Evolution and origin of sympatric shallow-water morphotypes of Lake Trout, Salvelinus namaycush, in Canada's Great Bear Lake.

Range expansion in north-temperate fishes subsequent to the retreat of the Wisconsinan glaciers has resulted in the rapid colonization of previously unexploited, heterogeneous habitats and, in many situations, secondary contact among conspecific lineages that were once previously isolated. Such ecological opportunity coupled with reduced competition likely promoted morphological and genetic differentiation within and among post-glacial fish populations. Discrete morphological forms existing in sympatry, for example, have now been described in many species, yet few studies have directly assessed the association between morphological and genetic variation. Morphotypes of Lake Trout, Salvelinus namaycush, are found in several large-lake systems including Great Bear Lake (GBL), Northwest Territories, Canada, where several shallow-water forms are known. Here, we assess microsatellite and mitochondrial DNA variation among four morphotypes of Lake Trout from the five distinct arms of GBL, and also from locations outside of this system to evaluate several hypotheses concerning the evolution of morphological variation in this species. Our data indicate that morphotypes of Lake Trout from GBL are genetically differentiated from one another, yet the morphotypes are still genetically more similar to one another compared with populations from outside of this system. Furthermore, our data suggest that Lake Trout colonized GBL following dispersal from a single glacial refugium (the Mississippian) and support an intra-lake model of divergence. Overall, our study provides insights into the origins of morphological and genetic variation in post-glacial populations of fishes and provides benchmarks important for monitoring Lake Trout biodiversity in a region thought to be disproportionately susceptible to impacts from climate change.

Reliability and sensitivity of joint space measurements in hand radiographs using computerized image analysis.

OBJECTIVE: To establish the sensitivity and reliability of proximal interphalangeal (PIP) and metacarpophalangeal (MCP) mean joint space measurements using standard clinical radiographs of healthy subjects, in order to determine the limits at which a change in radiographic joint space could indicate a change in actual joint size. METHODS: Repeat hand radiographs of healthy subjects were taken using standard techniques at 3-5 day intervals with the hands flat (5 posteroanterior radiographs in 8 subjects) or in 6 different flexed positions on a single occasion (8 subjects). The mean joint space was determined using custom soft ware and was validated manually. Measurement reproducibility within subjects, within films, and between hand positions was assessed by analysis of variance. RESULTS: In repeat radiographs taken in the standard clinical position, the precision of individual join space measurements indicates that changes > 0.11 mm (approximately 7%) would represent an actual physical change in joint space width (with 95% probability). Averaging measurements across fingers for a single subject decreases the detectable change to 0.05 mm (approximately 3%). With increasing flexure, radiographic joint space tended to increase in MCP and decrease in PIP. CONCLUSION: Mean finger joint space measured from standard clinical radiographs is a reliable and sensitive measurement in healthy subjects even with some change in hand position. Work is required to establish whether the joint space change measured from serial radiographs of patients with arthritis over a period of 6-12 mo exceeds the detectable limits of change derived in this study.

Akt provides the CD28 costimulatory signal for up-regulation of IL-2 and IFN-gamma but not TH2 cytokines.

A region of the interleukin-2 (IL-2) promoter known as the RE/AP element is activated in concert by signals that originate from the T cell antigen receptor and the CD28 coreceptor. We show here that the serine-threonine kinase Akt can provide a costimulatory signal for RE/AP activation that is indistinguishable from the signal provided by CD28. This includes the ability of Akt, like antibodies to CD28, to synergize with protein kinase C theta (PKC-theta) in the induction of RE/AP. Retrovirus-mediated expression of activated Akt in primary T cells from CD28-deficient mice is capable of selectively restoring production of IL-2 and interferon gamma, but not IL-4 or IL-5. Our results provide evidence that CD28 costimulation of different cytokines is mediated by discrete signaling pathways, one of which includes Akt.

Non-traditional study designs to demonstrate average bioequivalence for highly variable drug products.

