Widespread monoclonal IgE antibody convergence to an immunodominant, proanaphylactic Ara h 2 epitope in peanut allergy.

BACKGROUND: Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. OBJECTIVE: We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. METHODS: We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. RESULTS: We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. CONCLUSIONS: Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.

Structure and stabilization of the Hendra virus F glycoprotein in its prefusion form.

Hendra virus (HeV) is one of the two prototypical members of the Henipavirus genus of paramyxoviruses, which are designated biosafety level 4 (BSL-4) organisms due to the high mortality rate of Nipah virus (NiV) and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released and, upon receptor-induced triggering, inserts into the host cell membrane. As F undergoes a dramatic refolding from its prefusion to postfusion conformation, the fusion peptide brings the host and viral membranes together, allowing entry of the viral RNA. Here, we present the crystal structure of the prefusion form of the HeV F ectodomain. The structure shows very high similarity to the structure of prefusion parainfluenza virus 5 (PIV5) F, with the main structural differences in the membrane distal apical loops and the fusion peptide cleavage loop. Functional assays of mutants show that the apical loop can tolerate perturbation in length and surface residues without loss of function, except for residues involved in the stability and conservation of the F protein fold. Structure-based disulfide mutants were designed to anchor the fusion peptide to conformationally invariant residues of the F head. Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation.

Relaxosome function and conjugation regulation in F-like plasmids - a structural biology perspective.

The tra operon of the prototypical F plasmid and its relatives enables transfer of a copy of the plasmid to other bacterial cells via the process of conjugation. Tra proteins assemble to form the transferosome, the transmembrane pore through which the DNA is transferred, and the relaxosome, a complex of DNA-binding proteins at the origin of DNA transfer. F-like plasmid conjugation is characterized by a high degree of plasmid specificity in the interactions of tra components, and is tightly regulated at the transcriptional, translational and post-translational levels. Over the past decade, X-ray crystallography of conjugative components has yielded insights into both specificity and regulatory mechanisms. Conjugation is repressed by FinO, an RNA chaperone which increases the lifetime of the small RNA, FinP. Recent work has resulted in a detailed model of FinO/FinP interactions and the discovery of a family of FinO-like RNA chaperones. Relaxosome components include TraI, a relaxase/helicase, and TraM, which mediates signalling between the transferosome and relaxosome for transfer initiation. The structures of TraI and TraM bound to oriT DNA reveal the basis of specific recognition of DNA for their cognate plasmid. Specificity also exists in TraI and TraM interactions with the transferosome protein TraD.

Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM.

The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM-sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM's N-terminal ribbon-helix-helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore.

Structural basis of specific TraD-TraM recognition during F plasmid-mediated bacterial conjugation.

F plasmid-mediated bacterial conjugation requires interactions between a relaxosome component, TraM, and the coupling protein TraD, a hexameric ring ATPase that forms the cytoplasmic face of the conjugative pore. Here we present the crystal structure of the C-terminal tail of TraD bound to the TraM tetramerization domain, the first structural evidence of relaxosome-coupling protein interactions. The structure reveals the TraD C-terminal peptide bound to each of four symmetry-related grooves on the surface of the TraM tetramer. Extensive protein-protein interactions were observed between the two proteins. Mutational analysis indicates that these interactions are specific and required for efficient F conjugation in vivo. Our results suggest that specific interactions between the C-terminal tail of TraD and the TraM tetramerization domain might lead to more generalized interactions that stabilize the relaxosome-coupling protein complex in preparation for conjugative DNA transfer.

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Chronic arterial insufficiency of the upper extremity.

