Symptom control in chronic rhinosinusitis is an independent predictor of productivity loss.

AIMS: Sinonasal symptoms cause significant productivity losses in patients with chronic rhinosinusitis (CRS). Patient-perceived CRS symptom control is a longitudinal measure of CRS symptomatology and is directly associated with general health-related quality of life (QOL) in patients with CRS. The aim of this study was to better understand the relationship between symptom control and productivity loss in CRS. MATERIALS AND METHODS: Prospective cross-sectional cohort study of 200 patients with CRS. Patients categorized their CRS symptom control as "Not at all", "A little", "Somewhat", "Very", and "Completely". Lost productivity was assessed by determining the number of work and/or school days missed in the last 3 months due to CRS symptoms. Sinonasal symptom severity was measured using the 22-item Sinonasal Outcomes Test (SNOT-22). Associations were sought between lost productivity and patient-perceived CRS symptom control. OBJECTIVE: To determine the association between patient-perceived longitudinal symptom control and productivity in patients with CRS. RESULTS: A total of 200 participants (48% male, 52% female), with a mean age of 52 years (Standard Deviation [SD]: 16) were enrolled. The mean SNOT-22 score of participants was 33.5 (SD: 22.4). Participants missed a mean of 3 days (SD: 10) of work or school due to CRS. CRS symptom control classified as "not at all" was associated with 11 days of lost productivity due to CRS on univariate analysis (ß=11.16, 95% CI: 5.39-16.94, P<0.001) and 8 days of lost productivity on multivariate analysis (ß=8.02, 95% CI: 1.92-14.13, P=0.011). None of the other categories of patient-reported CRS symptom control were associated with lost productivity due to CRS. CONCLUSIONS: Patient-perceived control of CRS symptoms, an important metric previously shown to be significantly associated with QOL in CRS patients, is independently associated with lost productivity. These results motivate longitudinal studies to determine if improvement of CRS symptom control may reduce losses in productivity.

Depressed mood is associated with loss of productivity in allergic rhinitis.

Allergic rhinitis (AR) is associated with significant decreases in quality of life and productivity losses. We hypothesized that symptoms of AR may differentially associate with lost productivity due to AR. We performed a cross-sectional cohort study of 105 prospectively recruited patients with persistent AR. AR control, severity of depressed mood, and sinonasal symptoms were assessed with the Rhinitis Control Assessment Test (RCAT), Patient Health Questionnaire (PHQ-2), and the 22-item Sinonasal Outcome Test (SNOT-22), respectively. Lost productivity was assessed by asking the number of days of work/school missed due to AR in the last 3 months. Patients missed a mean of 1.5 days (SD:2.9) of work or school. Lost productivity was associated with PHQ-2 (adjusted linear regression coefficient [ß] = .68, 95% CI: 0.20-1.15, P = .007) analysis but not SNOT-22 or RCAT scores. Productivity losses due to AR are associated with severity of depressed mood rather than classic nasal or extra-nasal symptoms of AR.

Chronic rhinosinusitis severity is associated with need for asthma-related systemic corticosteroids.

BACKGROUND: Chronic rhinosinusitis (CRS) is highly prevalent in patients with asthma. However, no study has evaluated the effect of CRS severity on asthma-related oral corticosteroid use - a marker of poor asthma control and prognosis. We therefore sought to evaluate the association between CRS severity and asthma-related oral corticosteroid use. METHODOLOGY: Prospective cross-sectional study of 110 adult asthmatic CRS patients. CRS severity was measured using the 22-item Sinonasal Outcomes Test (SNOT-22) and Lund-Kennedy endoscopy score. Number of asthma-related courses of oral corticosteroids in the past year was queried at enrollment. Association was sought between metrics for CRS severity and asthma-related oral corticosteroids use in the last year. Receiver operating characteristic (ROC) curves defined whether SNOT-22 or endoscopy scores could be used for detecting asthma-related oral corticosteroid use. RESULTS: The mean SNOT-22 score was 44.9 (standard deviation [SD] : 23.3) and mean endoscopy score was 4.1 (SD: 3.0). The mean number of asthma-related oral corticosteroid courses taken in the last year was 1.1 (SD: 1.9). SNOT-22, but not endoscopy score, was associated with requiring at least one course of asthma-related oral corticosteroids in the last year (odds ratio = 1.03, 95%CI: 1.02 - 1.06, p=0.003), which translates to an odds ratio of 2.0 for a 21-point increase in SNOT-22. ROC analysis identified equally optimal SNOT-22 scores of greater than 32 (sensitivity: 88.1%, specificity: 41.2%) or greater than 65 (sensitivity: 38.1%, specificity: 91.2%) for detecting the need for at least one course of oral corticosteroids within the past year. CONCLUSIONS: CRS symptom severity is associated with past asthma-related oral corticosteroid use. SNOT-22 scores may be used as a versatile tool to screen for past asthma-related oral corticosteroid use in asthmatic CRS patients - i.e. those at greatest risk from their asthma - with either high sensitivity or high specificity.

