Biomedical subjects
Publications and source records attributed to J F Miller.
The thymus has been an enigmatic organ for centuries. Its true function was revealed only in 1961 when it was shown to be responsible for the proper development of the immune system. This finding has revolutionized the science of immunology and has led to a better understanding of the mechanisms involved in resistance to infections and of the pathogenesis of autoimmune and immunodeficiency diseases.
Diabetes was dramatically accelerated in non-obese diabetic (NOD) transgenic mice that expressed interleukin-2 (IL-2) in their beta cells. A single cross to C57BL/6 completely prevented this effect and a further backcross to the NOD genetic background showed that at least two diabetes susceptibility loci (Idd1s and Idd3/10s) were required for the diabetes acceleration T cells activated to islet antigens were not circulating in the mice. The accelerating effect of IL-2 was present; but decreased, in NOD mice that lacked CD8+ T cells as well as in NOD SCID mice. The implications are that in the NOD genetic background, the production of cytokines, such as IL-2, by islet-specific CD4+ T cells can lead to beta cell damage and diabetes and that CD8+ T cells may have a role in accelerating diabetes onset.
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Experiments with transgenic mice expressing genes encoding both antigens in defined tissues and T-cell receptor genes of known specificities have enhanced our understanding of the mechanisms involved in the pathogenesis of autoimmune states. They have also shed light on the means by which potentially autoreactive cells may be prevented from exerting their autoaggressive potential. The value of the transgenic approach is that it can overcome the low frequency of peptide-specific T cells occurring in normal animals, and also provide a tissue-specific, cognate antigen that is absent in controls. These factors allow reactive T cells to be isolated or quantified by flow cytometry and their responses to antigen in vitro and in vivo be defined.
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This longitudinal investigation charted the course of cognitive and language development from the prelinguistic period to multiword productions in 19 typically developing (TD) toddlers and 4 toddlers who were subsequently identified as having late onset of expressive language. Assessments were conducted at 3-month intervals over a 21-month period, with 12 months of prospective assessment data comparing the TD and late talker (LT) subjects (mean CA: 13 to 25 months) and 9 months of follow-up data (mean CA: 25-34 months) on the 4 subjects who were identified as late talkers at age 2. Three different developmental profiles were identified, and predictors of expressive language outcomes for the LT subjects were evaluated.
Transgenic mice that expressed a single-copy IL-2 transgene in their pancreatic beta cells were previously shown to develop a massive inflammation in and around the islets, but did not progress to diabetes. When these mice were made homozygous for the transgene, diabetes did ensue in most animals by 200 days. Analysis of the T cells present in the pancreatic infiltrates of single-copy and homozygous rat insulin promoter IL-2 mice showed a predominance of CD4+ cells which was especially apparent in the very young mice. Furthermore, many of the CD4+ T cells in young mice displayed a memory-like phenotype in that they expressed higher levels of adhesion molecules and the IL-2R p55 marker. When the IL-2 transgene was introduced into nude mice, an almost identical pathology of inflammation was seen except that the infiltrating cells were mostly B cells. Expression of the same transgene in scid mice also resulted in an inflammatory response and in some situations it was sufficient to induce diabetes. From these results it appears that the T cell product, IL-2, in the absence of antigen-specific T or B cells can induce an inflammatory response of sufficient intensity to cause diabetes.
Members of the Bordetella genus alternate between two distinct phenotypic phases in response to changes in their environment. This switch, termed phenotypic modulation, is mediated by the BvgAS sensory transduction system. We developed an animal model based on the interaction of Bordetella bronchiseptica with one of its natural hosts, the rabbit. To investigate the importance of BvgAS signal transduction, we constructed constitutive (RB53) and Bvg- (RB54) phase-locked derivatives of a wild-type strain, RB50. RB50 and RB53, but not RB54, established respiratory infections in B. bronchiseptica-free rabbits with an intranasal 50% infective dose of less than 200 organisms, and the course of the infection closely resembled that observed with naturally infected rabbits. Bacteria were recovered from the nasal cavity, larynx, trachea, and lungs in similar numbers from RB50- and RB53-infected rabbits, yet no pathology was detected by histological examination of lung and tracheal sections. The antibody responses in rabbits inoculated with RB50 or RB53 were quantitatively and qualitatively indistinguishable; high titers of antibodies were generated primarily against Bvg(+)-phase-specific antigens. No response against flagella, a Bvg- phase factor, was detected. Assessment of bacteria associated with alveolar macrophages indicated that only a small percentage of bacteria, if any, appear to be residing within lung macrophages. We also tested the ability of these strains to survive in a nutrient poor environment, conditions which may be encountered within certain niches in the host or in an environmental reservoir. The Bvg- phase was advantageous for growth under these conditions. Our results indicate the Bvg+ phase is sufficient for establishment of respiratory tract infection in the rabbit and the normal BvgAS-mediated response to environmental signals is not required during initial colonization. The Bvg- phase may play a role at later stages of infection, including persistence, transmission, or survival in the environment.
