understanding the hemodynamic-metabolic correlates of cortical auditory processing. To date, such knowledge has shown broad clinical applications in the diagnosis, treatment, and intervention procedures in disorders affecting auditory processing; however, exploration of the hemodynamic response to auditory tasks is yet incomplete. This holds particularly true in the context of auditory event-related fNIRS experiments, where preliminary work has shown the presence of valid responses while leaving the need for more comprehensive explorations of the hemodynamic correlates of event-related auditory processing. In this study, we apply an individual-specific approach to characterize fNIRS-based hemodynamic changes during an auditory task in healthy adults. Oxygenated hemoglobin (HbO2) concentration change time courses were acquired from eight participants. Independent component analysis (ICA) was then applied to isolate individual-specific class discriminative spatial filters, which were then applied to HbO2 time courses to extract auditory-related hemodynamic features. While six of eight participants produced significant class discriminative features before ICA-based spatial filtering, the proposed method identified significant auditory hemodynamic features in all participants. Furthermore, ICA-based filtering improved correlation between trial labels and extracted features in every participant. For the first time, this study demonstrates hemodynamic features important in experiments exploring auditory processing as well as the utility of individual-specific ICA-based spatial filtering in fNIRS-based feature extraction techniques in auditory experiments. These outcomes provide insights for future studies exploring auditory hemodynamic characteristics and may eventually provide a baseline framework for better understanding auditory response dysfunctions in clinical populations.

Disruptions of cortico-kinematic interactions in Parkinson's disease.

The cortical role of the motor symptoms reflected by kinematic characteristics in Parkinson's disease (PD) is poorly understood. In this study, we aim to explore how PD affects cortico-kinematic interactions. Electroencephalographic (EEG) and kinematic data were recorded from seven healthy participants and eight participants diagnosed with PD during a set of self-paced finger tapping tasks. Event-related desynchronization (ERD) was compared between groups in the α (8-14 Hz), low-ß (14-20 Hz), and high-ß (20-35 Hz) frequency bands to investigate between-group differences in the cortical activities associated with movement. Average kinematic peak amplitudes and latencies were extracted alongside Sample Entropy (SaEn), a measure of signal complexity, as variables for comparison between groups. These variables were further correlated with average EEG power in each frequency band to establish within-group interactions between cortical motor functions and kinematic motor output. High ß-band power correlated with mean kinematic peak latency and signal complexity in the healthy group, while no correlation was found in the PD group. Also, the healthy group demonstrated stronger ERD in the broad ß-band than the PD participants. Our results suggest that cortical ß-band power in healthy populations is graded to finger tapping latency and complexity of movement, but this relationship is impaired in PD. These insights could help further enhance our understanding of the role of cortical ß-band oscillations in healthy movement and the possible disruption of that relationship in PD. These outcomes can provide further directions for treatment and therapeutic applications and potentially establish cortical biomarkers of Parkinson's disease.

An fNIRS-Based Motor Imagery BCI for ALS: A Subject-Specific Data-Driven Approach.

OBJECTIVE: Functional near-infrared spectroscopy (fNIRS) has recently gained momentum in research on motor-imagery (MI)-based brain-computer interfaces (BCIs). However, strikingly, most of the research effort is primarily devoted to enhancing fNIRS-based BCIs for healthy individuals. The ability of patients with amyotrophic lateral sclerosis (ALS), among the main BCI end-users to utilize fNIRS-based hemodynamic responses to efficiently control an MI-based BCI, has not yet been explored. This study aims to quantify subject-specific spatio-temporal characteristics of ALS patients' hemodynamic responses to MI tasks, and to investigate the feasibility of using these responses as a means of communication to control a binary BCI. METHODS: Hemodynamic responses were recorded using fNIRS from eight patients with ALS while performing MI-Rest tasks. The generalized linear model (GLM) analysis was conducted to statistically estimate and evaluate individualized spatial activation. Selected channel sets were statistically optimized for classification. Subject-specific discriminative features, including a proposed data-driven estimated coefficient obtained from GLM, and optimized classification parameters were identified and used to further evaluate the performance using a linear support vector machine (SVM) classifier. RESULTS: Inter-subject variations were observed in spatio-temporal characteristics of patients' hemodynamic responses. Using optimized classification parameters and feature sets, all subjects could successfully use their MI hemodynamic responses to control a BCI with an average classification accuracy of 85.4% ± 9.8%. SIGNIFICANCE: Our results indicate a promising application of fNIRS-based MI hemodynamic responses to control a binary BCI by ALS patients. These findings highlight the importance of subject-specific data-driven approaches for identifying discriminative spatio-temporal characteristics for an optimized BCI performance.