Parainfluenza virus type 3 Ab in allogeneic hematopoietic cell transplant recipients: factors influencing post-transplant Ab titers and associated outcomes.

Parainfluenza virus type 3 (PIV-3) can cause severe respiratory illness among hematopoietic cell transplantation (HCT) recipients. Factors associated with PIV-3-specific Ab level, and the association between PIV-3 Ab levels and clinical outcomes in HCT recipients who acquire PIV-3 infection, are unknown. We evaluated PIV-3-specific hemagglutination inhibition Ab levels and clinical outcomes among 172 patients with PIV-3 infection following HCT. In a multivariable linear regression model, high post-transplantation Ab levels were independently associated with higher pre-transplantation recipient titer (mean difference 0.38 (95% confidence interval (CI), 0.26, 0.50), P<0.001). Significant associations between pre-HCT Ab titers in both patients and donors and occurrence of lower respiratory tract disease (LRD) after HCT were not observed. In conclusion, low pre-transplantation titers are associated with low Ab levels after HCT. The relationship between PIV-3 Ab levels and outcomes remain uncertain. Further study is needed to prospectively evaluate the dynamics of PIV-3-specific Ab responses and the relative contribution of PIV-3-specific Ab to protection from infection acquisition and progression to LRD.

Temozolomide induced liver injury.

A 62-year female received radiotherapy over six weeks with daily 75 mg/m2 Temozolomide (TMZ) for Glioblastoma (GB). At the last week of radiotherapy, her liver enzymes and serum bilirubin started deteriorating. TMZ was discontinued. The histopathology demonstrated the features of acute cholestasis and focal parenchymal inflammation. A range of investigations failed to show any other contributory cause of hepatitis. She required in-hospital care for a prolonged period for a grade three hepatic failure. The liver functions very slowly recovered over 40 weeks, but her general condition continues to deteriorate. TMZ may cause a mild temporary rise in the liver enzymes and has been reported to reactivate hepatitis B. In few other cases concomitant medications were the possible causes of hepatitis. However, searching the Medline and other bibliographic database, we have not come across any case of TMZ-induced liver injury (TMZ-DILI). Histopathology and pattern of liver enzyme elevation suggest that unlike Dacarbazine, which causes veno-occlusive type liver damage, TMZ in this patient caused mainly cholestasis type liver injury. On Naranjo Adverse Drug Reaction (ADR) probability scale, this case falls in probable grade (Scale 7).

Comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients.

BACKGROUND: Sensitive detection of respiratory viruses is important for early diagnosis of infection in patients following hematopoietic cell transplantation (HCT). To evaluate the relative sensitivity of respiratory virus detection in specimens from HCT recipients, we compared the results of conventional and quantitative molecular methods. METHODS: We tested 688 nasal wash samples collected prospectively from 131 patients during the first 100 days after HCT by viral culture, fluorescent antibody staining (FA), and real-time quantitative reverse transcription-polymerase chain reaction (PCR) assay for detection of respiratory syncytial virus (RSV), influenza virus types A (FluA) and B (FluB), and parainfluenza virus types 1 (PIV1) and 3 (PIV3). Testing for human metapneumovirus (MPV) was performed only by PCR. Data regarding 10 respiratory symptoms were collected with each sample. RESULTS: By any method 37 specimens were positive for a respiratory virus; 34 were positive by PCR, 15 by culture, and 6 by FA. Four specimens were positive by all 3 methods (3 RSV, 1 FluA). One specimen was positive for PIV1, and 2 were positive for rhinovirus by culture alone. Specimens positive by PCR alone included 2 RSV, 2 PIV1, 8 PIV3, and 8 MPV. In 10 specimens positive for RSV, PIV, or influenza virus collected from patients reporting no respiratory symptoms, 9, 4, and 1 specimen were positive by PCR, culture, and FA, respectively. Overall, specimens positive only by PCR had significantly fewer viral copies/mL (mean log(10)=4.32) than specimens positive by both PCR and culture (mean log(10)=5.75; P=0.002) or PCR and FA (mean log(10)=6.83; P<0.001). CONCLUSIONS: FA testing alone did not detect a significant proportion of respiratory virus-positive samples in HCT recipients, especially in patients with no respiratory symptoms and patients with PIV detection. PCR increased the yield of positive specimens 2 times relative to culture and more than 4 times relative to FA. Detection of respiratory viruses by PCR alone was associated with lower virus quantities and with fewer reported respiratory symptoms compared with concomitant detection by both PCR and conventional methods, indicating that PCR may be important to detect asymptomatic or mildly symptomatic stages of respiratory viral infections.

