Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Decreased immunosuppression associated with antitumor activity of 5-deoxy-5-fluorouridine compared to 5-fluorouracil and 5-fluorouridine.

5-Fluorouracil (5-FUra), 5-deoxy-5-fluorouridine (5'dFUrd), and 5-fluorouridine were compared for their relative antitumor activity, their capacity to inhibit leukocyte exudation and macrophage (macrophage) killing of tumor cells in vivo and in vitro, and their ability to induce leukopenia and monocytopenia. 5'dFUrd was less toxic than 5-FUra and exhibited anti-Ehrlich ascites activity over a wider range of drug doses. Inflammatory exudates induced by thioglycollate or pyran were inhibited up to 91% by prior 5-FUra injection but were inhibited not more than 62% by 5'dFUrd. Pyran-induced macrophage inhibition of Ehrlich ascites proliferation in vivo was diminished up to 5-fold by 5-FUra but was never diminished more than 2-fold by 5'dFUrd, while neither agent suppressed in vitro macrophage cytotoxicity of in vivo pyran-activated macrophage. At high doses, 5-FUra reduced white blood cell counts 73%, in contrast to the 8% reduction caused by 5'dFUrd, while at their optimal anti-Ehrlich ascites doses, 5-FUra and 5'dFUrd both lowered white blood cell counts by only 20%. However, 5-FUra caused a severe monocytopenia not seen in animals given injections of comparable doses of 5'dFUrd. Therefore, 5-FUra appeared to inhibit the inflammatory response and antitumor activity by inhibiting the influx of immature macrophage into the peritoneal cavity, not by inhibiting the function of mature effector cells.

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features.

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.

Evaluation of proliferating cell nuclear antigen (PCNA) as an endogenous marker of cell proliferation in rat liver: a dual-stain comparison with 5-bromo-2'-deoxyuridine.

Proliferating cell nuclear antigen (PCNA) was evaluated as a marker of cell proliferation in formalin-fixed rat liver tissue through a comparative study with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). The comparison was conducted through the introduction of a dual immunohistochemical procedure that allows the simultaneous detection of the two antigens. The results of this study suggest that although statistically similar indexes for each can be achieved, what has been reported to be the "S-phase fraction" of PCNA-labeled nuclei is significantly different from the population of cells marked by BrdU. The data also suggest that the reason for this difference is that the "S-phase fraction" of PCNA-labeled nuclei is the population of cells in late G1- and early S-phases. BrdU, by comparison, is incorporated into cells only during DNA synthesis. Therefore, although BrdU and PCNA labeling techniques may both be effective for evaluating cell proliferation rates, it must be recognized that labeling indices derived from each are not entirely synonymous. The method presented here for the simultaneous labeling of PCNA and BrdU antigens may have utility in studies of cell cycle perturbations.

Immunoregulation of the mixed lymphocyte reaction by macrophage-derived factors: functional and biochemical separation of enhancing and inhibitory factors.

Positive and negative immunoregulation of the mixed lymphocyte reaction (MLR) occurred through release of macrophage(MO)-derived, soluble enhancing and inhibitory factors. Macrophages, sonicated or cultured in low concentrations, produced nondialyzable, soluble factor(s) capable of enhancing the MLR; but the culture supernatant had no biologically detectable levels of Interleukin 1, Interleukin 2, or interferon. Production of enhancing supernatants was not affected by pretreatment of MO with trypsin or anti-Thy 1 antibody plus complement. In contrast, MO cultured in high concentrations yielded an inhibitory supernatant factor(s) which suppressed MLR reactivity even when MO were cultured in the presence of indomethacin. Culturing MO with proteolytic enzyme inhibitors increased the yields of the inhibitory and enhancing factors. Both factors were precipitable with ammonium sulfate and could be separated into several biologically active fractions using anion exchange chromatography.

Mechanism of cytotoxic activity of 5'-deoxy-5-fluorouridine.