OBJECTIVE: To demonstrate average bioequivalence, the ninety-percent confidence intervals (CI) on the ratio of geometric means for area under the concentration-time curve (AUC) and maximum observed plasma concentration (Cmax) must lie within 0.80-1.25. Demonstration of average bioequivalence (ABE) for highly variable drug products requires large numbers of subjects in a standard, adequately powered, two-period crossover. METHODS: Application of non-traditional study designs can help to meet this hurdle. Study design and analysis for replicate and group sequential-replicate study designs are presented and illustrated using examples. It is demonstrated how to use such approaches to meet the difficult regulatory hurdle of average bioequivalence for a highly variable drug product. RESULTS: To illustrate, data are provided from three separate ABE studies for a highly variable drug product at three dosage strengths. In all three studies, a replicate study design was used to compensate for high intrasubject variation. Additionally, for the last study, a group sequential study design was imposed to provide early evidence of conclusive results. CONCLUSION: Replicate designs and group-sequential designs in bioequivalence should be used to demonstrate average bioequivalence for highly variable drug products or when uncertain of true intrasubject variability in order to ensure conclusive study results.

A heterogeneous outbreak of Enterobacter cloacae and Serratia marcescens infections in a surgical intensive care unit.

OBJECTIVE: To investigate an outbreak of invasive disease due to Enterobacter cloacae and Serratia marcescens in a surgical intensive care unit (ICU). DESIGN: Pulsed-field gel electrophoresis (PFGE) analysis of restriction fragments was used to characterize the outbreak isolate genotypes. A retrospective cohort study of surgical ICU patients was conducted to identify risk factors associated with invasive disease. Unit staffing data were analyzed to compare staffing levels during the outbreak to those prior to and following the outbreak. SETTING: An urban hospital in San Francisco, California. PATIENTS: During the outbreak period, December 1997 through January 1998, there were 52 patients with a minimum ICU stay of > or = 72 hours. Of these, 10 patients fit our case definition of recovery of E. cloacae or S. marcescens from a sterile site. RESULTS: PFGE analysis revealed a highly heterogeneous population of isolates. Bivariate analysis of patient-related risk factors revealed duration of central lines, respiratory colonization, being a burn patient, and the use of gentamicin or nafcillin to be significantly associated with invasive disease. Both respiratory colonization and duration of central lines remained statistically significant in a multivariate analysis. Staffing data suggested a temporal correlation between understaffing and the outbreak period. CONCLUSIONS: Molecular epidemiological techniques provided a rapid means of ruling out a point source or significant cross-contamination as modes of transmission. In this setting, patient-related risk factors, such as respiratory colonization and duration of central lines, may provide a focus for heightened surveillance, infection control measures, and empirical therapy during outbreaks caused by common nosocomial pathogens. In addition, understaffing of nurses may have played a role in this outbreak, highlighting the importance of monitoring staffing levels.

The roles of CD28 and CD40 ligand in T cell activation and tolerance.

Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28-/- T cells showed a greater reduction in proliferative responses to Ag than did CD40L-/- T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L-/- T cells were defective in Th1 development. In vivo, CD28-/- T cells failed to expand upon immunization, whereas CD40L-/- T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and CD40L:CD40 interactions has an additive effect in inhibiting T cell responses.

Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin.

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.

Evening dosing is associated with higher plasma concentrations of pranlukast, a leukotriene receptor antagonist, in healthy male volunteers.

AIMS: To study the magnitude of differences in the pharmacokinetics of pranlukast, after morning and evening administration. METHODS: Pranlukast (300 mg) was administered to 12 healthy male volunteers on two separate occasions, either in the morning or evening. Both doses were given 30 min after a standard high fat content meal. Blood samples were collected up to 18 h postdose. Plasma was assayed by high performance liquid chromatography. Standard pharmacokinetic and statistical analyses were performed. RESULTS: Statistically significant (P < 0.05) increases were noted in AUC(o,t) (56%) and tmax (2.5 h) after evening administration. Cmax was 14% higher after evening dosing (95% C.I. 0.71-1.84). CONCLUSIONS: Pranlukast bioavailability is apparently increased after evening dosing as compared with morning administration. Higher night-time and early morning plasma concentrations may confer additional therapeutic benefit at a time when asthmatics are at greatest risk of developing bronchospasm.

Effect of pranlukast, an oral leukotriene receptor antagonist, on leukotriene D4 (LTD4) challenge in normal volunteers.