1. Steady-state postsynaptic potentials (PSPs) were generated by prolonged (approximately 1 s) high-frequency (100-200 Hz) electrical stimulation of nerves in the cat hindlimb. The characteristics of these steady-state PSPs were compared for two polysynaptic afferent pathways (ipsilateral cutaneous sural vs. contralateral peroneal nerves), two animal preparations (decerebrate vs. chloralose), and two motoneuron pools (medial gastrocnemius vs. lateral gastrocnemius-soleus). 2. PSPs from both nerves usually (36 of 51 cases) contained a mixture of depolarizing and hyperpolarizing components. In all 36 cases where the PSP contained a hyperpolarizing component, a consistent qualitative pattern emerged during prolonged stimulation: the hyperpolarization reached a peak approximately 20 ms after stimulation onset and then decayed with a biphasic time course that consisted of an initial rapid phase (20-40 ms) and a later slower phase (200-400 ms) before the steady-state value was reached. This pattern occurred regardless of the differences in polysynaptic afferent pathways, animal preparations, and motoneuron pools. 3. The consistency of this overall pattern was remarkable, given the existence of several quantitative differences among the PSPs. These differences include the following: hyperpolarizing components were least common in the sural and peroneal PSPs in the decerebrate preparation. And only these sural and peroneal PSPs tended to have prolonged afterpotentials after stimulus cessation. The steady-state sural PSPs in the decerebrate preparation tended to generate the largest PSPs and, moreover, these PSPs did not follow the overall trend of having a statistically significant relation between the amplitude of the initial hyperpolarization and the amount of its decay. Finally, transient sural PSPs in lateral gastrocnemius-soleus motoneurons in the decerebrate preparation tended to have the largest hyperpolarizations. 4. To determine whether the decay of the hyperpolarization and the subsequent dominance of depolarization was due to a decreased inhibition or an increased excitation, injected current pulses were utilized to measure the changes in the cell's input resistance during the course of the synaptic input. A strong decrease in input resistance accompanied the initial period of maximal hyperpolarization (50% with respect to the resting input resistance). Input resistance then returned toward resting values as hyperpolarization faded and depolarization became dominant. However, there remained a persistent decrease in input resistance during the final phase of the PSP that amounted to < 10% of the initial decrease. These findings indicated that much of the reduction in hyperpolarization reflected a progressive decrease in synaptic efficacy for the inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)
The association of major histocompatibility complex genes with autoimmune diseases is firmly established, but the mechanisms by which these genes confer resistance or susceptibility remain controversial. The controversy extends to the nonobese diabetic (NOD) mouse that develops disease similar to human insulin-dependent diabetes mellitus. The transgenic incorporation of certain class II major histocompatibility complex genes protects NOD mice from diabetes, and clonal deletion or functional silencing of autoreactive T cells has been proposed as the mechanism by which these molecules provide protection. We show that neither thymic deletion nor anergy of autoreactive T cells occurs in NOD mice transgenic for I-Ak. Autoreactive T cells are present, functional, and can transfer diabetes to appropriate NOD-recipient mice.
CD8+ T cells taken directly from mice expressing a Kb-specific T cell receptor (TCR) transgene expressed the transgenic TCR in a bimodal profile as detected by flow cytometric analysis using a clonotype-specific monoclonal antibody. Those cells expressing the lower density of the transgenic TCR expressed the transgenic beta chain and two different alpha chains on their surface. One alpha chain was the product of the alpha transgene, whereas the other was derived by endogenous rearrangement. This report provides the first demonstration that T cells isolated directly from mice may express two different TCR clonotypes on their surface. The potential consequences of this finding for studies using TCR transgenic mice and for the induction of autoimmunity are discussed.
The immune system must not only fight off infections, but also ensure that it does not react against its own body tissues. Since clones of lymphocytes have predetermined reactivities, some will be self-reactive and have the potential to cause damage. They should therefore be neutralized in some way. In a system as complex and important as that governing self-tolerance, many mechanisms must exist to neutralize autoaggressive lymphocytes. They may be classified under two main groups. In one the tolerant state arises from the physical or functional silencing of potentially autoaggressive lymphocytes after antigen encounter. This may involve clonal deletion, clonal abortion or clonal anergy. In the second, regulatory mechanisms of the immune system itself may hold autoreactive lymphocytes in check, for example through the operation of idiotypic network interactions and the action of specialized suppressor cells. Much evidence has accumulated for the physical deletion of autoreactive T cells as they mature in the thymus. The fate of any that escape thymus censorship has been the subject of recent research and is discussed here. Under certain conditions, self-tolerance must also be imposed at the B-cell level to prevent the production of potentially damaging autoantibodies. Although the mechanisms which silence self-reactive lymphocytes are very efficient, self-tolerance can break down, and autoimmunity will thus ensue. The main factors responsible for this are briefly described here.