The purpose of these studies was to characterize the effect of the new fluoropyrimidine nucleoside 5'-deoxy-5-fluorouridine (5'dFUrd) on macromolecular processes in correlation with its cytotoxicity in Ehrlich ascites tumor cells. Following a 2-h exposure, 5'-dFUrd exhibited an LD50 (as determined by clonogenicity) of 48 microM. In cells supplemented with 10 microM dThd, the LD50 for 5'-dFUrd increased to 660 microM. DNA synthesis was markedly and rapidly suppressed by all cytotoxic concentrations of 5'-dFUrd. There was no apparent direct measurable effect of 5'-dFUrd on either RNA or protein synthesis, although both were suppressed 24 h after the drug exposure. Thymidylate synthetase activity was completely inhibited by all cytotoxic concentrations of 5'-dFUrd. FUra incorporation into RNA was also measured and appeared to correlate with the dThd-nonreversible toxicity of 5'-dFUrd. These studies indicate that the mechanism of 5'-dFUrd cytotoxicity is directly analogous to that reported for 5-fluorouracil. The inhibition of thymidylate synthetase leading to an inhibition of DNA synthesis was the most potent cytotoxic mechanism (i.e., dThd-reversible) for 5'-dFUrd, and was found to be highly time-dependent. Higher concentrations of 5'-dFUrd resulted in dThd-nonreversible toxicity, which appeared to be related to the incorporation of FUra into RNA.

Diagnostic laryngeal electromyography: The Wake Forest experience 1995-1999.

BACKGROUND: Laryngeal electromyography (LEMG) is a valuable diagnostic/prognostic test for patients with suspected laryngeal neuromuscular disorders. OBJECTIVE: To report our experience with diagnostic LEMG at the Center for Voice Disorders of Wake Forest University and to evaluate the impact of LEMG on clinical management. METHODS: Retrospective chart review of 415 patients who underwent diagnostic LEMG over a 5-year period (1995-1999). RESULTS: Of 415 studies, 83% (346 of 415) were abnormal, indicating a neuropathic process. LEMG results altered the diagnostic evaluation (eg, the type of radiographic imaging) in 11% (46 of 415) of the patients. Unexpected LEMG findings (eg, contralateral neuropathy) were found in 26% (107 of 415) of the patients, and LEMG results differentiated vocal fold paralysis from fixation in 12% (49 of 415). Finally, LEMG results altered the clinical management (eg, changed the timing and/or type of surgical procedure) in 40% (166 of 415) of the patients. CONCLUSIONS: LEMG is a valuable diagnostic test that aids the clinician in the diagnosis and management of laryngeal neuromuscular disorders.

Avoiding intubation in the injured subglottis: the role of heliox therapy.

Intubation in the child presenting with severe viral tracheobronchitis or prior subglottic injury can be detrimental to the child and the subglottis. Intubation may lead to further mucosal ischemia, scar, subglottic stenosis, or failed extubation requiring a tracheotomy. Heliox is a combination of helium and oxygen that produces less-dense gas exchange. Its use leads to a decrease in turbulent airflow, which may obviate the need for intubation. Here we report our experience using heliox as initial therapy in 14 consecutive children presenting with severe airway distress and the need for intubation. (Five had viral tracheobronchitis, 5 had inflammatory exacerbation of subglottic stenosis, and 4 had acute iatrogenic subglottic injury.) In 10 of the 14 children, intubation, which can lead to mucosal injury and scarring, was avoided by the use of heliox therapy. Of the 4 children in whom heliox therapy failed, 3 had a prior history of subglottic stenosis. Heliox is a relatively safe and reliable alternative to intubation of children with severe subglottic edema or injury. Heliox should be considered before intubation for selected children with subglottic inflammation and severe airway distress.

Elective extracorporeal membrane oxygenation: an improved perioperative technique in the treatment of tracheal obstruction.