Multimodal exploration of non-motor neural functions in ALS patients using simultaneous EEG-fNIRS recording.

OBJECTIVE: Despite the high prevalence of non-motor impairments reported in patients with amyotrophic lateral sclerosis (ALS), little is known about the functional neural markers underlying such dysfunctions. In this study, a new dual-task multimodal framework relying on simultaneous electroencephalogram (EEG) and functional near-infrared spectroscopy (fNIRS) recordings was developed to characterize integrative non-motor neural functions in people with ALS. APPROACH: Simultaneous EEG-fNIRS data were recorded from six subjects with ALS and twelve healthy controls. Through a proposed visuo-mental paradigm, subjects performed a set of visuo-mental arithmetic operations. The data recorded were analyzed with respect to event-related changes both in the time and frequency domains for EEG and de/oxygen-hemoglobin level (HbR/HbO) changes for fNIRS. The correlation of EEG spectral features with fNIRS HbO/HbR features were then evaluated to assess the mechanisms of ALS on the electrical (EEG)-vascular (fNIRS) interrelationships. MAIN RESULTS: We observed overall smaller increases in EEG delta and theta power, decreases in beta power, reductions in HbO responses, and distortions both in early and later EEG event-related potentials in ALS subjects compared to healthy controls. While significant correlations between EEG features and HbO responses were observed in healthy controls, these patterns were absent in ALS patients. Distortions in both electrical and hemodynamic responses are speculated to be associated with cognitive deficits in ALS that center primarily on attentional and working memory processing. SIGNIFICANCE: Our results highlight the important role of ALS non-motor dysfunctions in electrical and hemodynamic neural dynamics as well as their interrelationships. The insights obtained through this study can enhance our understanding of the underlying non-motor neural processes in ALS and enrich future diagnostic and prognostic techniques.

An exploration of neural dynamics of motor imagery for people with amyotrophic lateral sclerosis.

OBJECTIVE: Studies of the neuropathological effects of amyotrophic lateral sclerosis (ALS) on the underlying motor system have investigated abnormalities in the magnitude and timing of the event-related desynchronization (ERD) and synchronization (ERS) during motor execution (ME). However, the spatio-spectral-temporal dynamics of these sensorimotor oscillations during motor imagery (MI) have not been fully explored for these patients. This study explores the neural dynamics of sensorimotor oscillations for ALS patients during MI by quantifying ERD/ERS features in frequency, time, and space. APPROACH: Electroencephalogram (EEG) data were recorded from six patients with ALS and 11 age-matched healthy controls (HC) while performing a MI task. ERD/ERS features were extracted using wavelet-based time-frequency analysis and compared between the two groups to quantify the abnormal neural dynamics of ALS in terms of both time and frequency. Topographic correlation analysis was conducted to compare the localization of MI activity between groups and to identify subject-specific frequencies in the µ and ß frequency bands. MAIN RESULTS: Overall, reduced and delayed ERD was observed for ALS patients, particularly during right-hand MI. ERD features were also correlated with ALS clinical scores, specifically disease duration, bulbar, and cognitive functions. SIGNIFICANCE: The analyses in this study quantify abnormalities in the magnitude and timing of sensorimotor oscillations for ALS patients during MI tasks. Our findings reveal notable differences between MI and existing results on ME in ALS. The observed alterations are speculated to reflect disruptions in the underlying cortical networks involved in MI functions. Quantifying the neural dynamics of MI plays an important role in the study of EEG-based cortical markers for ALS.

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.

Synthesis of immunogenic hepatitis A virus particles by recombinant baculoviruses.

Recombinant baculoviruses were constructed which contained the hepatitis A virus (HAV) open reading frame (ORF) under the control of the polyhedrin promoter. Northern blot analysis with an HAV-specific oligonucleotide probe demonstrated a single transcript large enough to include the HAV ORF in Spodoptera frugiperda cells infected with these recombinants. Immunoblots revealed a 220 kDa protein representing the HAV polyprotein. In addition, proteins which co-migrated with HAV capsid proteins, and several proteins of intermediate size were present, consistent with processing intermediates. HAV antigen was present in cells infected with the recombinant baculoviruses when assessed by solid-phase radioimmunoassay. This HAV antigen had a buoyant density in caesium chloride gradients similar to HAV empty capsids, and elicited HAV neutralizing antibodies in mice.

