Biomedical subjects

J F Miller

Publications and source records attributed to J F Miller.

At least 37 records · Page 2Linked to original sources

PubMed2000

Multiple roles for Bordetella lipopolysaccharide molecules during respiratory tract infection.

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B. pertussis), a trisaccharide plus an O-antigen-like repeat (B. bronchiseptica), or an altered trisaccharide plus an O-antigen-like repeat (B. parapertussis). Deletion of the wlb locus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide. We have used wlb deletion (Deltawlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly. Although the Deltawlb mutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5(-/-) mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects. B. pertussis and B. parapertussis Deltawlb mutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, the B. bronchiseptica Deltawlb strain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deficient mice, suggesting that wlb-dependent LPS modifications in B. bronchiseptica modulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.

Animals↗

PubMed2000

Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response.

Fimbriae are filamentous, cell surface structures which have been proposed to mediate attachment of Bordetella species to respiratory epithelium. Bordetella bronchiseptica has four known fimbrial genes: fim2, fim3, fimX, and fimA. While these genes are unlinked on the chromosome, their protein products are assembled and secreted by a single apparatus encoded by the fimBCD locus. The fimBCD locus is embedded within the fha operon, whose genes encode another putative adhesin, filamentous hemagglutinin (FHA). We have constructed a Fim(-) B. bronchiseptica strain, RB63, by introducing an in-frame deletion extending from fimB through fimD. Western blot analysis showed that RB63 is unable to synthesize fimbriae but is unaffected for FHA expression. Using this mutant, we assessed the role of fimbriae in pathogenesis in vitro and in vivo in natural animal hosts. Although RB63 was not significantly defective in its ability to adhere to various tissue culture cell lines, including human laryngeal HEp-2 cells, it was considerably altered in its ability to cause respiratory tract infections in rats. The number of DeltafimBCD bacteria recovered from the rat trachea at 10 days postinoculation was significantly decreased compared to that of wild-type B. bronchiseptica and was below the limit of detection at 30 and 60 days postinoculation. The number of bacteria recovered from the nasal cavity and larynx was not significantly different between RB63 and the wild-type strain at any time point. The ability of fimbriae to mediate initial attachment to tracheal tissue was tested in an intratracheal inoculation assay. Significantly fewer RB63 than wild-type bacteria were recovered from the tracheas at 24 h after intratracheal inoculation. These results demonstrate that fimbriae are involved in enhancing the ability of B. bronchiseptica to establish tracheal colonization and are essential for persistent colonization at this site. Interestingly, anti-Bordetella serum immunoglobulin M (IgM) levels were significantly lower in animals infected with RB63 than in animals infected with wild-type B. bronchiseptica at 10 days postinoculation. Even at 30 days postinoculation, RB63-infected animals had lower serum anti-Bordetella antibody titers in general. This disparity in antibody profiles suggests that fimbriae are also important for the induction of a humoral immune response.

Animals↗

PubMed2000

Information characteristics and the structure of landscapes.

Various techniques for statistical analysis of the structure of fitness landscapes have been proposed. An important feature of these techniques is that they study the ruggedness of landscapes by measuring their correlation characteristics. This paper proposes a new information analysis of fitness landscapes. The underlying idea is to consider a fitness landscape as an ensemble of objects that are related to the fitness of neighboring points. Three information characteristics of the ensemble are defined and studied. They are termed: information content, partial information content, and information stability. The information characteristics of a range of landscapes with known correlation features are analyzed in an attempt to reveal the advantages of the information analysis. We show that the proposed analysis is an appropriate tool for investigating the structure of fitness landscapes.

Biological Evolution↗

PubMed1999

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose.

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8(+) T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8(+) T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8(+) T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8(+) T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8(+) T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8(+) T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

Adoptive Transfer↗

PubMed1999

A central role for thymic emigrants in peripheral T cell homeostasis.

After initial seeding by thymic emigrants, homeostatic regulation of the T cell pool has been thought to occur entirely within the periphery. Here we report that the degree of thymic emigration directly affects the number and the CD4/CD8 ratio of peripheral T cells. We demonstrate that the increase in T cell pool size caused by the engraftment of 2, 6, or 9 thymic lobes correlates almost exactly with the number of emigrants exported from those grafts in the previous 3 weeks, regardless of how long the graft has been in place. The extent of the increase supports the concept of a 3-week period after thymic export in which emigrant T cells are exempt from peripheral T cell homeostasis. This apparent exclusion of recent thymic emigrants from the niche-based regulation of peripheral T cell numbers ensures repertoire turnover throughout adult life and provides the basis of a direct and previously unrecognized role for the thymus in the regulation of peripheral T cell homeostasis.

