Biomedical subjects

John D Clements

Publications and source records attributed to John D Clements.

29 records · Page 2Linked to original sources

PubMed2003

Presynaptic Ca2+ channels: a functional patchwork.

A key step in the release of neurotransmitter is the entry of Ca(2+) into the presynaptic terminal via voltage-activated Ca(2+) channels. N-type and P/Q-type Ca(2+) channels play a predominant role but, surprisingly, their distribution across presynaptic terminals lacks any apparent order. They form a patchwork: at some terminals only N-type channels contribute to transmitter release and in others only P/Q-type channels contribute, but in many terminals both sub-types are active. The physiological implications of this non-uniform distribution are starting to emerge. Recent studies reveal that G-protein-mediated depression of N-type channels is stronger than that of P/Q-type channels, whereas voltage-dependent relief of inhibition is more pronounced for P/Q-type channels. The patchwork distribution of Ca(2+) channel subtypes might therefore enable terminal-specific modulation of transmitter release, enhancing the power of synaptic processing.

Animals↗

PubMed2003

Evidence for peripheral clearance of cerebral Abeta protein following chronic, active Abeta immunization in PSAPP mice.

Immunization with amyloid-beta (Abeta) peptide in mouse models of Alzheimer's disease has been reported to decrease cerebral Abeta levels and improve behavioral deficits. Several mechanisms have been proposed, including antibody-induced phagocytosis of Abeta by cerebral microglia and increased efflux of Abeta from the brain to the periphery. The latter mechanism was suggested in mice undergoing acute, passive transfer of an Abeta monoclonal antibody. Here, PSAPP transgenic mice were actively immunized by a single intraperitoneal injection of synthetic Abeta followed by chronic intranasal administration of Abeta with the mucosal adjuvant, Escherichia coli heat-labile enterotoxin, LT, twice weekly for 8 weeks. Serum from Abeta-immunized mice had an average of 240 microg/ml of anti-Abeta-specific antibodies; these antibodies had epitope(s) within Abeta1-15 and were of immunoglobulin (Ig) isotypes IgG2b, IgG2a, and IgG1. Immunization led to a 75% decrease in plaque number (P < 0.0001) and a 58% decrease in Abetax-42 levels (P < 0.026) in brain, and gliosis and neuritic dystrophy were diminished. No pathological effects of the immunization were observed in kidney, spleen, or snout. Serum Abeta levels increased 28-fold in immunized mice (53.06 ng/ml) compared to controls (1.87 ng/ml). Most of the Abeta in the serum of the immunized mice was bound to antibodies. We conclude that following active immunization, anti-Abeta antibodies sequester serum Abeta and may increase central nervous system to serum Abeta clearance.

Amyloid beta-Peptides↗

PubMed2003

Oral immunogenicity of human papillomavirus-like particles expressed in potato.

Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.

Administration, Oral↗

PubMed2002

Binding site stoichiometry and the effects of phosphorylation on human alpha1 homomeric glycine receptors.

The kinetic properties of the human alpha1 homomeric glycine receptor were investigated. Receptors were expressed in HEK 293 cells, and glycine was applied to outside-out membrane patches with sub-millisecond solution exchange. The activation time course of the glycine response was used to investigate receptor stoichiometry. The unbinding of three strychnine molecules and the cooperative binding of two glycine molecules were required to activate the channel. The effects of phosphorylation on glycine receptor kinetics were investigated by pretreating cells with phosphorylators or with phosphatases. Phosphorylation accelerated desensitisation, but slowed deactivation and recovery from desensitisation. A chemical-kinetic model was developed that reproduced the experimental observations. The model suggests that only three binding sites on the glycine channel are functional, while the remaining two binding sites are 'silent', possibly due to strong negative cooperativity.

Binding Sites↗

PubMed2002

Intranasal or oral immunization of inbred and outbred mice with murine or human rotavirus VP6 proteins protects against viral shedding after challenge with murine rotaviruses.

Intranasal (i.n.) administration of an Escherichia coli-expressed chimeric VP6 protein from the EDIM strain of murine rotavirus to adult BALB/c (H-2(d)) mice along with LT(R192G), an attenuated mutant of the mucosal adjuvant E. coli heat-labile toxin, has been found to consistently stimulate ca. 99% reductions in rotavirus shedding after subsequent EDIM challenge. This study was designed to determine the robustness of this protection, i.e. can VP6 immunization consistently protect against shedding in this model, thus, providing an indication of its potential as a vaccine. Intranasal immunization with two 8.8 microg doses of EDIM VP6 and 10 microg of LT(R192G) was found to stimulate 99% reductions in EDIM shedding in four additional strains of inbred mice belonging to three haplotypes, i.e. DBA/2 (H-2(d)), C57BL/6 (H-2(b)), 129 (H-2(b)) and C3H (H-2(k)). Protection stimulated against EDIM antigen shedding following i.n. immunization with VP6 from the human CJN strain was less (P=0.02) than induced by EDIM VP6 (86% versus 99%), but no further loss of protection was observed when the dose of CJN VP6 was reduced 100-fold. Protection against EDIM shedding was also maintained after i.n. immunization of three strains of outbred mice (CF-1, CD-1 and Swiss Webster) with either EDIM or CJN VP6, i.e. EDIM VP6 immunization reduced EDIM shedding by 99% while CJN VP6 immunization produced reductions of 86-96%. Protection stimulated by oral immunization of BALB/c mice with two 8.8 microg doses of either VP6 chimera plus LT(R192G) was not significantly different from that induced by i.n. immunization. Finally, protection found after either oral or i.n. immunization with EDIM or CJN VP6 was no different when the mice were challenged with McN, another strain of murine rotavirus. These results support further evaluation of VP6 as a vaccine.

Administration, Intranasal↗

PubMed2002

Presynaptic and postsynaptic mechanisms underlie paired pulse depression at single GABAergic boutons in rat collicular cultures.

Paired pulse depression (PPD) is a common form of short-term synaptic plasticity. The aim of this study was to characterise PPD at the level of a single inhibitory bouton. Low-density collicular cultures were loaded with the Ca2+ indicator Oregon Green-1, active boutons were stained with RH414, and action potentials were blocked with TTX. Evoked IPSCs (eIPSCs) and presynaptic Ca2+ transients were recorded in response to direct presynaptic depolarisation of an individual bouton. The single bouton eIPSCs had a low failure rate (< 0.1), large average quantal content (3-6) and slow decay (tau(1) = 15 ms, tau(2) = 81 ms). The PPD of eIPSCs had two distinct components: PPD(fast) and PPD(slow) (tau = 86 ms and 2 s). PPD(slow) showed no dependence on extracellular Ca2+ concentration, or on the first eIPSC's failure rate or amplitude. Most probably, it reflects a release-independent inhibition of exocytosis. PPD(fast) was only observed in normal or elevated Ca2+. It decreased with the failure rate and increased with the amplitude of the first eIPSC. It coincided with paired pulse depression of the presynaptic Ca2+ transients (tau = 120 ms). The decay of the latter was accelerated by EGTA, which also reduced PPD(fast). Therefore, a suppressive effect of residual presynaptic Ca2+ on subsequent Ca2+ influx is considered the most likely cause of PPD(fast). PPD(fast) may also have a postsynaptic component, because exposure to a low-affinity GABA(A) receptor antagonist (TPMPA; 300 microM) counteracted PPD(fast), and asynchronous IPSC amplitudes were depressed for a short interval following an eIPSC. Thus, at these synapses, PPD is produced by at least two release-independent presynaptic mechanisms and one release-dependent postsynaptic mechanism.

Animals↗

PubMed2002

Role of B7 costimulatory molecules in the adjuvant activity of the heat-labile enterotoxin of Escherichia coli.

Much interest has been directed at understanding the adjuvant properties of the heat-labile enterotoxin of Escherichia coli (LT). In this study, we have assessed how LT compared with the nonenzymatic mutant LT (E112K) affect the level of B7-1 and B7-2 expression on APCs, and we determined how these costimulatory molecules influence their adjuvant properties. Analysis of B7-1 and B7-2 expression on B cells revealed that LT enhanced B7-2 but not B7-1, while LT (E112K) had no effect on the expression of either costimulatory molecule. Treatment of macrophage or dendritic cells with LT resulted in a predominant enhancement of B7-2, while LT (E112K) induced mainly B7-1 expression. Analysis of LT- and LT (E112K)-treated B cells, macrophage, and dendritic cells also revealed significant differences in their ability to enhance anti-CD3-stimulated CD4(+) T cell proliferative responses via B7-1 and B7-2. Furthermore, the ability of LT to enhance both Ab and CD4(+) T cell responses to a coadministered Ag was severely abrogated in B7-2- but not B7-1-deficient mice. In contrast, the in vivo adjuvant properties of LT (E112K) appeared to be mediated by both B7-1 and B7-2 for optimal CD4(+) T cell responses, while B7-1 appeared to be the predominant B7 molecule involved in the ability of LT (E112K) to augment Ab responses to a coadministered Ag. These findings demonstrate distinct differences in the ability of LT and LT (E112K) to enhance B7-1 and B7-2 on APC, as well as a dependence upon these costimulatory molecules for their adjuvant properties.

Adjuvants, Immunologic↗

PubMed2002

The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant.

Intranasal (i.n.) immunization of BALB/c mice with chimeric murine rotavirus EDIM (epizootic diarrhea of infant mice) VP6 and attenuated E. coli heat-labile toxin (LT), LT(R192G), stimulated >99% protection against rotavirus shedding after EDIM challenge. Here, we evaluated other potential adjuvants with chimeric VP6 administered by two mucosal routes: i.n. and oral. Besides LT(R192G), the adjuvants examined included Adjumer, CpG oligodeoxynucleotides (CpG ODN), chimeric A1 subunit of cholera toxin (CTA1)-DD, and QS-21. All except QS-21 significantly (P<0.05) increased VP6-specific serum IgG responses after i.n. immunization, but none significantly increased these responses when administered orally. The i.n. delivery of chimeric VP6 alone induced both rotavirus IgG1 and IgG2a whose relative titers suggested a skewed Th2-like response. Inclusion of Adjumer greatly increased Th2-like responses, while CpG ODN shifted the response to a less Th2-like response. The adjuvants CTA1-DD, LT(R192G), QS-21 had no significant effect on ratios of IgG1/IgG2a titers. Following EDIM challenge of mice immunized i.n. with chimeric VP6 and either LT(R192G), CTA1-DD, Adjumer or CpG ODN, shedding was reduced >99, 95, 80, 74, respectively, relative to that found in unimmunized mice (P<0.05). QS-21 induced less protection (43%, not significant (N.S.)) while immunization with chimeric VP6 alone reduced shedding by only 16% (N.S.). Oral immunization with chimeric VP6 and all selected adjuvants except QS-21 was less effective than after i.n. immunization, with protection levels of 94 (P<0.05), 71 (P<0.05), 55, 35 and 28% for LT(R192G), QS-21, CpG ODN, CTA1-DD, and Adjumer, respectively, while immunization with chimeric VP6 alone gave no protection. Thus, different adjuvants induced different degrees of protection and oral immunization was generally less effective then the i.n. route.

Adjuvants, Immunologic↗

PubMed2002

Identification of novel membrane proteins by searching for patterns in hydropathy profiles.

A technique has been developed to search a proteome database for new members of a functional class of membrane protein. It takes advantage of the highly conserved secondary structure of functionally related membrane proteins. Such proteins typically have the same number of transmembrane domains located at similar relative positions in their polypeptide sequence. This gives rise to a characteristic pattern of peaks in their hydropathy profiles. To conduct a search, each member of a polypeptide database is converted to a hydropathy profile, peaks are automatically detected, and the pattern of peaks is compared with a template. A template was designed for the acetylcholine (ACh) and glycine receptors of the cys-loop receptor superfamily. The key feature was a closely spaced triplet of hydropathy peaks bracketed by deep valleys. When applied to the human proteome the search procedure retrieved 153 profiles with a receptor-like triplet of peaks. The approach was highly selective with 70% of the retrieved profiles annotated as known or putative receptors. These included ACh, glycine, gamma-amino butyric acid and serotonin receptors, which are all related by sequence. However, ionotropic glutamate receptors, which have almost no sequence homology with ACh receptors, were also retrieved. Thus, the strategy can find members of a functional class that cannot be identified by sequence alignment. To demonstrate that the strategy can easily be extended to other membrane protein families, a template was developed for the neurotransmitter/Na+ symporter family, and similar results were obtained. This approach should prove a useful adjunct to sequence-based retrieval tools when searching for novel membrane proteins.

Databases, Protein↗

PubMed2002

CD4 T cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric VP6 protein and the adjuvant LT(R192G).

Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on alphabeta T cells but mice lacking gammadelta T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, alphabeta CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 x 10(6) CD8 T cells had no effect on shedding, while transfer of 2 x 10(5) CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).

Adjuvants, Immunologic↗

PubMedDate not supplied

Intranasal immunotherapy for the treatment of Alzheimer's disease: Escherichia coli LT and LT(R192G) as mucosal adjuvants.

Alzheimer's disease (AD) is the most common form of dementia worldwide, yet there is currently no effective treatment or cure. Extracellular deposition of amyloid-beta protein (Abeta) in brain is a key neuropathological characteristic of AD. In 1999, Schenk et al. first reported that an injected Abeta vaccine given to PDAPP mice, an AD mouse model displaying Abeta deposition in brain, led to the lowering of Abeta levels in brain. In 2000, we demonstrated that intranasal (i.n.) immunization with human synthetic Abeta1-40 peptide for 7 months led to a 50-60% reduction in cerebral Abeta burden in PDAPP mice; serum Abeta antibody titers were low (approximately 26 microg/ml). More recently, we have optimized our i.n. Abeta immunization protocol in wild-type (WT) mice. When low doses Escherichia coli heat-labile enterotoxin (LT) were given as a mucosal adjuvant with Abeta i.n., there was a dramatic 12-fold increase in Abeta antibody titers in WT B6D2F1 mice treated two times per week for 8 weeks compared to those of mice receiving i.n. Abeta without adjuvant. A non-toxic form of LT, designated LT(R192G), showed even better adjuvanticity; anti-Abeta antibody titers were 16-fold higher than those seen in mice given i.n. Abeta without adjuvant. In both cases, the serum Abeta antibodies recognized epitopes within Abeta1-15 and were of the immunoglobulin (Ig) isotypes IgG2b, IgG1, IgG2a and low levels of IgA. This new and improved Abeta vaccine protocol is now being tested in AD mouse models with the expectation that higher Abeta antibody titers may be more effective in reducing cerebral Abeta levels.

Adjuvants, Immunologic↗