An animal model of human-type memory loss based on aging, lesion, forebrain ischemia, and drug studies with the rat

The goals of this research were to develop a within-subject test of spatial working memory and performance for the rat in a T-maze, based on a delayed alternation, or "win-shift" foraging strategy. Using this model, specific aims were to compare the effects of: (1) age, (2) basal forebrain, medial septal, and amygdala lesions, (3) four vessel occlusion (4-VO), forebrain ischemia, and (4) physostigmine, scopolamine, arecoline, piracetam, and clonidine on memory and performance of young middle-aged, and old rats. Aging significantly impaired working memory and performance of Long-Evans rats. Memory of septal and basal forebrain, but not of amygdala lesioned rats was significantly impaired without effects on performance. Transient, 4-VO forebrain ischemia produced significant memory impairment, without effects on performance, and highly selective CA1 cell loss in the hippocampus. Physostigmine enhanced working memory in middle-aged and old rats. Scopolamine impaired memory in young, middle-aged, and old rats. Physostigmine reversed the scopolamine impairments of working memory. Arecoline enhanced memory in old rats without effects on performance. Piracetam and clonidine had no direct effects on memory, but piracetam increased and clonidine decreased speed of performance. From the aging, lesion, ischemia, and drug studies it was concluded that there was a convergence of evidence from 4 different approaches for a critical role for the hippocampus, particularly the CA1 fields, in spatial working memory.     

A phage display system for detection of T cell receptor-antigen interactions

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCRα chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCRα chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the α chains were derived. Cross-specificity analysis using two antigen-binding TCRα chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCRα-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.     

Analysis of Host Cells Associated with the Spv-Mediated Increased Intracellular Growth Rate of Salmonella typhimurium in Mice

The 90-kb virulence plasmid of Salmonella typhimurium encodes five spv genes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P. A. Gulig and T. J. Doyle, Infect. Immun. 61:504–511, 1993). The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae. To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice. Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv⁺ or Spv⁻ S. typhimurium strains at 5 days postinoculation. When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv⁺ versus Spv⁻ S. typhimurium was not changed after oral inoculation. In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv⁺ and Spv⁻ salmonellae. To examine if the spv genes affected the growth of salmonellae in nonphagocytic cells, an invA::aphT mutation was transduced into Spv⁺ and Spv⁻ S. typhimurium strains. InvA⁻ Spv⁺ salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA⁻ Spv⁻ salmonellae were only slightly attenuated relative to InvA⁺ Spv⁻ salmonellae. Invasion-defective salmonellae still exhibited the Spv phenotype. Therefore, infection of nonphagocytes is not involved with the Spv virulence function. Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv⁻ S. typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.     

Physical fitness of blind and sighted children

Summary Twenty-seven children (age 7–17 years) with varying degrees of blindness but with no other known disorder were assessed for physical fitness. Twenty-seven randomly selected children with normal eyesight were also assessed. Maximum oxygen uptake ( ) was measured directly during a progressive exercise test on a treadmill. There was a significant and substantial reduction in in totally blind children (mean ± standard deviation 35.0±7.5 ml · min^–1 · kg^–1) compared with normal children (45.9±6.6 ml · min^–1 · kg^–1). Partially sighted children had a significant but smaller reduction in . Fitness assessed by a step-test was significantly reduced in the visually impaired children, and skin-fold thickness was also significantly greater in totally blind children.The level of habitual physical activity for each child, as assessed by a questionnaire, correlated with (r=0.53,p<0.0001). Blind children were significantly less active than normal children, and the difference between mean for blind and normal children became non-significant when their different activity levels were taken into account. It is concluded that totally blind children are less fit than other children at least partly because of their lower level of habitual activity.     

Lymphocyte transformation response of calves to respiratory syncytial virus

Virus-specific cell-mediated immunity, as determined by in vitro lymphocyte transformation (LT), was demonstrated in calves following infection and vaccination with respiratory syncytial virus (RSV). After experimental infection, 4 of 6 gnotobiotic calves and 6 of 21 conventional calves developed a significant LT response to RSV. By means of a whole blood assay, the LT responses of calves were examined after vaccination with an inactivated vaccine, which consisted of glutaraldehyde-fixed bovine nasal mucosa cells persistently infected with a bovine strain of RSV (GC), a live modified bovine strain of RSV (MV), or a live temperature-sensitive mutant of a human strain of RSV (ts-l). Three weeks after vaccination, a virus-specific LT response was detected in 6 of 6 calves given the GC vaccine, 0 of 4 calves given the MV vaccine, and 2 of 4 calves given the ts-l vaccine. The magnitude of the response was greatest in those animals given the GC vaccine. There was no significant correlation between the magnitude of the LT response and levels of serum neutralising antibody. However, the LT response did correlate with serum antibody measured by the single radial haemolysis test 3 weeks after the first vaccination. LT activity to RSV was associated with T and not B lymphocytes. The development of a virus-specific LT response in calves given an inactivated RSV vaccine was not associated with an increase in respiratory disease following challenge with live virus, but rather was related to increased resistance to RSV infection.     