Animals↗

PubMed1999

A comparison of T cell memory against the same antigen induced by virus versus intracellular bacteria.

Cytotoxic T cell (CTL) memory was analyzed after infection with lymphocytic choriomeningitis virus (LCMV) and recombinant Listeria monocytogenes (rLM) expressing the complete nucleoprotein of LCMV (rLM-NP(actA)) or only the immunodominant epitope of H-2(d) mice (rLM-NP(118-126)). Immunization with LCMV and rLM induced a long-lived increased CTL precursor (CTLp) frequency specific for the viral (NP(118-126)) and for the bacterial (LLO(91-99)) epitope, respectively. However, after infection with rLM memory, CTLs were less protective against an intravenous LCMV challenge infection than a comparable number of LCMV-induced memory T cells. LCMV, but not recombinant Listeria-induced memory T cells were able to protect against lethal choriomeningitis by LCMV or a subsequent peripheral infection with recombinant vaccinia virus expressing LCMV-NP. The protective memory after viral and after rLM immunization was paralleled by evidence of LCMV but not rLM antigen persistence on day 15 and 30 after vaccination. These results document a striking difference in protective T cell memory between viral and bacterial vaccines and indicate that rapid T cell-dependent immune protection correlates with antigen persistence.

Animals↗

PubMed1999

Signalling through CD30 protects against autoimmune diabetes mediated by CD8 T cells.

Autoantigens found on pancreatic islets can move to draining lymph nodes, where they are able to cause the activation and consequent deletion of autoreactive T cells by a mechanism termed cross-tolerance. This deletion depends on signalling through CD95 (also known as Fas), a member of the superfamily of tumour-necrosis-factor receptors. Here we describe a new mechanism that protects against autoimmunity: this mechanism involves another member of this superfamily, CD30, whose function was largely unknown. CD30-deficient islet-specific CD8-positive T cells are roughly 6,000-fold more autoaggressive than wild-type cells, with the transfer of as few as 160 CD30-deficient T cells leading to the complete destruction of pancreatic islets and the rapid onset of diabetes. We show that, in the absence of CD30 signalling, cells activated but not yet deleted by the CD95-dependent cross-tolerance mechanism gain the ability to proliferate extensively upon secondary encounter with antigen on parenchymal tissues, such as the pancreatic islets. Thus, CD30 signalling limits the proliferative potential of autoreactive CD8 effector T cells and protects the body against autoimmunity.

Adoptive Transfer↗

PubMed1999

CD30 prevents T-cell responses to non-lymphoid tissues.

Self antigens can induce T-cell tolerance via a mechanism termed cross-tolerance. This involves the transfer of peripheral tissue antigens to professional APC for presentation in the draining lymph nodes. In this site, CD8+ T cells are activated, proliferate, and are slowly deleted by a CD95-dependent mechanism. Prior to their deletion, some activated cells leave the lymph nodes and encounter antigens on peripheral parenchymal tissues. Without functional CD30, these cells proliferate extensively and cause substantial tissue damage. Thus, CD30 limits autoreactivity, acting as a 'brake' on T-cell proliferation after recognition of autoantigens on parenchymal tissues.

Animals↗

PubMed1999

Existing antilisterial immunity does not inhibit the development of a Listeria monocytogenes-specific primary cytotoxic T-lymphocyte response.

Infection of BALB/c mice with Listeria monocytogenes stimulates an antilisterial immune response evident by the appearance of H2-Kd-restricted CD8(+) cytotoxic T lymphocytes (CTLs) specific for the nanomer peptides amino acids (aa) 91 to 99 of listeriolysin O (LLO 91-99) and aa 217 to 225 of the p60 molecule (p60 217-225). We have introduced point mutations at anchor residues within LLO 91-99 (92F) or p60 217-225 (218F), and BALB/c mice infected with L. monocytogenes strains containing these point mutations do not develop CTLs specific for LLO 91-99 or p60 217-225, respectively. We have used these strains to test whether primary CTL responses against L. monocytogenes-derived determinants can be stimulated within an environment of existing antilisterial immunity. We found that the development of a primary L. monocytogenes-specific CTL response is not altered by existing immunity to L. monocytogenes. For example, primary immunization with the p60 218F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of p60 217-225-specific CTLs at primary response levels and LLO 91-99-specific effectors at levels consistent with a memory CTL response. Similarly, primary immunization with the 92F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of LLO 91-99-specific CTLs at primary response levels and p60 217-225-specific effectors at levels consistent with a memory CTL response. These results provide additional support for the use of L. monocytogenes as a recombinant vaccine vector and show that antivector immunity does not inhibit the development of a primary CTL response when the epitope is delivered by L. monocytogenes as the vaccine strain.