BACKGROUND: There is increasing evidence to show that leukotrienes are important mediators in asthma. Leukotriene receptor antagonists protect against antigen and exercise challenges in patients with chronic asthma. A study was undertaken to investigate the activity of the leukotriene receptor antagonist pranlukast (SB 205312, ONO-1078) in blocking bronchoconstriction induced by leukotriene D4 (LTD4) inhalation. The selectivity of pranlukast was evaluated using histamine challenge. METHODS: Pranlukast, 450 mg twice daily, was given to eight healthy non-smoking men for five days in a randomised, double blind, placebo controlled, crossover study. The specific airways conductance (sGaw) was measured before and after bronchial provocation with inhaled LTD4 at 3.5 hours after the first dose and at 3.5 and 9.5 hours after the last dose of pranlukast on the morning of day 5. The concentration of LTD4 required to produce a fall in sGaw of 35% (PC35) was calculated. Subjects also underwent a histamine challenge 3.5 hours after a single dose of pranlukast, 450 mg, or placebo. RESULTS: A single dose of pranlukast produced a 10.6 fold increase in PC35sGaw (95% confidence interval (CI) 4.4 to 25.5; p < 0.001) for LTD4 at 3.5 hours after dosing compared with placebo. Three and a half hours after the morning dose of pranlukast on day 5 the PC35sGaw for LTD4 was increased 25.9 fold (95% CI 10.8 to 62.2; p < 0.001) and was still increased sevenfold (95% CI 2.9 to 16.7; p < 0.001) relative to placebo 9.5 hours after administration of the morning dose. No significant differences were noted for the PC35sGaw to histamine for pranlukast compared with placebo. CONCLUSIONS: This study shows that pranlukast is a potent and selective LTD4 receptor antagonist in humans which blocks LTD4 challenge after initial and repeated administration when given twice daily for five days.

DAB389 interleukin-2 receptor binding domain mutations. Cytotoxic probes for studies of ligand-receptor interactions.

Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB389 interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB389 IL-2(T439P), was 300-fold less cytotoxic than wild type DAB389 IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB389 IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB389 IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB389 IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells.

The transmembrane (T) domain of diphtheria toxin (DT) comprises nine alpha-helices and has been shown to play an essential role in the efficient delivery of the catalytic (C) domain of DT across the eukaryotic cell membrane and into the cytosol. We have demonstrated recently that the first three amphipathic helixes of the T domain, although not necessary for either channel formation or receptor binding, are required for the efficient transmembrane delivery of the C domain. In the present study, we have performed a detailed structure-function analysis of T domain helix 1 (TH1) of the DT-related fusion protein DAB389IL-2. We performed exchange and site-directed mutagenesis of TH1 and the resulting mutant fusion toxins were analyzed by gel electrophoresis and tested for their efficiencies in the delivery of the C domain to the cell cytosol. We demonstrate that the overall charge distribution and hydrophobicity of amino acids in the amphipathic helix TH1, rather than a specific amino acid sequence, are critical for the function of this helix. The insertion of a charged residue in the hydrophobic face of TH1 abolishes cytotoxic activity, whereas replacement of a hydrophobic residue by a charged amino acid in the hydrophilic face of the helix has little, if any, effect on cytotoxic activity. In addition, we have identified Ser220 by site-directed mutagenesis as a residue that appears to be critical for correct folding of the fusion toxin. Mutations in this position result in fusion proteins that are extremely sensitive to proteolytic attack.

Resistance and susceptibility to cyclosporin A as CD3-4-8- human thymocytes differentiate in vitro.

Human T-cell development appears to be relatively resistant to cyclosporin A (CsA). Children exposed to CsA in utero as part of kidney transplant maintenance have few abnormalities. The objective of the study described here was to analyse the effects of CsA on the development in vitro of human multinegative (MN) (CD3-4-8-) thymocytes as a model system for thymic progenitor development in vivo. MN thymocytes, prepared by depletion methods, differentiated in vitro to acquire CD3 and undergo transitions in CD45 isoform expression analogous to those postulated to occur in vivo. In this work MN thymocytes were cultured with IL-2 and on thymic epithelial cells (TEC) with or without IL-2, either in the presence or absence of CsA. For many thymocyte preparations, differentiation in the presence of CsA resulted in almost complete inhibition of the acquisition of CD3 and of the low Mr isoform CD45R0. Expression of CD45RA and of total CD45 were reduced but not eliminated and the density of CD29 was unaffected. For others, neither CD3 nor CD45 expression was affected, but selective inhibition of TCR delta expression TCR delta expression occurred. At all doses of CsA (0.1-100 micrograms/ml), MN thymocytes continued to cycle indicating a CsA-resistant generative compartment. Treatment of peripheral blood T cells with CsA had no effect on surface expression of CD3 or CD45 isoforms but did reduce the amount of de novo-synthesized CD45R0 mRNA. Culture of MN thymocytes on TEC rendered them virtually resistant to the negative effects of CsA. CD3 acquisition was unhindered and total CD45 remained high, but the transition from CD45RA to CD45R0 appeared to be delayed. In the absence of TEC, expression of both TCR alpha beta and of TCR delta was inhibited, but on TEC, TCR delta was actually up-regulated in some conditions. The effects of CsA on human thymocyte development appeared to be modulated by the physiological state of the donor and the growth conditions to which the cells were subjected. Conditions which most closely approximated those manifest in vivo rendered thymocytes most resistant to the negative effects of CsA. The amount of CsA required to affect differentiation in vitro was significantly higher than could be attained in vivo suggesting that the immunomodulatory effects of CsA in the maintenance of organ transplants may derive from an as yet uncharacterized mechanism.