The surgical management of children with tracheal stenosis and obstruction is complicated by the perioperative needs of pressure ventilation and indwelling endotracheal tubes. These factors predispose to surgical failure and anastomotic breakdown, restenosis. and pneumomediastinum. The use of extracorporeal membrane oxygenation (ECMO) to manage ventilation during tracheal repair allows better visualization at the surgical site and obviates the need for indwelling endotracheal tubes and high-pressure ventilation. Six children were treated with elective ECMO at a tertiary care hospital. All 6 underwent successful surgical repair, and 4 of the 6 were ultimately extubated. There were no significant complications at the surgical site. There was 1 death from postoperative complications, and 2 patients required tracheotomy. One tracheotomy was performed for upper airway obstruction secondary to retrognathia, and this patient was subsequently decannulated. Medical complications were confined to 2 patients and included sepsis, hyperbilirubinemia, seizure disorder, renal failure, intracranial hemorrhage, and hydrocephalus. Elective ECMO provides a reliable perioperative technique for airway management of children with tracheal stenosis or obstruction. This technique offers the advantage of improved visibility at the operative site and eliminates the need for high-pressure ventilation, thereby likely reducing the risk of perioperative morbidity.

Enhancement of T cell MLR reactivity by addition of macrophage supernatants from tumor-bearing mice.

One-way mixed lymphocyte reactions (MLR) were used to establish a correlation between loss of in vitro T cell reactivity and in vivo tumor growth. Though supernatants from anti-Thy 1 treated macrophages (M phi) greatly enhanced MLR activity in normal T cells, such addition had little effect in reversing the inability of T cells from 2-week palpable tumor-bearing mice (TBM) to recognize in vitro foreign histocompatibility antigens. Since normal T cells, exposed to TBM M phi supernatants, exhibited no decrease in proliferative response, TBM T cell loss of MLR reactivity could not be ascribed to tumor-induced suppressor M phi.

Effect of auranofin on plasma fibronectin, C reactive protein, and albumin levels in arthritic rats.

Auranofin, a member of a class of compounds with disease modifying activity, was given to arthritic rats to determine if it could reverse the abnormal plasma concentrations of fibronectin (Fn), C reactive protein (CRP), and albumin, which were unaffected by treatment with non-steroidal anti-inflammatory drugs (NSAIDs). When auranofin was orally administered for two weeks to adjuvant induced arthritic rats it significantly inhibited swelling of the injected and non-injected paws at doses of 3 and 10 mg/kg. Rocket electroimmunoassay measurement of plasma proteins in normal, arthritic, and auranofin treated arthritic rats indicated that auranofin at 10 mg/kg significantly decreased (by 77%) the abnormally high concentration of arthritic rat plasma Fn, though it had no effect on Fn concentrations when administered to normal rats. CRP, which was raised approximately twofold above normal in arthritic rats, was reduced by 56% after treatment of arthritic rats with auranofin at 10 mg/kg, though CRP concentrations in normal rats were unaffected by auranofin treatment. Depressed albumin concentrations in arthritic rats were significantly enhanced (by 30%) by dosing with 10 mg/kg of auranofin. At the 3 mg/kg dose, auranofin did not significantly change plasma concentrations of Fn, CRP, and albumin in arthritic rats. At a dose of 10 mg/kg, however, auranofin, in addition to inhibiting chronic systemic paw inflammation, also altered abnormal concentrations of plasma Fn, CRP, and albumin in the adjuvant arthritic rat, thus distinguishing auranofin from standard NSAIDs we have previously tested.

Examination of interleukin-1 activity, the acute phase response, and leukocyte subpopulations in rats with adjuvant-induced arthritis.

