Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara.

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present "gold standard" of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.

Suicide induced by cytolytic activity controls the differentiation of memory CD8(+) T lymphocytes.

Cytotoxic T lymphocytes (CTL) confer protection against intracellular pathogens, yet the mechanism by which some escape activation induced cell death (AICD) and give rise to long-lived memory cells is unclear. We studied the differentiation of transgenic TCR CD8(+) cells into CTL and memory cells using a novel system that allowed us to control cytolytic activity. The perforin/granzyme granules used to lyse targets induced the apoptosis of CTL in a fratricide-independent manner. After adoptive transfer to antigen-free mice, the ability of CTL to give generate memory cells was determined. We found that the extent of cytolysis by a common pool of CTL controlled the differentiation into memory cells, which were only generated under conditions of minimal cytolytic activity. Thus, the differentiation of naive CD8(+) cells into memory cells may not depend on the presence on a subset of committed CTL precursors, but rather is controlled by the extent of granule-mediated cytolysis.

Generation and characterization of recombinant vascular targeting agents from hybridoma cell lines.

Vascular targeting agents (VTAs) can be produced by linking antibodies or antibody fragments directed against endothelial cell markers to effector moieties. So far, it has been necessary to produce the components of VTAs (antibody, antibody fragment, linker, and effector) separately and, subsequently, to conjugate them by biochemical reactions. We devised a cloning and expression system to allow rapid generation of recombinant VTAs from hybridoma cell lines. The VTAs consist of a single chain Fv antibody fragment as a targeting moiety and either truncated Pseudomonas exotoxin (resulting in immunotoxins) or truncated human tissue factor (resulting in coaguligands) as effectors. The system was applied to generate recombinant immunotoxins and coaguligands directed against endoglin, vascular endothelial growth factor (VEGF):VEGF receptor (VEGFR) complex and vascular cell adhesion molecule 1 (VCAM-1). The fusion proteins exhibited similar functional activity to analogous biochemical constructs. This is the first report to describe the generation and characterization of recombinant coaguligands.

Conceptual implicit memory performance in Alzheimer's disease.

Alzheimer's disease (AD) patients often exhibit deficits on conceptual implicit memory tests such as category exemplar generation and word association. However, these tests rely on word production abilities, which are known to be disrupted by AD. The current study assessed conceptual implicit memory performance in AD patients and elderly control participants using a conceptual priming task that did not require word production (i.e., semantic decision). Memory performance was also examined using a category exemplar generation test (i.e., a conceptual priming task that required word production) and a recognition memory test. AD patients exhibited deficits on the semantic decision task, the category exemplar generation task, and the recognition memory task. The results indicate that the conceptual memory deficits observed in AD patients cannot be attributed completely to word production difficulties.

Affinity of thymic self-peptides for the TCR determines the selection of CD8(+) T lymphocytes in the thymus.

Experiments with synthetic antigen peptides have suggested that a critical parameter that determines the developmental fate of an immature thymocyte is the affinity of interaction between TCR and self-peptide/MHC expressed on thymic stromal cells. To test the physiological relevance of this model for thymocyte development, we determined the affinity of the anti-HY TCR (B6.2.16) expressed on CD8(+) cells for thymic self-peptide/H-2D(b) tetramers, then examined the ability of these self-peptides to determine the outcome of B6.2.16 CD8 cell selection in the thymus. The B6.2.16 TCR bound the male HY self-antigen with high affinity. Thymic self-peptides, which are highly abundant on the surface of thymic epithelial cells, bound the B6.2.16 TCR with low affinity. The ability of self-peptides to trigger positive or negative selection of B6.2.16 CD8 cells in cultured fetal thymi was determined by the relative affinity of self-peptide/H-2D(b) for the B6.2.16 TCR. High-affinity binding of the HY self-peptide resulted in B6.2.16 TCR complex zeta chain phosphorylation and the negative selection of B6.2.16 CD8 cells. Low-affinity binding of thymic self-peptides to B6.2.16 TCR resulted in the positive selection of B6.2.16 CD8 cells. Differences between the binding affinities of self-peptides to B6.2.16 TCR accounted for the self-peptide specificity of B6.2.16 CD8 cell positive selection. We conclude that the relative affinity of TCR for thymic self-peptide/class I MHC is a critical parameter in determining fate of CD8(+) cells during thymic selection.