Gestational immunosuppression is mediated by specific Lyt 2+ T cells.

Female BALB/c mice were tested during the first week of pregnancy for their lymphocyte-mediated cytotoxic response to paternal alloantigens. Spleen or uterine regional lymph node cells were not spontaneously cytotoxic against concanavalin A-activated paternal target lymphocytes. Female mice immunized i.p. with paternal H-2-matched or third-party allogeneic cells on the fifth day and tested on the 12th day of pregnancy demonstrated total suppression of cell-mediated cytotoxicity to paternal alloantigens and partial suppression to third-party alloantigens. A generalized non-specific immunosuppression to alloantigens seems to be associated with pregnancy, which may indicate that soluble factors were involved in mediating the suppressive effect. Cocultures of spleen cells from virgin mice and the whole population of spleen or regional lymph node cells from allogeneic pregnant female mice demonstrated specifically suppressed responses to alloantigens. Similar cocultures with Thy 1.2- and Lyt 2.2-depleted populations restored the cytotoxicity levels of activated spleen cells. We conclude that antigen-specific Lyt 2+ T cells were activated during pregnancy to regulate the female T-cell response to paternal alloantigens.     

Patterns of aneuploidy in stage IV clear cell renal cell carcinoma revealed by comparative genomic hybridization and spectral karyotyping

We report the use of spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to describe the numerous genomic imbalances characteristic of stage IV clear cell renal cell carcinoma (CCRCC). SKY and CGH were performed on 10 cell lines established from nephrectomy specimens, and CGH on uncultured material from five of the primary renal tumors. The mutational status of VHL (3p25) and MET (7q31), genes implicated in renal carcinogenesis, were determined for each case. Each case showed marked aneuploidy, with an average number of copy alterations of 14.6 (+/-2.7) in the primary tumors and 19.3 (+/-4.6) in the cell lines. Both whole-chromosome and chromosome-segment imbalances were noted by CGH: consistent losses or gains included +5q23-->ter (100%), -3p14-->ter (80%), and +7 (70%). All VHL mutations and 83% of the genomic imbalances found in the primary tumors were also found in the cell lines derived from them. SKY showed many complex structural rearrangements that were undetected by conventional banding analysis in these solid tumors. All cases with VHL inactivation had 3p loss and 5q gain related primarily to unbalanced translocations between 3p and 5q. In contrast, gains of chromosome 7 resulted primarily from whole-chromosome gains and were not associated with mutations of MET. SKY and CGH demonstrated that genomic imbalances in advanced RCC were the result of either segregation errors [i.e., whole chromosomal gains and losses (7.8/case)] or chromosomal rearrangements (10.7/case), of which the majority were unbalanced translocations.     

Death of peripheral CD8+ T cells in the absence of MHC class I is Fas-dependent and not blocked by Bcl-xL

Productive immune responses require an appropriate environment to support peripheral CD8(+) T cell survival. Although host MHC class I molecules appear to be required for this process, the cellular and molecular requirements have not been comprehensively studied. Using adoptive transfer of 2C/recombinase-activating gene-2 (RAG-2)(-/-) TCR-transgenic T cells, we found that the survival of both naive and effector CD8(+) T cells was dependent upon host expression of the same MHC class I alleles that supported thymic selection. Expression of appropriate MHC class Iby either bone marrow- or non-bone-marrow-derived cells was sufficient, suggesting that professional antigen-presenting cells were not mandatory. In contrast to MHC class I, neither T cell expression of CD28 nor host expression of ICAM-1 was required for peripheral T cell survival. Finally, T cell death in the absence of appropriate host MHC class I was overcome by elimination of Fas signaling but not by overexpression of Bcl-x(L) by CD8(+) T cells. These results suggest that, in the absence of a survival signal provided by engagement of host MHC/self peptide complexes, CD8(+) T cells die via a Fas-dependent, mitochondria-independent pathway.