This investigation compared the effectiveness of two language treatment methods, modeling versus modeling plus evoked production, in promoting productive vocabulary in three toddlers identified as late talkers. A single-subject alternating treatments design was employed in this study in which different sets of words were taught under the two treatment methods during group and individual instruction. Some evidence of differential patterns of response to the treatment types was found for two subjects, but the subjects differed as to which particular treatment method was associated with better performance. Neither treatment method was effective for the third subject. Dynamic assessment measures were only marginally useful in predicting overall lexical learning potential in these subjects, and did not predict response to the two teaching methods. Implications of these results are discussed with regard to the role of language intervention for late talkers.
Samples of conversational language were elicited with a standardized interview protocol from 24 children, aged 2:6 to 7:8, half with specific language impairment (SLI), half with normally developing language (LN), matched for language level. Samples were analyzed to determine whether there were associations between adult questioning and children's use of ellipsis. For the SLI children, but not the LN children, increased proportions of questions were significantly correlated to increased proportions of ellipsis. This finding has implications for the use of MLU measures in clinical and research practice.
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The products of the bvgAS locus activate expression of a majority of the known Bordetella virulence factors but also exert negative control over a class of genes called vrg genes (bvg-repressed genes). BvgAS negatively controls the production of flagella and the phenotype of motility in Bordetella bronchiseptica. In this study flaA, the flagellin gene, was cloned and characterized to facilitate studies of this negative control pathway. An internal flaA probe detected hybridizing sequences on genomic Southern blots of Bordetella pertussis, Bordetella parapertussis, and Bordetella avium, although B. pertussis and B. parapertussis are nonmotile. FlaA is similar to the FliC flagellins of Salmonella typhimurium and Escherichia coli, and flaA complemented an E. coli flagellin mutant. Insertional inactivation of the chromosomal flaA locus eliminated motility, which was restored by complementation with the wild-type locus. Analysis of flaA mRNA production by Northern (RNA) blotting and primer extension indicated that negative regulation by BvgAS occurs at the level of transcription. The transcriptional start site of flaA mapped near a consensus site for the alternative sigma factor, sigma F, encoded by fliA in E. coli and S. typhimurium. Consistent with a role for a fliA analog in B. bronchiseptica, transcriptional activation of a flaA-lacZ fusion in E. coli required fliA and a flaA-linked locus designated frl.frl also efficiently complemented mutations in the flagellar master regulatory locus, flhDC, of E. coli. Our analysis of the motility phenotype of B. bronchiseptica suggests that the Bordetella virulence control system mediates transcriptional control of flaA through a regulatory hierarchy that includes the frl locus and an alternative sigma factor.
We have recently reported a phoA expression vector, termed pMG, which, like TnphoA, is useful in identifying genes encoding membrane-spanning sequences or signal peptides. This cloning system has been modified to facilitate the distinction of outer membrane and periplasmic alkaline phosphatase (AP) fusion proteins from inner membrane AP fusion proteins by transforming pMG recombinants into Escherichia coli KS330, the strain utilized in the "blue halo" assay first described by Strauch and Beckwith (Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988). The lipoprotein mutation lpp-5508 of KS330 results in an outer membrane that is leaky to macromolecules, and its degP4 mutation greatly reduces periplasmic proteolytic degradation of AP fusion proteins. pMG AP fusions containing cleavable signal peptides, including the E. coli periplasmic protein beta-lactamase, the E. coli and Chlamydia trachomatis outer membrane proteins OmpA and MOMP, respectively, and Tp 9, a Treponema pallidum AP recombinant, diffused through the leaky outer membrane of KS330 and resulted in blue colonies with blue halos. In contrast, inner membrane AP fusions derived from E. coli proteins, including leader peptidase, SecY, and the tetracycline resistance gene product, as well as Tp 70, a T. pallidum AP recombinant which does not contain a signal peptide, resulted in blue colonies without blue halos. Lipoprotein-AP fusions, including the Borrelia burgdorferi OspA and T. pallidum Tp 75 and TmpA showed halo formation, although there was significantly less halo formation than that produced by either periplasmic or outer membrane AP fusions. In addition, we applied this approach to screen recombinants constructed from a 9.0-kb plasmid isolated from the B31 virulent strain of B. burgdorferi. One of the blue halo colonies identified produced an AP fusion protein which contained a signal peptide with a leader peptidase I cleavage recognition site. The pMG/KS330r- cloning and screening approach can identify genes encoding proteins with cleavable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors.
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