Conformational studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU.

Among the various proposals that have been made in attempting to explain the ability of thermophiles to reproduce at high temperatures, there is no doubt that obligate and extreme thermophiles synthesize proteins (and other molecules) that have sufficient intrinsic molecular stability to withstand increased thermal stress. In contrast, the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU has been shown to be quite thermolabile in vitro. Thermal inactivation is not due to loss of bound NAD+. It has also been shown that the enzymatic activity can be thermostabilized in vitro by increased ionic strength. As previously reported [J. W. Crabb, A. L. Murdock, and R. E. Amelunxen (1975) Biochem. Biophys. Res. Commun. 62, 627; (1977) Biochemistry 16, 4840], the enzyme loses 94-97% of enzymatic activity after heat treatment at 55 degrees C for 5 min in 0.05 M sodium phosphate buffer (pH 7.1); however, by increasing the ionic strength to 1.8, complete protection was conferred at this temperature. Gel-filtration chromatography has been used to study the initial dissociation and subsequent aggregation of the glyceraldehyde-3-phosphate dehydrogenase after thermal inactivation. Aggregation occurs when the enzyme is heated at 50 degrees or 55 degrees C. Loss of enzymatic activity is correlated with changes in the tertiary structure as measured by the near-uv CD spectrum of the enzyme following heat inactivation, with essential disappearance of the peaks at 263 and 296 nm, and a blue shift of the far-uv spectrum, which is a measure of secondary structure. Estimation of secondary structure of the unheated protein from the far-uv CD data showed the enzyme contains approximately 26% alpha-helix, approximately 21% beta-structure, and approximately 53% disordered structure. Heat treatment at various temperatures resulted in only slight changes of the estimated secondary structure. Increased ionic strength prevents thermal alteration of the CD spectrum in both near- and far-uv regions. The data support the previous proposal that thermolabile enzymes such as the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile B. coagulans are thermostabilized in vivo mainly by the intracellular charged macromolecular environment.

Expression of foreign epitopes on recombinant occlusion bodies of baculoviruses.

Recombinant polyhedrin proteins of the baculovirus Autographa californica nuclear polyhedrosis virus were constructed to serve as immunologic carriers of foreign epitopes. Several recombinants containing an influenza haemagglutinin epitope were obtained. Three of the five recombinants formed occlusion bodies (OBs) and two did not. All of the recombinant polyhedrin proteins reacted with a monoclonal antibody (mAb) specific for an influenza epitope in Western blots. Presentation of the foreign epitope on the surface of recombinant OBs was demonstrated by specific immunoprecipitation of the anti-influenza mAb with recombinant OBs. The recombinant polyhedrin-influenza epitope fusion protein stimulated an influenza-specific immune response in rabbits.

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli.

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R . Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.

Antigenic and immunogenic properties of recombinant hepatitis A virus 14S and 70S subviral particles.

Hepatitis A virus (HAV) has an immunodominant neutralization antigenic site. By using a panel of monoclonal antibodies targeted against the HAV neutralization antigenic site, it was shown that three epitopes within this site are present on 14S subunits (pentamers of the structural unit). In contrast, two other epitopes within this site are formed upon assembly of 14S subunits into capsids. Thus, the epitopes recognized by these two monoclonal antibodies are formed either by a conformational change in the antigenic site or by the juxtaposition of epitope fragments present on different 14S subunits during assembly of 14S into 70S particles. Both 14S and 70S particles elicited HAV-neutralizing antibodies in mice; thus, these particles may be useful for HAV vaccine development.

Characterization of hepatitis G virus (GB-C virus) particles: evidence for a nucleocapsid and expression of sequences upstream of the E1 protein.