Amino Acid Substitution↗

PubMed1999

Pregenomic comparative analysis between bordetella bronchiseptica RB50 and Bordetella pertussis tohama I in murine models of respiratory tract infection.

We describe here a side-by-side comparison of murine respiratory infection by Bordetella pertussis and Bordetella bronchiseptica strains whose genomes are currently being sequenced (Tohama I and RB50, respectively). B. pertussis and B. bronchiseptica are most appropriately classified as subspecies. Their high degree of genotypic and phenotypic relatedness facilitates comparative studies of pathogenesis. RB50 and Tohama I differ in their abilities to grow in the nose, trachea, and lungs of BALB/c mice and to induce apoptosis, lung pathology, and an antibody response. To focus on the interactions between the bacteria and particular aspects of the host immune response, we used mice with specific immune defects. Mice lacking B cells and T cells were highly susceptible to B. bronchiseptica and were killed by intranasal inoculation with doses as low as 500 CFU. These mice were not killed by B. pertussis, even when doses as high as 10(5) CFU were delivered to the lungs. B. bronchiseptica, which was highly resistant to naive serum in vitro, caused bacteremia in these immunodeficient mice, while B. pertussis, which was highly sensitive to naive serum, did not cause bacteremia. B. bronchiseptica was, however, killed by immune serum in vitro, and adoptive transfer of anti-Bordetella antibodies protected SCID-beige mice from B. bronchiseptica lethal infection. Neutropenic mice were similarly killed by B. bronchiseptica but not B. pertussis infection, suggesting neutrophils are critical to the early inflammatory response to the former but not the latter. B. bronchiseptica was dramatically more active than B. pertussis in mediating the lysis of J774 cells in vitro and in inducing apoptosis of inflammatory cells in mouse lungs. This side-by-side comparison describes phenotypic differences that may be correlated with genetic differences in the comparative analysis of the genomes of these two highly related organisms.

Animals↗

PubMed1999

Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity.

We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria. SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin.

Adenylate Cyclase Toxin↗

PubMed1999

Discovering the origins of immunological competence.

Work done in the late 1950s and in the 1960s revealed the role of the thymus in virus-induced leukemia in mice. Thymectomizing mice at birth to test whether the virus first multiplied in thymus tissue and then spread elsewhere ultimately led to the conclusion that the thymus was essential to the normal development of the immune system. Subsequent testing to try to understand how the thymus contributes to the pool of immunocompetent lymphocytes opened a new chapter in immunology and required a reappraisal of many immunological phenomena and an understanding of the molecular interactions that take place during cell-to-cell interactions.

Allergy and Immunology↗

PubMed1998

B cells directly tolerize CD8(+) T cells.

This report investigates the response of CD8(+) T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the Kb-restricted ovalbumin (OVA) determinant OVA257-264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro-activated CD8(+) T cells from the Kb-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8(+) T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.

Animals↗

PubMed1998

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents.

We have identified a novel series of antidiabetic N-(2-benzoylphenyl)-L-tyrosine derivatives which are potent, selective PPARgamma agonists. Through the use of in vitro PPARgamma binding and functional assays (2S)-3-(4-(benzyloxy)phenyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)+ ++amin o)propionic acid (2) was identified as a structurally novel PPARgamma agonist. Structure-activity relationships identified the 2-aminobenzophenone moiety as a suitable isostere for the chemically labile enaminone moiety in compound 2, affording 2-((2-benzoylphenyl)amino)-3-(4-(benzyloxy)phenyl)propionic acid (9). Replacement of the benzyl group in 9 with substituents known to confer in vivo potency in the thiazolidinedione (TZD) class of antidiabetic agents provided a dramatic increase in the in vitro functional potency and affinity at PPARgamma, affording a series of potent and selective PPARgamma agonists exemplified by (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(methylpyridin-2-ylamino+ ++)ethoxy ]phenyl¿propionic acid (18), 3-¿4-[2-(benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propanoic acid (19), and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (20). Compounds 18 and 20 show potent antihyperglycemic and antihyperlipidemic activity when given orally in two rodent models of type 2 diabetes. In addition, these analogues are readily prepared in chiral nonracemic fashion from L-tyrosine and do not show a propensity to undergo racemization in vitro. The increased potency of these PPARgamma agonists relative to troglitazone may translate into superior clinical efficacy for the treatment of type 2 diabetes.