Plasma concentrations and urinary excretion of histamine after inhalation and subcutaneous injection of histamine.

1. Increased histamine concentrations are found in the plasma and urine following allergen challenge in allergic subjects. This study compared a controlled challenge with clinically relevant doses of inhaled and injected histamine, as indicative of an allergic response, in an attempt to validate the use of urinary histamine or 1-methylhistamine measurements as an objective, non-invasive diagnostic test. 2. Inhalation of histamine produced peripheral vasodilation, increased heart rate, a fall in partial expiratory flow rate (pEFR) and blood pressure, 'tight chest' and cough. Subcutaneous injection produced vasodilation and headache but no change in heart rate or blood pressure. 3. Plasma histamine concentrations were similar in the two studies. Inhalation of increasing doses of histamine through a nebuliser (output 0.13 ml min-1) resulted in an increase from a mean of 0.30 to 1.65 ng ml-1, with return towards baseline within 20 min. Injection of 1 mg histamine s.c. produced an increase from 0.32 to 1.4 ng ml-1 within 5 min, remaining above 1 ng ml-1 for 30 min. 4. There was a significant increase of 15.2 ng mg-1 creatinine in urinary histamine concentration following the injection of histamine (P = 0.04) and an increase of 11.4 ng mg-1 creatinine when histamine was given by inhalation (P = 0.18). Histamine excretion rate increased by 108 ng min-1 (P = 0.04) after inhalation and by 37.2 ng min-1 (P = 0.09) after injection. Urinary 1-methylhistamine concentrations were significantly raised following both histamine inhalation (+ 238 ng mg-1 creatinine; P = 0.013) and injection (+ 180 ng mg-1 creatinine; P = 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Human T cell responses to human papillomavirus type 16 L1 and E6 synthetic peptides: identification of T cell determinants, HLA-DR restriction and virus type specificity.

Four T cell determinants in the major capsid protein of human papillomavirus (HPV) type 16 L1 and one in the E6 protein associated with cellular transformation were defined using synthetic peptides to stimulate peripheral blood mononuclear cells from asymptomatic individuals. HLA-DR restriction was defined using murine L cells transfected with HLA-DR genes to present antigen. Responses to two of the five determinants by T cell lines and clones were shown to be specific for HPV-16 based on the lack of cross-recognition of the corresponding sequences of other known papillomavirus sequences (types 1a, 5, 6b, 8, 11, 18 and 33). The T cells raised against two of the other peptides cross-reacted with corresponding peptides from other strains to varying extents, depending on their structural homology. The implications of these results regarding the prevalence of HPV-16 infection in the population and the possible diagnostic role of these responses in papillomavirus infection is discussed.

Degenerate binding of immunogenic peptides to HLA-DR proteins on B cell surfaces.

Binding of linear fragments of protein antigens to class I or class II molecules of the MHC is necessary for the stimulation of a cellular immune response. This report describes the binding of a biotinylated T cell determinant from influenza hemagglutinin to class II proteins on the surface of Epstein-Barr virus-transformed B lymphocytes. The rapid, simple, and quantitative binding assay involves flow cytometric analysis of transformed B cells stained with fluoresceinated streptavidin following incubation with the biotinylated peptide. Binding of the biotinylated peptide required cell surface expression of human class II molecules, and was inhibited by an anti-HLA-DR monoclonal antibody as well as the unbiotinylated natural determinant. Rates of association and dissociation of the peptide were similar to those reported for purified MHC class II proteins, and the peptide bound only approximately 1% of the DR molecules expressed on the cell surface. When assayed on many different DR-homozygous B cell lines, the biotinylated hemagglutinin T cell determinant bound to HLA-DR on each cell line. The degeneracy of peptide binding to B cell lines was not unique to the hemagglutinin peptide because three other biotinylated T cell determinants failed to bind to class II deficient B-lymphoblastoid cells but bound to varying degrees to multiple DR-homozygous lines.