Rats with adjuvant-induced arthritis (AA) were used to determine whether chronic systemic paw inflammation was accompanied by appearance of the acute phase response, an increase in splenic interleukin-1 (IL-1) production, and a reduction of nonhelper T cells in the spleen and peripheral blood. Two weeks after injection of adjuvant, the rats developed significant (P less than or equal to 0.01) swelling of the noninjected paw indicative of systemic inflammation. The rats with AA also exhibited signs of the acute phase response, as measured by a significant increase in plasma C-reactive protein (138% above normal) and a significant decrease in plasma albumin (47% below normal), zinc (30% below normal), and iron (58% below normal). IL-1 production from spleen cells of rats with AA was increased 115% compared with normals. Results from immunofluorescence studies with W3/25 and OX8 antibodies to distinguish rat T-helper-inducer (TH) spleen cells from suppressor-cytotoxic (nonhelper T) spleen cells indicated no significant difference between the percentage of OX8+ cells in spleens of arthritic rats compared with OX8+ cells in spleens of normal rats. The W3/25+-to-OX8+ ratio of 1.6 +/- 0.2 for normal rat spleen cells (33% to 21%) was not significantly different from the arthritic rat ratio of 1.9 +/- 0.1 (36% to 19%). When the phenotypes of peripheral blood mononuclear cells were analyzed, the normal animals possessed a greater percentage of W3/25+(TH) cells than did rats with AA. Normal blood mononuclear cell samples were composed of 51% +/- 3% W3/25+ cells and 22% +/- 2% OX8+ cells, and the samples from rats with AA contained 43% +/- 4% W3/25+ cells and 24% +/- 3% OX8+ cells. Thus, the helper-to-nonhelper ratio was higher for normal rats (2.3 +/- 0.1) than for arthritic rats (1.8 +/- 0.2). The data indicated that the appearance of the acute phase response and the abnormally high rate of splenic IL-1 production in the rats with AA did not stem from a subnormal percentage of OX8+ nonhelper T cells in the spleen or peripheral blood.

The effect of glucocorticoids on plasma fibronectin levels in normal and arthritic rats.

In vivo studies with normal and adjuvant-induced arthritic rats were undertaken in order to measure the effects of glucocorticoids on paw inflammation and plasma fibronectin (Fn) levels. Dexamethasone, methylprednisolone, and corticosterone all enhanced plasma Fn levels in normal animals. All drugs also significantly decreased inflammation in arthritic rats as measured by paw swelling. Of the three glucocorticoids, only corticosterone did not significantly enhance Fn levels in arthritic rats, possibly due to its lesser potency and narrow therapeutic window.

Single-step purification of rat C-reactive protein and generation of monospecific C-reactive protein antibody.

Although Sepharose-phosphorylcholine affinity chromatography has been used extensively to purify some acute phase proteins, the operation has usually been a laborious multi-step procedure. By modifying previously described multi-step protein purification assays, centigram quantities of pure rat C-reactive protein (CRP) could be obtained in a single chromatographic step using affinity chromatography. Rat serum was passed over a column of p-aminophenylphosphorylcholine and extraneous proteins eluted with Tris-saline-Ca2+ buffer. Similar to other purification procedures, CRP was eluted with phosphorylcholine in a Tris-saline-Ca2+ buffer. The technical detail which distinguished this procedure from others, was the use of a phosphorylcholine gradient shallow enough (0.95 mM-2.5 mM) to resolve the eluent into two peaks; the first peak was composed largely of the contaminant, serum amyloid protein (SAP), and the second was composed of CRP. Although there was some overlap between the first and second peak, pure CRP could be obtained by pooling fractions from the trailing shoulder of the second peak. Using this single step procedure, a greater than 25% yield of SAP-free, purified CRP could be obtained. The purified CRP was free of SAP contamination as measured by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis. Purified CRP was determined to be free of rat albumin, IgG and the C3 component of complement using immunoelectrophoresis. This one-step affinity column chromatography procedure provides a simple, efficient method for collecting large quantities of rat CRP pure enough to be used to obtain a monospecific goat, anti-rat CRP antibody.

NMR structure of the Tn916 integrase-DNA complex.

The integrase protein catalyzes the excision and integration of the Tn916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel beta-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-Ppol-DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent approximately 35 degrees and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon.

Site-specific DNA binding using a variation of the double stranded RNA binding motif.

The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast. The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy. The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD). The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded beta-sheet. This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.

Anti-inflammatory activity of salmeterol: down-regulation of cytokine production.

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.