When claudication or distal ischemia is significant, the treatment of choice for intrinsic arterial insufficiency of the upper extremity caused by atherosclerotic occlusive disease of the subclavian, axillary, or brachial artery with patent distal circulation is direct arterial surgery. Both endarterectomy and bypass procedures are utilized. In the 20 years from 1947 to 1967, 15 patients were operated on for such lesions and adequate circulation was restored in 12 patients treated by the direct method. Nine patients available for 1-year follow-up were cured of their symptoms; of the remaining three patients, one had amputation of an arm and two had sympathectomy with improvement. No hospital death occurred and morbidity was minimal. Although the diagnosis can be established on clinical grounds, arteriography is essential to ascertain the surgical procedure needed. Peripheral diseases involving the small arteries are clinically a more common cause of arterial insufficiency in the upper extremity; progress is slow and complications are infrequent. When ischemic complications exist, direct arterial surgery is not feasible and cervicodorsal sympathectomy is the usual form of treatment. Of 76 patients operated on for such disease, 74 underwent sympathetic neurectomy. The results were excellent, good, or satisfactory in 50 (78%) of the 64 traced patients. The remaining 14 patients had persistent or recurrent symptoms after operation. Removal of the stellate ganglion in addition to the second ganglion yielded better results and is indicated in recurrent or more cases.

Selection and preparation of high-risk patients for repair of abdominal aortic aneurysms.

OBJECTIVE: To discuss the most important risk factors in patients who undergo surgical repair of an abdominal aortic aneurysm (AAA). DESIGN: This update in vascular surgical repair highlights the criteria that identify high-risk patients, the useful preoperative tests, and the perioperative measures that can aid surgical recovery. MATERIAL AND METHODS: In elective repair of AAAs, high-risk patients are those with severe coronary or valvular heart disease, decompensated chronic obstructive pulmonary disease, severe cerebrovascular disease, chronic renal failure, hepatic cirrhosis with portal hypertension, and chronic hematologic disorders associated with bleeding dysfunction. Patients with unstable or severely symptomatic heart disease should undergo preoperative coronary angiography and ventriculography. Pharmacologic stress testing is recommended for patients with clinical markers of serious coronary artery disease and other medical or physical factors that prevent any type of standard exercise stress testing. RESULTS: Our experience with high-risk patients supports conventional repair of AAAs. Our preference for the midline abdominal incision in high-risk patients is substantiated by an operative mortality rate of 5.7% in comparison with a reported 7% mortality rate for nonresective therapy. Approximately one in three high-risk patients will have a serious postoperative complication, the most common of which is a cardiac event. Most patients recover after a slightly prolonged hospital stay. CONCLUSION: Despite an increased operative risk, patients with a stable medical condition and an AAA larger than 6 cm in diameter should be considered for elective repair. High-risk patients with smaller aneurysms (5 to 6 cm in diameter) should undergo efforts to stabilize or to improve their general medical condition before elective operation.

Clinical features and differential diagnosis of aortic dissection: experience with 236 cases (1980 through 1990).

Acute aortic dissection is the most common fatal condition that involves the aorta; nevertheless, despite major advances in noninvasive diagnosis, the correct antemortem diagnosis is made in less than half the cases. To promote continued improvement in the prompt recognition of aortic dissection, we present a review of the Mayo Clinic experience with 235 patients who had 236 substantiated aortic dissections. At the time of initial assessment, 158 patients (67%) had acute and 78 patients (33%) had chronic aortic dissection. Hypertension was the most common predisposing factor (78% of patients overall). The acute onset of severe chest pain was the most common initial complaint (74%), but 33 patients (15%) had painless aortic dissection and abnormal chest roentgenographic findings. Less common manifestations included congestive heart failure, syncope, cerebrovascular accident, shock, paraplegia, and lower extremity ischemia. The initial clinical impression was aortic dissection in 62% of patients overall. In 17 patients (28%), the correct diagnosis was not made before postmortem examination. Although the clinical features of aortic dissection have gained wider appreciation, the diagnosis still remains unsuspected in a substantial number of patients. In a patient who has a catastrophic illness and unexplained symptoms that could be of vascular origin, especially in the presence of chest pain, aortic dissection should always be included in the differential diagnosis.

Buerger's disease (thromboangiitis obliterans).

Thromboangiitis obliterans is a progressive, often relentless and devastating, vasculitis causing significant loss of digits and limbs in a youthful population of tobacco users. Whereas the specific pathogenetic mechanism has not been defined, tobacco use is clearly a trigger for what appears to be an autoimmune mechanism in a given group of patients. Its cessation almost always prevents further tissue damage. Medical and surgical therapy palliate accrued damage, but only complete abstinence from tobacco use allows stabilization of the process. An appreciation of the characteristic clinical, angiographic, and histopathologic features allows specific diagnosis and differentiation from premature atherosclerosis and other mechanisms of distal and microcirculatory deficits. There is a pressing need for the evaluation of agents that might interrupt this process in the face of continued tobacco use; such an agent would be helpful in combating proliferative arterial change in other types of vasculitis.