A role for the region encompassing the c" strand of a TCR V alpha domain in T cell activation events.

The distinct strand topology of TCR V alpha domains results in a flatter surface in the region encompassing the c" strand than the corresponding region in Ig V domains. In the current study a possible role for this region in T cell activation has been investigated by inserting a potential glycosylation site at V alpha residue 82. This residue is in proximity to the c" strand and distal to the putative interaction site for cognate peptide:MHC ligand. An additional N-linked carbohydrate at this position would create a protrusion on the V alpha domain surface, and this may interfere with TCR aggregation and/or recruitment of signaling molecules. The modified TCR has been expressed in transfected T cells, and the phenotype following stimulation has been compared with that of cells expressing the wild-type TCR. The mutation has significant effects on activation-induced cell death and TCR internalization, but, unexpectedly, does not affect IL-2 secretion. Furthermore, analyses with tetrameric, peptide:MHC class II complexes suggest that the mutation decreases the ability of the TCR to aggregate into a configuration compatible with avid binding by these multivalent ligands.

The porcine humoral immune response against pseudorabies virus specifically targets attachment sites on glycoprotein gC.

High titers of virus-neutralizing antibodies directed against glycoprotein gC of Pseudorabies virus (PRV) (Suid herpesvirus 1) are generally observed in the serum of immunized pigs. A known function of the glycoprotein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains (HBDs). Therefore, it was suggested that the virus-neutralizing activity of anti-PRV sera is directed against HBDs on gC. To address this issue, sera with high virus-neutralizing activity against gC were used to characterize the anti-gC response. Epitope mapping demonstrated that amino acids of HBDs are part of an antigenic antibody binding domain which is located in the N-terminal part of gC. Binding of antibodies to this antigenic domain of gC was further shown to interfere with the viral attachment. Therefore, these results show that the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance induction against self-proteins, which utilize similar HBDs to promote host protein-protein interactions. The findings indicate that the host's immune system can specifically block the attachment function of PRV gC. Since HBDs promote the attachment of a number of herpesviruses, the design of future antiherpesvirus vaccines should aim to induce a humoral immune response that prevents HBD-mediated viral attachment.

Naturally occurring and experimentally induced tip-of-the-tongue experiences in three adult age groups.

Tip-of-the-tongue (TOT) experiences were examined in 30 young (ages 18-24 years), 30 young-old (ages 60-74), and 30 old-old (ages 80-92) adults. In Study 1, TOT experiences were experimentally induced with definitions of to-be-retrieved targets. If the target was not retrieved, orthographic or semantic cues were provided. Age-related increases in the occurrence of TOT experiences and in the time needed to resolve TOT experiences were found for young versus young-old and young-old versus old-old groups; all comparisons were significant except for young versus young-old TOT occurrence, which approached significance. In Study 2, the same participants recorded naturally occurring TOT experiences in structured diaries during a 4-week interval. Both the number of TOT experiences and the resolution time for TOT experiences increased with age. However, the percentage of TOT experiences resolved was equal across age groups; given enough time, even the oldest participants resolved virtually all TOT experiences.

Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.

Independent frontal-system deficits in schizophrenia: cognitive, clinical, and adaptive implications.

This study examined whether frontal-system impairments in schizophrenia occur independently of one another and whether they have distinct implications for information processing, symptom severity, and adaptive functioning. We assessed 26 medication-free schizophrenic outpatients and 18 normal control subjects on eight frontally mediated tasks, semantic information processing, IQ, the BPRS, and long-term psychosocial adaptation. Schizophrenic subjects showed three types of deficits, which were uncorrelated with one another: (1) Executive dysfunction (inflexible problem solving) was related to decreased use of expectancy during controlled semantic priming, lower intelligence, more severe negative symptoms and stereotyped mannerisms. (2) Disinhibition of responses (to irrelevant stimuli) was associated with increased automatic priming, a trend for more severe hallucinations, and was unrelated to intelligence. (3) Motor dyscoordination (inaccurate, dysfluent motor sequencing) was not related to semantic processing, intelligence, or symptoms. Furthermore, all three impairments were unrelated to generalized slowness, age, sex, illness length, or pre-washout neuroleptic dose. Two deficits accounted for aspects of long-term psychosocial adaptation, even after statistical correction for IQ: Executive dysfunction was associated with younger illness onset, poor purposefulness and planning, impaired social relations, and lower global functioning. Motor dyscoordination was associated with poor treatment outcome and restricted educational advancement. Furthermore, executive and motor deficits interacted significantly; subjects who had both deficits showed the least favorable treatment outcome. These findings are neither consistent with generalized impairment nor with a unitary 'frontal syndrome' in schizophrenia. They provide preliminary evidence for at least three frontal-system deficits (dorsolateral, orbital, and premotor), which are dissociable from one another, can occur without general intellectual impairment, and have distinct implications for long-term adaptive functioning.