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.

Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili.

Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal alpha-helix. Engineering of a truncated pilin from Pseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an alpha-helix at the N terminus that lies across a 4-stranded antiparallel beta-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.

Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide.

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P. aeruginosa.

Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy.

The backbone dynamics of a 15N-labeled recombinant PAK pilin peptide spanning residues 128-144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys128-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Se r-Lys144) were probed by measurements of 15N NMR relaxation. This PAK(128-144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The 15N longitudinal (T1) and transverse (T2) relaxation rates and the steady-state heteronuclear [1H]-15N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 degrees C) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the 'model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153-162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II beta-turns spanning residues Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.

Glycosylations versus conformational preferences of cancer associated mucin core.

Synthetic oligosaccharide vaccines based on core STn (sialyl alpha2-6 GalNAc) carbohydrate epitopes are being evaluated by a number of biopharmaceutical firms as potential immunotherapeutics in the treatment of mucin-expressing adenocarcinomas. The STn carbohydrate epitopes exist as discontinuous clusters, O-linked to proximal serine and threonine residues within the mucin sequence. In an effort to probe the structure and dynamics of STn carbohydrate clusters as they may exist on the cancer-associated mucin, we have used NMR spectroscopy and MD simulations to study the effect of O-glycosylation of adjacent serine residues in a repeating (Ser)n sequence. Three model peptides/glyco-peptides were studied: a serine trimer containing no carbohydrate groups ((Ser)3 trimer); a serine trimer containing three Tn (GalNAc) carbohydrates alpha-linked to the hydroxyls of adjacent serine sidechains ((Ser.Tn)3 trimer); and a serine trimer containing three STn carbohydrates alpha-linked to the hydroxyls of adjacent serine sidechains ((Ser.STn)3 trimer). Our results demonstrate that clustering of carbohydrates shifts the conformational equilibrium of the underlying peptide backbone into a more extended and rigid state, an arrangement that could function to optimally present the clustered carbohydrate antigen to the immune system. Steric effects appear to drive these changes since an increase in the size of the attached carbohydrate (STn versus Tn) is accompanied by a stronger shift in the equilibrium toward the extended state. In addition, NMR evidence points to the formation of hydrogen bonds between the peptide backbone NH protons and the proximal GalNAc groups in the (Ser.Tn)3 and (Ser.STn)3 trimers. The putative peptide-sugar hydrogen bonds may also play a role in influencing the conformation of the underlying peptide backbone, as well as the orientation of the O-linked carbohydrate. The significance of these results will be discussed within the framework of developing clustered STn-based vaccines, capable of targeting the clustered STn epitopes on the cancer-associated mucin.

Engineering out motion: a surface disulfide bond alters the mobility of tryptophan 22 in cytochrome b5 as probed by time-resolved fluorescence and 1H NMR experiments.

In the accompanying paper [Storch et al. (1999) Biochemistry 38, 5054-5064] equilibrium denaturation studies and molecular dynamics (MD) simulations were used to investigate localized dynamics on the surface of cytochrome b5 (cyt b5) that result in the formation of a cleft. In those studies, an S18C:R47C disulfide mutant was engineered to inhibit cleft mobility. Temperature- and urea-induced denaturation studies revealed significant differences in Trp 22 fluorescence between the wild-type and mutant proteins. On the basis of the results, it was proposed that wild type populates a conformational ensemble that is unavailable to the disulfide mutant and is mediated by cleft mobility. As a result, the solvent accessibility of Trp 22 is decreased in S18C:R47C, suggesting that the local environment of this residue is less mobile due to the constraining effects of the disulfide on cleft dynamics. To further probe the structural effects on the local environment of Trp 22 caused by inhibition of cleft formation, we report here the results of steady-state and time-resolved fluorescence quenching, differential phase/modulation fluorescence anisotropy, and 1H NMR studies. In Trp fluorescence experiments, the Stern-Volmer quenching constant increases in wild type versus the oxidized disulfide mutant with increasing temperature. At 50 degrees C, KSV is nearly 1.5-fold greater in wild type compared to the oxidized disulfide mutant. In the reduced disulfide mutant, KSV was the same as wild type. The bimolecular collisional quenching constant, kq, for acrylamide quenching of Trp 22 increases 2.7-fold for wild type and only 1.8-fold for S18C:R47C, upon increasing the temperature from 25 to 50 degrees C. The time-resolved anisotropy decay at 25 degrees C was fit to a double-exponential decay for both the wild type and S18C:R47C. Both proteins exhibited a minor contribution from a low-amplitude fast decay, consistent with local motion of Trp 22. This component was more prevalent in the wild type, and the fractional contribution increased significantly upon raising the temperature. The fast rotational component of the S18C:R47C mutant was less sensitive to increasing temperature. A comparison of the 1H NMR monitored temperature titration of the delta-methyl protons of Ile 76 for wild type and oxidized disulfide mutant, S18C:R47C, showed a significantly smaller downfield shift for the mutant protein, suggesting that Trp 22 in the mutant protein experiences comparatively decreased cleft dynamics in core 2 at higher temperatures. Furthermore, comparison of the delta'-methyl protons of Leu 25 in the two proteins revealed a difference in the ratio of the equilibrium heme conformers of 1.2:1 for S18C:R47C versus 1.5:1 for wild type at 40 degrees C. The difference in equilibrium heme orientations between wild type and S18C:R47C suggests that the disulfide bond affects heme binding within core 1, possibly through damped cleft fluctuations. Taken together, the NMR and fluorescence studies support the proposal that an engineered disulfide bond inhibits the formation of a dynamic cleft on the surface of cyt b5.

Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study.

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sönnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor and antibody. These results pave the way for future isotope-edited NMR studies of the labeled recombinant PAK pilin peptide bound to antibody and receptor, studies integral to the design of an effective synthetic peptide vaccine.

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design.

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-turn.

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.

Altered expression of TGF alpha and TGF beta 1 in the mucosa of the functioning pelvic ileal pouch.

The mucosa of the functioning pelvic ileal pouch undergoes loss of villous height and an increase in crypt cell proliferation as an adaptive response to its new luminal environment. These changes can occur in the absence of inflammation and could be mediated by growth factors such as transforming growth factors alpha and beta 1 (TGF alpha and TGF beta 1). Expression of TGF alpha and TGF beta 1 messenger RNA (mRNA) and protein was determined by in situ hybridization and immunohistochemistry in sections of terminal ileum taken at the time of pouch formation and of subsequent pouch biopsies from 14 patients (total of 90 specimens). Crypt cell proliferation was assessed using the monoclonal antibody MIB-1. As ileal pouch mucosa underwent loss of villous height and crypt hyperplasia, epithelial expression of TGF alpha mRNA and protein decreased. In contrast, TGF beta 1 mRNA and protein were abundant in both normal and flat mucosa. Epithelial expression of TGF beta 1 protein was maximal in flat, inflamed biopsies. These results suggest that although altered expression of TGF alpha and TGF beta 1 mRNA and protein may play some part in the regulation of the adaptive response in ileal pouch mucosa, TGF alpha does not have a direct, positive role in the regulation of crypt cell proliferation.

Lactam bridge stabilization of alpha-helices: the role of hydrophobicity in controlling dimeric versus monomeric alpha-helices.

A series of lactam-bridged and linear 14 residue amphipathic alpha-helical peptides based on the sequence Ac-EXEALKKEXEALKK-amide were prepared in order to determine the effect of decreasing the hydrophobicity of the nonpolar face to helical content and stability. This was done by substituting position X by Ile, Val, and Ala. Lactam bridges spaced i to i + 4 were formed between the side chains of Glu3 and Lys7 and Glu10 and Lys14 while the linear noncyclized peptides could potentially form i to i + 4 salt bridges with the same residues. It was found that in all cases the lactam-bridged peptides were substantially more helical than the corresponding linear peptides as determined by CD spectroscopy. Moreover, the helical content approached 100% for the lactam-bridged peptides X = Ile and Ala and was greater than 80% for X = Val. For X = Ile and Val, this was partly due to the ability of the lactam bridges to enhance interchain interactions relative to the linear versions of the same sequence. Size-exclusion chromatography demonstrated that the Ile-based peptide associates as a dimer. The alanine-based lactam-bridged peptide was found to be monomeric as determined by concentration dependency studies and size-exclusion chromatography. Thermal denaturation studies in benign media indicated that the lactam-based peptides were very stable. The conformation of the Ala-based lactam peptide was further characterized by two-dimensional NMR spectroscopy and was found to be highly helical. The results demonstrate the ability of lactam bridges to stabilize the helical conformation and enhance dimerization of peptides based on a 3,4 hydrophobic heptad repeat. The substitution of Ala residues in the hydrophobic face of the alpha-helix can prevent dimerization and specify monomeric helical structure.