Splenic artery aneurysms.

The records of 100 patients with documented splenic artery aneurysms seen between January 1960 and January 1980 were reviewed. Eighty-seven were women and 13 men. Ages ranged from 16 to 81 years (mean 58.2 years). Eighty women (92%) had been pregnant. The number of pregnancies ranged from 1 to 16 (mean 4.5); 21 women (24.1%) had been pregnant six or more times. Aneurysm diameter ranged from 0.6 to 30 cm (mean 2.1 cm). The aneurysm was calcified in 72 patients. Most aneurysms (78%) were located in the distal third of the splenic artery and were saccular. Multiple aneurysm occurred in 29 patients. Seventeen patients were symptomatic at the time of diagnosis; all presented with abdominal pain. Rupture occurred in three patients, one of whom was pregnant; two survived. Atherosclerotic occlusive disease occurred in 21 patients. Other arterial aneurysms occurred in 12 patients. Eighty-one patients had surgical treatment. Aneurysmectomy with splenectomy was the most common procedure. The elective operative mortality rate was 1.3%. The 19 patients not undergoing surgery were followed from 1.0 to 19 years (mean 7.4 years). No ruptures or other complications of the aneurysm occurred in these patients. This study supports the view that patients who are pregnant or who present with a symptomatic splenic artery aneurysm should undergo surgical resection.

Arteriomegaly: classification and morbid implications of diffuse aneurysmal disease.

Ninety-one patients with arteriomegaly and diffuse aneurysmal disease below the level of the renal arteries have been classified according to the extent and location of aneurysmal change. There exists a high incidence of thrombotic and embolic complications, and treatment entails increased rates of morbidity and mortality when compared to surgical treatment of simple abdominal aneurysms of peripheral artery aneurysms. Complete revascularization at the initial operation would appear to give the best result, but this approach must be tempered by the individual patient risk factors and the urgency of the mode of presentation of the patient.

Thoracic aortic aneurysms: a population-based study.

Thoracic aortic aneurysms were detected in 72 residents (44 women and 28 men) in a stable midwestern community over a 30-year period, for an age- and sex-adjusted incidence of 5.9 new aneurysms per 100,000 person-years. The incidence was equal in both sexes and decreased slightly over the 30 years. Ages ranged from 47 to 93 years (median 65 years for men and 77 years for women). The ascending aorta was involved in 37 patients, the aortic arch in 8, and the descending aorta in 27. Pathologic examination was performed in 51 patients. The cause was aortic dissection in 27 patients (53%), atherosclerosis in 15 (29%), aortitis in 4 (8%), cystic medial necrosis in 3 (6%), and syphilis in 2 (4%). All autopsied patients had pathologic evidence of significant hypertension. Eleven patients (25%) had concomitant abdominal aortic aneurysms. Rupture occurred in 53 patients (74%) and 50 died. Thirty-seven of these patients had no prior diagnosis of aneurysm. The median interval between diagnosis and rupture in the 16 remaining patients was 2 years (range 1 month to 16 years). Ninety-five percent of aortic dissections ruptured and 51% of nondissecting aneurysms ruptured. The actuarial 5-year survival for all 72 patients was 13%; for patients with aortic dissection, 7% and for patients without dissection, 19.2%.

Doppler-determined segmental pressures and wound-healing in amputations for vascular disease.

One hundred and two diabetic and non-diabetic patients with severe vascular disease of the lower extremities were studied in the vascular laboratory at the Mayo Clinic prior to amputation. The level of amputation was chosen on the basis of clinical findings alone. Sixty-seven of the amputations were the classic below-the-knee type. The tests performed included determinations of segmental systolic pressures of the arm, thigh, calf, and ankle determined with the Doppler ultrasonic velocity detector. Contrary to other reports, we found no demonstrable relationship between calf pressure or calf-arm pressure index and the degree of healing of below-the-knee amputations in diabetic or non-diabetic patients.

Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM.

The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH ß-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.