Linear differentiation of cytotoxic effectors into memory T lymphocytes.

A central question in immunology is the origin of long-lived T cell memory that confers protection against recurrent infection. The differentiation of naïve T cell receptor transgenic CD8+ cells into effector cytotoxic T lymphocytes (CTLs) and memory CD8+ cells was studied. Memory CD8+ cells that were generated after strong antigenic stimulation were the progeny of cytotoxic effectors and retained antigen-specific cytolytic activity 10 weeks after adoptive transfer to antigen-free recipient mice. Thus, potential vaccines based on CTL memory will require the differentiation of naïve cells into post-effector memory T cells.

Well-organized conceptual domains in Alzheimer's disease.

We used a novel apparatus called the flags board to elicit similarity judgments from 32 Alzheimer's disease (AD) patients and 32 elderly normal (EN) controls for two 12-member conceptual domains, ANIMALS and (musical) INSTRUMENTS. Based on Pathfinder and multidimensional scaling (MDS) analyses, performance by AD patients was nearly identical to that of EN controls for ANIMALS. Performance differed for INSTRUMENTS, but the AD group's Pathfinder network was found to agree with the intuitions of a panel of 18 raters as well as the EN group's. MDS analysis showed no deficit on abstract dimensions for the AD group, for either domain. The results are discussed in the context of degradation versus preservation of semantic memory in AD.

Predictors of perceived memory impairment: do they differ in Alzheimer's disease versus normal aging?

Predictors of perceived memory impairment were investigated in 40 elderly normal adults and 28 individuals with Alzheimer's disease. Measures of perceived memory impairment, global cognitive functioning, memory, use of memory strategies, memory strategy efficacy, and depressive symptomatology were obtained for all participants. The elderly normal and Alzheimer's disease groups did not differ in the extent to which they reported perceived memory impairment. For both participant groups, more frequent use of memory strategies and lower perceived memory strategy efficacy were significant predictors of perceived memory impairment. Depressive symptomatology was an additional, significant determinant of perceived memory impairment for the elderly normal group.

Slower and more variable reaction times in schizophrenia: what do they signify?

Extensive research has demonstrated that schizophrenic subjects are slower than normal comparison subjects on a range of reaction-time tasks. Some investigators have also observed that schizophrenic patients exhibit larger intraindividual variability in reaction times when performing these tasks than do normal comparison subjects. This study, using a lexical decision choice reaction time (CRT) task, explored the relation of mean CRT and it intra-individual variability (CRT-SD) to psychiatric symptoms and to performance on executive-motor tasks in 26 medication-free schizophrenic out-patients and 17 normal comparison subjects. Schizophrenic subjects had both significantly slower and more variable CRTs which were unrelated to general intellectual abilities (IQ). Among schizophrenic subjects, both CRT and CRT-SD were significantly related to severity of psychotic symptoms, failure to maintain cognitive set, and poorer motor coordination and global functioning. After controlling for mean CRT, CRT-SD showed unique covariation with clinical symptoms (positive, disorganized and tension/hostility). Conversely, mean CRT showed unique covariation with the failure to maintain cognitive set and with stereotypic mannerisms, independent of CRT-SD. These results suggest that slower CRT and increased intra-individual variability in CRT, while not fully independent of one another, may reflect separate aspects of symptomatic and cognitive dysfunction in schizophrenia.

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases.

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.

Vaccine-induced, pseudorabies virus-specific, extrathymic CD4+CD8+ memory T-helper cells in swine.

Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4+CD8+ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4+CD8+ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4+CD8+ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4+CD8+ memory T lymphocytes and showed that CD4+CD8+ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4+CD8+ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.

Automatic versus controlled semantic priming in schizophrenia.

Schizophrenic individuals (n = 31), including paranoid and nonparanoid diagnostic subgroups, and normal controls (n = 20) participated in a semantic priming experiment involving a single-choice lexical decision task. For the automatic priming blocks, a 260-ms stimulus onset asynchrony (SOA) was used; for the controlled priming blocks, 1,000-ms SOA was used. The paranoid subgroup showed significantly less priming than did the control group. The nonparanoid subgroup showed a decrease in priming compared with the control group that approached significance. There was an increased priming effect for the controlled compared with the automatic priming condition; this difference was not modulated by participant group. Nonsignificant semantic priming (equal to 0) occurred only for schizophrenic subgroups and only in automatic priming conditions.

Recombinant T cell receptor molecules can prevent and reverse experimental autoimmune encephalomyelitis: dose effects and involvement of both CD4 and CD8 T cells.

Autoimmune diseases are often characterized by spontaneous remission followed by relapses. Although the mechanism(s) controlling pathogenic self-reactive T cells are not fully understood, recent data in experimental autoimmune encephalomyelitis (EAE), a prototype for CD4 T cell-mediated autoimmune diseases, indicate that spontaneous recovery is mediated by regulatory T cells (Treg) specific for peptides derived from the beta-chain of the TCR. Here we have tested whether recombinant single-chain TCRs (scTCRs) containing Vbeta domains can be used as vaccines for efficient priming of Treg. A single injection of mice with these recombinant proteins leads to efficient in vivo priming of Treg and almost complete protection from Ag-induced EAE. Significantly, administration of scTCRs during ongoing disease at a 10-fold lower dose than that required for prophylactic treatment also reverses established EAE. However, if a higher dose of scTCR is administered during ongoing disease, paralytic symptoms become exacerbated and the majority of treated animals die from severe and chronic EAE. Furthermore, we demonstrate that regulatory determinants are processed and presented from scTCRs resulting in the recruitment of both CD4 and CD8 regulatory T cells which are required for efficient regulation induced by scTCR. Reversal of established disease following an optimum dose of recombinant TCRs suggests that proteins expressing appropriate Vbeta domains could be used for the treatment of a variety of T cell-mediated pathologic conditions.

Clinical and neurocognitive aspects of source monitoring errors in schizophrenia.

OBJECTIVE: Source monitoring, an aspect of memory that involves judgments about the origin of information, has been found to be more prone to errors in schizophrenic subjects than in normal persons. To examine the precise nature of such errors and their relationship to clinical and neurocognitive variables, the authors compared schizophrenic and normal subjects. METHOD: Schizophrenic subjects who had been medication free for 1 week (N = 26) and demographically matched normal subjects (N = 21) performed a source monitoring task and were assessed on current psychiatric symptoms, IQ, and frontal lobe functioning. RESULTS: The schizophrenic subjects had normal recognition memory of target words (recognition hits) and a normal generation effect but made more errors than the comparison subjects in identifying the source of target words. Specifically, the schizophrenic subjects made more errors in remembering the source of new and self-generated items, and they tended to attribute items to an external source. In 11 retested subjects, these errors were stable and independent from medication status after a 2-year interval. Secondary analyses suggested that certain source monitoring errors may be associated with hostility and lower IQ. When the effect of IQ was controlled, correlations with frontal dysfunction were not significant. CONCLUSIONS: Schizophrenic subjects make significantly more source monitoring errors than normal subjects, but not because of problems with recognition memory hits or with the generation effect. This tendency may be trait like and may be related to hostility. Lower IQ in schizophrenia plays a partial role in these errors, but frontal dysfunction does not.

Methodological control of semantic priming in Alzheimer's disease.

We conducted a lexical-decision, semantic priming experiment that included 250- and 1000-ms stimulus onset asynchronies (SOAs) with 32 probable Alzheimer's disease (AD) and 40 older normal persons. Attention-based, controlled processes are assumed to occur only at the longer of the 2 SOAs. The AD group showed greater than normal priming in the long-SOA but not the short-SOA condition. We conclude that greater than normal AD priming is a function of controlled processing rather than semantic network degradation.