Hepatitis G virus (HGV or GB-C virus) is a newly described virus that is closely related to hepatitis C virus (HCV). Based on sequence analysis and by evaluation of translational initiation codon preferences utilized during in vitro translation, HGV appears to have a truncated or absent core protein at the amino terminus of the HGV polyprotein. Consequently, the biophysical properties of HGV may be very different from those of HCV. To characterize HGV particle types, we evaluated plasma from chronically infected individuals with and without concomitant HCV infection by using sucrose gradient centrifugation, isopycnic banding in cesium chloride, and saline density flotation centrifugation. Similar to HCV, HGV particles included an extremely-low-density virion particle (1.07 to 1.09 g/ml) and a nucleocapsid of approximately 1.18 g/ml. One major difference between the particle types was that HGV was consistently more stable in cesium chloride than HCV. Plasma samples from chronically HGV-infected individuals and controls were assessed by a synthetic peptide-based immunoassay to determine if they contained HGV antibody specific for a conserved region in the coding region upstream of the E1 protein. Chronically HGV-infected individuals contained antibody to the HGV core protein peptide, whereas no binding to a hepatitis A virus peptide control was observed. Competitive inhibition of binding to the HGV peptide confirmed the specificity of the assay. These data indicate that HGV has a nucleocapsid and that at least part of the putative core region of HGV is expressed in vivo.

A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma.

Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease. Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany. Southern analysis using a human progesterone receptor (hPR) cDNA probe identified a germline TaqI restriction fragment length polymorphism (RFLP) defined by two alleles: T1, represented by a 2.7 kb fragment; and T2, represented by a 1.9 kb fragment and characterised by an additional TaqI restriction site with respect to T1. An over-representation of T2 in ovarian cancer patients compared with controls in the pooled Irish/German population (P < 0.025) was observed. A difference (P < 0.02) in the distribution of the RFLP genotypes between Irish and German control populations was also observed. The allele distributions could not be shown to differ significantly from Hardy-Weinberg distribution in any subgroup. Using hPR cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene.

Sequence homology in the amino-terminal and active-site regions of thermolabile glyceraldehyde-3-phosphate dehydrogenase from a thermophile.

The unusual thermolability of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU (Crabb et al., Biochemistry 16:4840-4847, 1977) has provided the first opportunity to study a homologous enzyme from the same genus that exhibits a marked difference in thermostability. In pursuit of the structural bases for the thermostability of proteins, the sequences of the amino terminus (residues 1 through 27) and the active-site cysteine cyanogen bromide peptide (residues 130 through 167) of this enzyme have been determined and compared with sequences of the enzyme from other sources. The importance of comparing phylogenetically related proteins is evident from the 87% identity found between these sequences in the enzyme from B. coagulans and Bacillus stearothermophilus, versus only 45% identity for all other known sequences. The marked sequence identity of the enzyme from the two Bacillus species drew attention to the variable region (residues 138 through 140a) which is exposed to the exterior of the quaternary structure of this enzyme. Based on the reported crystallographic structures of the enzyme from lobster muscle and B. stearothermophilus and space-filling models of the variable region, the segment Asp-Pro-Lys-Ala in B. stearothermophilus should be more thermostable than the analogous sequence, Asp-Ala-Ala-Asn, from B. coagulans. In addition, the space-filling models suggested that the spatial relationship of an amino acid side chain and its potential for close packing and interactions with neighboring side chains may be more important than the type of amino acid substituted.

Expression of human plasminogen cDNA in a baculovirus vector-infected insect cell system.

A cDNA that encodes the human plasminogen (HPg) amino acid sequence has been inserted adjacent to the polyhedrin promoter in the genome of the baculovirus, Autographa californica nuclear polyhedrosis virus, which was then used to infect cultured cells of the farm armyworm, Spodoptera frugiperda. Under the conditions of cell growth employed, recombinant (rec)-HPg was secreted into the medium after 24 h postinfection (p.i.), at which point virtually no rec-HPg antigen remained inside the cells. At 48 h p.i., a maximal level of intact rec-HPg was present in the medium, which underwent substantial proteolytic digestion after that time. The rec-HPg produced by this expression system possessed a molecular weight equivalent to that of plasma [Glu1]-plasminogen. In addition, the rec-HPg adsorbed to Sepharose-lysine, and was eluted with epsilon-aminocaproic acid (EACA). The recombinant protein also interacted with polyclonal antibodies generated to plasma HPg, as well as with a monoclonal antibody directed against a distinct region (kringle 1-3) of the plasma HPg molecule. Finally, the insect-expressed rec-HPg was activatable to plasmin (HPm) by urokinase. The results demonstrate that this expression system produces a full-length functional single-chain rec-HPg, which can be isolated intact from the culture medium, with some consideration for the temporal events that occur in secretion and longer-term degradation of the protein. The fact that this rec-HPg was converted to HPm with a plasminogen activator, and that it interacted with anti-plasma HPg polyclonal and monoclonal antibodies, as well as with the ligand, EACA, indicates that the molecule retains many of its important functional properties and is folded in an integral manner.