Peptides recognized by class I restricted T cells also bind to MHC class II molecules.

The mechanisms of antigen recognition employed by both class I and class II MHC-restricted T cells are very similar, yet many of the T cell determinants described to date are recognized in the context of a single class of MHC molecules, and generally with only one or a very few different MHC alleles. To determine whether this might be due to a structural difference between class I and class II restricted T cell determinants, peptides previously shown to be recognized in the context of MHC class I proteins by mouse or human CD8+ T lymphocytes were tested for their capacity to bind to HLA-DR molecules on the surface of B lymphoblastoid cell lines (B-LCL). Four out of five class I restricted T cell determinants tested bound to a panel of B-LCL, and the binding was inhibited by anti-HLA-DR mAb. The peptides did not bind to the class II-negative B-LCL RJ2.2.5 nor to mouse L cells, but did bind to L cells transfected with HLA-DR1.

Structural analysis of a peptide--HLA class II complex: identification of critical interactions for its formation and recognition by T cell receptor.

An assay for the binding of peptides to major histocompatibility complex (MHC) class II proteins on the surface of cells has been used to determine the relative importance of the amino acids composing an influenza haemagglutinin T cell determinant in binding. The important contact residues were identified by the effect substitution of each residue with biotinylated lysine had on the ability of the peptide to bind. The spacing of the critical residues within the peptide sequence was consistent with the central core, of approximately eight amino acids, adopting a helical conformation. The terminal residues were less constrained and might not be part of a regular conformation. Increasing the helical propensity of the determinant, by simply acetylating and amidating the peptide, resulted in an analogue that was able to stimulate a specific T cell clone at significantly lower concentrations than the natural sequence. A potential location for the peptide in the binding site was postulated based on the presence of complementary amino acids in the class II molecule and supported by screening a large number of peptide analogues for their ability either to bind the restriction element or to stimulate T cell proliferation.

Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle.

Diabetes-associated peptide has recently been isolated and characterized from the amyloid of the islets of Langerhans in type 2 (non-insulin-dependent) diabetics, and immunoreactivity with antibodies to the peptide has been demonstrated in islet B cells of both normal and type 2 diabetic subjects. In view of the evidence presented in this paper that this 37-amino acid peptide may be a hormone present in normal individuals, we now propose the name "amylin" to replace "diabetes-associated peptide." Because increased amylin, deposited as amyloid within the islets of Langerhans, is characteristic of type 2 diabetes, the study below was performed to examine the possible effects of amylin on peripheral glucose metabolism. Whole amylin was synthesized by using solid-phase techniques, with formation of the disulfide linkage by oxidation in dilute aqueous solution and recovery of the peptide by lyophilization. The effects of amylin on glucose metabolism were studied in two preparations in vitro, isolated rat soleus muscle strips and isolated rat adipocytes. In skeletal muscle exposed to 120 nM amylin for 1 hr, there was a marked decrease in both basal and submaximally insulin-stimulated rates of glycogen synthesis, which resulted in significant reduction in the rates of insulin-stimulated glucose uptake. In muscles treated with amylin there was no response at the concentration of insulin required to stimulate glucose uptake half-maximally in untreated (control) muscles. In marked contrast, amylin had no effect on either basal or insulin-stimulated rates of glucose incorporation into either CO2 or triacylglycerol in isolated adipocytes. Therefore, amylin may be a factor in the etiology of the insulin resistance in type 2 diabetes mellitus, as both deposition of the peptide in islet amyloid and decreased rates of glucose uptake and glycogen synthesis in skeletal muscle are characteristic of this condition.

Structural model of HLA-DR1 restricted T cell antigen recognition.

Two human helper T cell determinants in influenza have been identified, one in the hemagglutinin and the other in the matrix protein (M1). Both were shown to be DR1 restricted by using transfected L cells to present antigen. Comparison of the sequences of the two peptides revealed a similar pattern that could account for their DR1 specificity if the peptides adopt a helical conformation. The model was supported by the demonstration that hybrid peptides, composed of the amino acids that interact with DR1 from one determinant and the residues that interact with the T cell receptor from the other, were recognized by each clone. The generality of the motif was confirmed by the finding that DR1 individuals respond to a ragweed peptide containing the defined pattern.