Infection and allergy incidence in ambulatory surgery patients using white petrolatum vs bacitracin ointment. A randomized controlled trial.

OBJECTIVE: To assess the effect of white petrolatum vs bacitracin ointment on wound infection incidence, allergic contact dermatitis incidence, and healing characteristics. DESIGN: Randomized, double-blind, prospective trial comparing white petrolatum with bacitracin ointment in postprocedure wound care. SETTING: A general outpatient dermatology clinic and a tertiary referral advanced surgical procedure clinic at Walter Reed Army Medical Center, Washington, DC. PATIENTS: A total of 922 patients who had dermatologic surgery with a total of 1249 wounds. MAIN OUTCOME MEASURES: The incidence of infection and allergic contact dermatitis during a follow-up period of 4 weeks. Healing characteristics were secondary outcomes. RESULTS: Of the 922 patients enrolled, 440 in the white petrolatum group and 444 in the bacitracin group were evaluable for clinical response. The 2 treatment groups had comparable baseline characteristics. Thirteen patients developed postprocedure infection (1.5%), 9 (2.0%) in the white petrolatum group vs 4 (0.9%) in the bacitracin group (95% confidence interval for difference, -0.4% to 2.7%; P=.37). Eight infections (1.8%) in the white petrolatum group were due to Staphylococcus aureus vs none in the bacitracin group (P=.004). No patient in the group using white petrolatum developed allergic contact dermatitis vs 4 patients (0.9%) in the group using bacitracin (P=.12). Additionally, there were no clinically significant differences in healing between the treatment groups on day 1 (P=.98), day 7 (P=.86), or day 28 (P=.28) after the procedure. CONCLUSIONS: White petrolatum is a safe, effective wound care ointment for ambulatory surgery. In comparison with bacitracin, white petrolatum possesses an equally low infection rate and minimal risk for induction of allergy.

Cutaneous angiosarcoma arising in the radiation site of a congenital hemangioma.

We describe a patient in whom angiosarcoma developed at the site of a hemangioma that was treated during infancy with radiation for refractory thrombocytopenia. Our findings, along with those of the 10 reported cases from the world literature, are summarized. One third of angiosarcomas arise in the skin. They most often show one of three clinical patterns. First and most common is occurrence as a bruiselike lesion on the scalp or face of an elderly person. Second in frequency is the Stewart-Treves syndrome. Third and least common is angiosarcoma developing as a sequela of previous radiation therapy. The prognosis in general is poor, with a mean survival length of 24 months and a 5-year survival rate of 10%. