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Biomedical subjects

T Elliott

Publications and source records attributed to T Elliott.

At least 55 records · Page 3Linked to original sources
PubMed1999

A phase I/II study of the safety and pharmacokinetics of nevirapine in HIV-1-infected pregnant Ugandan women and their neonates (HIVNET 006).

OBJECTIVE: To determine the safety, pharmacokinetics, tolerance, antiretroviral activity, and infant HIV infection status after giving a single dose of nevirapine to HIV-1-infected pregnant women during labor and their newborns during the first week of life. DESIGN: An open label phase I/II study. SETTING: Tertiary care hospital, Kampala, Uganda. PATIENTS AND INTERVENTIONS: Nevirapine, 200 mg, was given as a single dose during labor to 21 HIV-1-infected pregnant Ugandan women. In cohort 1, eight infants did not receive nevirapine whereas in cohort 2, 13 infants received a single dose of nevirapine, 2 mg/kg, at 72 h of age. OUTCOMES: The number and type of adverse events; nevirapine concentrations in the plasma and breast milk; maternal plasma HIV-1 RNA copy number before and up to 6 weeks after delivery; and HIV-1 infection status of the infants were monitored. RESULTS: Nevirapine was well tolerated by women and infants; no serious adverse events that were related to nevirapine were observed. Median nevirapine concentration in the women at delivery was 1623 ng/ml (range 238-2356 ng/ml); median cord/maternal blood ratio of 0.75 (0.37-0.93). The median half-life in women was 61.3 h (27-90 h) and the transplacental nevirapine half-life in infants who did not receive a neonatal dose was 54 h. The median half-life after a single dose at 72 h in infants was 46.5 h. During the first week of life, the median colostrum/breast milk to maternal plasma nevirapine concentration was 60.5% (25-122%). The median nevirapine concentration in breast milk 1 week after delivery was 103 ng/ml (25-309 ng/ml). Plasma nevirapine concentrations were above 100 ng/ml in all infants from both cohorts tested at age 7 days. Maternal HIV-1 RNA levels decreased by a median of 1.3 logs at 1 week postpartum, and returned to baseline by 6 weeks postpartum. Detectable plasma HIV-1 RNA was observed in one out of 22 (4.5%) infants at birth; three out of 21 (14%) at 6 weeks; and four out of 21 (19%) at 6 months of age. CONCLUSION: The administration of a single dose of nevirapine to women during labor and to their newborns at 72 h was well tolerated and showed potent antiretroviral activity in the women at 1 week after dosing without rebound above baseline 6 weeks after a single dose. The nevirapine concentration was maintained above the target of 100 ng/ml in infants at age 7 days, even in those infants not receiving a neonatal dose. This regimen has promise as prophylaxis against intrapartum and early breast milk transmission in a breastfeeding population.

Anti-HIV Agents↗
PubMed1999

Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand.

Acetylglucosamine↗
PubMed1999

An assay for peptide binding to HLA-Cw*0102.

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) molecules is based on the ability of peptides to stabilize MHC class I molecules synthesized by transporter associated with antigen processing (TAP)-deficient cell. The TAP-deficient cell line T2 has previously been used in the assembly assay to analyze peptide binding to HLA-A*0201 and -B*5101. In this study, we have extended this technique to assay for peptides binding to endogenous HLA-Cw*0102 molecules. We have analyzed the peptide binding of 20 peptides with primary anchor motifs for HLA-Cw*0102. One-third of the peptides analyzed bound with high affinity, half of the peptides examined did not bind, whereas the remaining peptides displayed intermediate binding activity. Interest in HLA-C molecules has increased significantly in recent years, since it has been shown that HLA-C molecules both can present peptides to cytotoxic T lymphocytes (CTL) and in addition are able to inhibit natural killer (NK)-mediated lysis.

Animals↗
PubMed1999

RpoS synthesis is growth rate regulated in Salmonella typhimurium, but its turnover is not dependent on acetyl phosphate synthesis or PTS function.

The RpoS sigma factor of enteric bacteria is either required for or augments the expression of a number of genes that are induced during nutrient limitation, growth into stationary phase, or in response to stresses, including high osmolarity. RpoS is regulated at multiple levels, including posttranscriptional control of its synthesis, protein turnover, and mechanisms that affect its activity directly. Here, the control of RpoS stability was investigated in Salmonella typhimurium by the isolation of a number of mutants specifically defective in RpoS turnover. These included 20 mutants defective in mviA, the ortholog of Escherichia coli rssB/sprE, and 13 mutants defective in either clpP or clpX which encode the protease active on RpoS. An hns mutant was also defective in RpoS turnover, thus confirming that S. typhimurium and E. coli have identical genetic requirements for this process. Some current models predict the existence of a kinase to phosphorylate the response regulator MviA, but no mutants affecting a kinase were recovered. An mviA mutant carrying the D58N substitution altering the predicted phosphorylation site is substantially defective, suggesting that phosphorylation of MviA on D58 is important for its function. No evidence was obtained to support models in which acetyl phosphate or the PTS system contributes to MviA phosphorylation. However, we did find a significant (fivefold) elevation of RpoS during exponential growth on acetate as the carbon and energy source. This behavior is due to growth rate-dependent regulation which increases RpoS synthesis at slower growth rates. Growth rate regulation operates at the level of RpoS synthesis and is mainly posttranscriptional but, surprisingly, is independent of hfq function.

Acetic Acid↗
PubMed1999

A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.

The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positions at the HemA N terminus resulted in complete stabilization of the protein. By contrast, the addition of an N-terminal myc epitope tag did not affect turnover. This result confirms the importance of the N-terminal sequence for proteolysis of HemA. This region of the protein also contains a proline flanked by hydrophobic residues, a motif that has been suggested to be important for Lon-mediated proteolysis of UmuD. However, mutation of this motif did not affect the turnover of HemA protein. Cells expressing the stabilized HemA[KK] mutant protein display substantial defects in heme regulation.

ATP-Dependent Proteases↗
PubMed1999

Conditional stability of the HemA protein (glutamyl-tRNA reductase) regulates heme biosynthesis in Salmonella typhimurium.

In many bacteria, including the enteric species Salmonella typhimurium and Escherichia coli, heme is synthesized starting from glutamate by a pathway in which the first committed step is catalyzed by the hemA gene product, glutamyl-tRNA reductase (HemA). We have demonstrated previously that when heme limitation is imposed on cultures of S. typhimurium, HemA enzyme activity is increased 10- to 25-fold. Western (immunoblot) analysis with monoclonal antibodies reactive with HemA revealed that heme limitation results in a corresponding increase in the abundance of the enzyme. Similar regulation was also observed for E. coli. The near absence of regulation of hemA-lac operon fusions suggested a posttranscriptional control. We report here the results of pulse-labeling and immunoprecipitation studies of this regulation. The principal mechanism that contributes to elevated HemA abundance is protein stabilization. The half-life of HemA protein is approximately 20 min in unrestricted cells but increases to >300 min in heme-limited cells. Similar regulation was observed for a HemA-LacZ hybrid protein containing almost all of the HemA protein (416 residues). Sodium azide prevents HemA turnover in vivo, suggesting a role for energy-dependent proteolysis. This was confirmed by the finding that HemA turnover is completely blocked in a lon clpP double mutant of E. coli. Each single mutant shows only a small effect. The ClpA chaperone, but not ClpX, is required for ClpP-dependent HemA turnover. A hybrid HemA-LacZ protein containing just 18 amino acids from HemA is also stabilized in the lon clpP double mutant, but this shorter fusion protein is not correctly regulated by heme limitation. We suggest that the 18 N-terminal amino acids of HemA may constitute a degradation tag, whose function is conditional and modified by the remainder of the protein in a heme-dependent way. Several models are discussed to explain why the turnover of HemA is promoted by Lon-ClpAP proteolysis only when sufficient heme is available.

ATP-Dependent Proteases↗
PubMed1999

The human HLA-A*0201 allele, expressed in hamster cells, is not a high-affinity receptor for adenovirus type 5 fiber.

The coxsackie B virus and adenovirus receptor (CAR) and the major histocompatibility complex (MHC) class I alpha2 domain have been identified as high-affinity cell receptors for adenovirus type 5 (Ad5) fiber. In this study we show that CAR but not MHC class I allele HLA-A*0201 binds to Ad5 with high affinity when expressed on hamster cells. When both receptors are coexpressed on the cell surface of hamster cells, Ad5 fiber bind to a single high-affinity receptor, which is CAR.

Adenoviruses, Human↗
PubMed1999

Metabolic alterations in middle-aged and elderly obese patients with type 2 diabetes.

OBJECTIVE: We conducted this study to assess the metabolic alterations in middle-aged and elderly obese patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Healthy control subjects (9 middle-aged, aged 42 +/- 2 years, BMI 33 +/- 1 kg/m2; 10 elderly, aged 71 +/- 1 years, BMI 29 +/- 1 kg/m2) and patients with type 2 diabetes (11 middle-aged, aged 43 +/- 2 years, BMI 34 +/- 2 kg/m2; 23 elderly, aged 73 +/- 1 years; BMI 30 +/- 1 kg/m2) underwent a 3-h oral glucose tolerance test (OGTT), a 2-h hyperglycemic glucose clamp, and a 3-h euglycemic glucose clamp study with tritiated glucose methodology to measure hepatic glucose production and peripheral disposal rates. RESULTS: Middle-aged and elderly control subjects and patients with diabetes were similar in percentage of body fat. Waist-to-hip ratio was greater in elderly patients with diabetes than in elderly control subjects (P < 0.01), but was similar in both middle-aged groups. VO2max was less in control subjects than in both middle-aged and elderly patients with diabetes (P < 0.05). Insulin responses during the OGTT were similar in elderly control subjects and patients with diabetes, but were less in middle-aged patients with diabetes than in control subjects (305 +/- 49 vs. 690 +/- 136 pmol/l, P < 0.01). Patients with type 2 diabetes had absent first-phase insulin responses during the hyperglycemic clamp. Second-phase (80-120 min) insulin values were similar in elderly patients and control subjects, but were reduced in middle-aged patients with diabetes compared with control subjects (285 +/- 35 vs. 894 +/- 143 pmol/l, P < 0.0001). During the euglycemic clamp, basal and steady-state (150-180 min) hepatic glucose output values were less in middle-aged control subjects than in patients with diabetes (basal, 3.03 +/- 0.10 vs. 3.69 +/- 0.09 mg.kg-1 lean body mass.min-1, P < 0.0001; steady-state, 0.72 +/- 0.10 vs. 1.84 +/- 0.20 mg.kg-1 lean body mass.min-1, P < 0.0001). Basal and steady-state hepatic glucose output values were similar in elderly patients and control subjects. Finally, steady-state (150-180 min) glucose disposal rates were higher in control subjects than in patients with diabetes in both the middle-aged (7.51 +/- 0.85 vs. 4.62 +/- 0.24 mg.kg-1 lean body mass.min-1, P < 0.01) and elderly (9.91 +/- 0.61 vs. 6.78 +/- 0.60 mg.kg-1 lean body mass.min-1, P < 0.01) groups. CONCLUSIONS: We conclude that type 2 diabetes in obese middle-aged subjects is characterized by impaired glucose-induced insulin release, altered regulation of hepatic glucose output, and resistance to insulin-mediated glucose disposal. In contrast, the primary defect in elderly obese patients with type 2 diabetes is resistance to insulin-mediated glucose disposal. Our findings may have important therapeutic implications for these patient populations.

Adult↗
PubMed1999

Hypoglycemia induced by hydroxychloroquine in a type II diabetic treated for polyarthritis.

A 77-year-old man with type II diabetes taking a stable dose of subcutaneous, twice daily human insulin developed symmetrical, inflammatory, rheumatoid factor positive polyarthritis. Within 2 weeks of starting therapy with prednisone 5 mg daily and hydroxychloroquine 400 mg daily he had 2 episodes of severe hypoglycemic coma requiring emergency care. His blood glucose became controlled again when his insulin was decreased by 37%. There are no reported cases of hypoglycemia in diabetic or nondiabetic patients treated with hydroxychloroquine. Hydroxychloroquine has been reported to reduce insulin requirements in refractory type II diabetes by an average of 30%. When hydroxychloroquine is initiated for the treatment of polyarthritis in a type II diabetic requiring insulin or sulfonylurea treatment, blood glucose levels should be monitored closely and the insulin dose may need to be reduced.

Aged↗
PubMed1998

Competition for neurotrophic factors: mathematical analysis.

Neurotrophic factors, particularly the neurotrophin gene family of neurotrophic factors, are implicated in activity-dependent anatomical plasticity in the visual cortex and at the neuromuscular junction. Accumulating evidence implicates neurotrophic factors as possible mediators of activity-dependent competition between afferents, leading to the segregation of afferents' arbors on the target space. We present a biologically plausible mathematical model of competition for neurotrophic factors. We show that the model leads to anatomical segregation, provided that the levels of neurotrophic factors released in an activity-independent manner, or the levels available by exogenous infusion, are below a critical value, which we derive. Above this critical value, afferent segregation breaks down. We also show that the model segregates afferents even in the presence of very highly correlated patterns of afferent activity. The model is therefore ideally suited for application to the development of ocular dominance columns in the kitten visual cortex.

Afferent Pathways↗
PubMed1998

DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription.

DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress sigma factor, RpoS. These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation. The third stem-loop, which behaves as a transcription terminator, can be substituted by the trp transcription terminator without loss of either DsrA function. The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of rpoS messenger RNA, suggesting that pairing of DsrA with the rpoS message might be important for translational regulation. Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation. We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.

Bacterial Proteins↗
PubMed1998

Glycan-regulated antigen processing of a protein in the endoplasmic reticulum can uncover cryptic cytotoxic T cell epitopes.

We and others have shown that influenza A nucleoprotein (NP) targeted to the secretory pathway cannot be processed to yield several cytotoxic T lymphocyte (CTL) epitopes in cell lines that lack the transporter associated with antigen processing (TAP). However, a large COOH-terminal fragment of NP is processed and presented in these cells. Full-length NP is cotranslationally glycosylated in the lumen of the endoplasmic reticulum at two sites distal to the major H2-Kk and H2-Db restricted CTL epitopes, and we show here that pharmacological or genetic inhibition of N-linked glycosylation, leads to the processing and presentation of both these epitopes in a TAP-independent way.

Animals↗
PubMed1998

Competition for neurotrophic factors: ocular dominance columns.

Activity-dependent competition between afferents in the primary visual cortex of many mammals is a quintessential feature of neuronal development. From both experimental and theoretical perspectives, understanding the mechanisms underlying competition is a significant challenge. Recent experimental work suggests that geniculocortical afferents might compete for retrograde neurotrophic factors. We show that a mathematically well-characterized model of retrograde neurotrophic interactions, in which the afferent uptake of neurotrophic factors is activity-dependent and in which the average level of uptake determines the complexity of the axonal arbors of afferents, permits the anatomical segregation of geniculocortical afferents into ocular dominance columns. The model induces segregation provided that the levels of neurotrophic factors available either by activity-independent release from cortical cells or by exogenous cortical infusion are not too high; otherwise segregation breaks down. We show that the model exhibits changes in ocular dominance column periodicity in response to changes in interocular image correlations and that the model predicts that changes in intraocular image correlations should also affect columnar periodicity.

Afferent Pathways↗
PubMed1998

A soluble major histocompatibility complex class I peptide-binding platform undergoes a conformational change in response to peptide epitopes.

Class I major histocompatibility complexes (MHC) are heterotrimeric structures comprising heavy chains (HC), beta2-microglobulin (beta2-m), and short antigenic peptides of 8-10 amino acids. These components assemble in the endoplasmic reticulum and are released to the cell surface only when a peptide of the appropriate length and sequence is incorporated into the structure. The binding of beta2-m and peptide to HC is cooperative, and there is indirect evidence that the formation of a stable heterotrimer from an unstable HC:beta2-m heterodimer involves a peptide-induced conformational change in the HC. Such a conformational change could ensure both a strong interaction between the three components and also signal the release of stably assembled class I MHC molecules from the endoplasmic reticulum. A peptide-induced conformational change in HC has been demonstrated in cell lysates lacking beta2-m to which synthetic peptides were added. Many features of this conformational change suggest that it may be physiologically relevant. In an attempt to study the peptide-induced conformational change in detail we have expressed a soluble, truncated form of the mouse H-2Db HC that contains only the peptide binding domains of the class I molecule. We have shown that this peptide-binding "platform" is relatively stable in physiological buffers and undergoes a conformational change that is detectable with antibodies, in response to synthetic peptides. We also show that the structural features of peptides that induce this conformational change in the platform are the same as those required to observe the conformational change in full-length HC. In this respect, therefore, the HC alpha1 and alpha2 domains, which together form the peptide binding site of class I MHC, are able to act independently of the rest of the molecule.

Animals↗
PubMed1998

Evidence for successive peptide binding and quality control stages during MHC class I assembly.

Intracellular antigens are continually presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules, which consist of a polymorphic 43 kDa heavy chain and a 12 kDa soluble subunit beta 2-microglobulin (beta 2m), and which bind an 8-10 amino-acid antigenic peptide. The assembly of this trimolecular complex takes place in the lumen of the endoplasmic reticulum (ER) and almost certainly requires cofactors. Most MHC class I molecules in the ER that have not yet acquired peptide are simultaneously bound to the transporter associated with antigen processing (TAP), to the 48 kDa glycoprotein tapasin and to the lectin-like chaperone calreticulin, in a multicomponent 'loading complex'. Previous studies have shown that a mutant MHC class I molecule T134K (in which Thr134 was changed to Lys) fails to bind to TAP. Here, we show that this point mutation also disrupted, directly or indirectly, the interaction between MHC class I molecules and calreticulin. T134K molecules did not present viral antigens to T cells even though they bound peptide and beta 2m normally in vitro. They exited the ER rapidly as 'empty' MHC class I complexes, unlike empty wild-type molecules which are retained in the ER and degraded. We show here that, paradoxically, the rapid exit of empty T134K molecules from the ER was dependent on a TAP-derived supply of peptides. This implies that MHC class I assembly is a two-stage process: initial binding of suboptimal peptides is followed by peptide optimisation that depends on temporary ER retention.

Antigen Presentation↗
PubMed1998

Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.

Animals↗
PubMed1998

Axonal processes and neural plasticity: a reply.

We examine the claim that a class of sprouting-and-retraction models is mathematically equivalent to a fixed-anatomy model. We accept, subject to important caveats, a narrow mathematical equivalence of the energy functions in both classes of model. We argue that this narrow equivalence of energy functions does not, however, entail equivalence of the models. Indeed, the claim of complete model equivalence hides significant dynamical differences between the approaches, which we discuss. We also disagree that our work demonstrates that subtractive constraint enforcement is natural in fixed-anatomy models.

Animals↗