Administration, Oral↗

PubMed1998

Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction.

Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I-restricted presentation to CD8+ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8+ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8+ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8+ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.

Animals↗

PubMed1998

The peripheral deletion of autoreactive CD8+ T cells induced by cross-presentation of self-antigens involves signaling through CD95 (Fas, Apo-1).

Recently, we demonstrated that major histocompatibility complex class I-restricted cross-presentation of exogenous self-antigens can induce peripheral T cell tolerance by deletion of autoreactive CD8+ T cells. In these studies, naive ovalbumin (OVA)-specific CD8+ T cells from the transgenic line OT-I were injected into transgenic mice expressing membrane-bound OVA (mOVA) under the control of the rat insulin promoter (RIP) in pancreatic islets, kidney proximal tubules, and the thymus. Cross-presentation of tissue-derived OVA in the renal and pancreatic lymph nodes resulted in activation, proliferation, and then the deletion of OT-I cells. In this report, we investigated the molecular mechanisms underlying this form of T cell deletion. OT-I mice were crossed to tumor necrosis factor receptor 2 (TNFR2) knockout mice and to CD95 (Fas, Apo-1) deficient mutant lpr mice. Wild-type and TNFR2-deficient OT-I cells were activated and then deleted when transferred into RIP-mOVA mice, whereas CD95-deficient OT-I cells were not susceptible to deletion by cross-presentation. Furthermore, cross-presentation led to upregulation of the CD95 molecule on the surface of wild-type OT-I cells in vivo, consistent with the idea that this is linked to rendering autoreactive T cells susceptible to CD95-mediated signaling. This study represents the first evidence that CD95 is involved in the deletion of autoreactive CD8+ T cells in the whole animal.

Animals↗

PubMed1998

Help for cytotoxic-T-cell responses is mediated by CD40 signalling.

Cytotoxic T lymphocytes (CTLs) which carry the CD8 antigen recognize antigens that are presented on target cells by the class I major histocompatibility complex. CTLs are responsible for the killing of antigen-bearing target cells, such as virus-infected cells. Although CTL effectors can act alone when killing target cells, their differentiation from naive CD8-positive T cells is often dependent on 'help' from CD4-positive helper T (TH) cells. Furthermore, for effective CTL priming, this help must be provided in a cognate manner, such that both the TH cell and the CTL recognize antigen on the same antigen-presenting cell. One explanation for this requirement is that TH cells are needed to convert the antigen-presenting cell into a cell that is fully competent to prime CTL. Here we show that signalling through CD40 on the antigen-presenting cells can replace the requirement for TH cells, indicating that T-cell 'help', at least for generation of CTLs by cross-priming, is mediated by signalling through CD40 on the antigen-presenting cell.

Animals↗

PubMed1998

The role of the thymus and recent thymic migrants in the maintenance of the adult peripheral lymphocyte pool.

The thymus is essential for the initial seeding of T cells to the periphery, but its role in maintaining the adult T cell pool remains poorly defined. We investigated whether changes to the rate of T cell export could form part of the mechanism(s) controlling the homeostatic regulation of the size and composition of the peripheral T cell pool. Using neonatal thymi grafted under the kidney capsule, we found that irrespective of whether the pool was oversupplied (by thymic grafts) or undersupplied (due to neonatal thymectomy), the thymic export rate was constant from both the host and graft thymus, and the periphery remained constant in size. Recent thymic emigrants (RTE) were also tracked to determine the extent of their acceptance into the T cell pool of a normal mouse. As a population, RTE are phenotypically mature, but were distinct from resident T cells in the periphery, being released in a CD4/CD8 ratio approximately twice that of established peripheral T cells. This export ratio is similar to that of T cells in the mature thymic compartment, but soon after entry into the periphery, the ratio falls, indicating separate thymic and peripheral regulation of the CD4/CD8 ratio. RTE may also be preferentially incorporated into the periphery, causing displacement of resident T cells, thus maintaining the size of the peripheral pool. Although not vital for the maintenance of a functional T cell pool, the acceptance of RTE in a "full" peripheral pool would ensure that the T cell receptor repertoire is kept diverse and that the T cell population encompasses a broad range of naive as well as memory T cells.

Animals↗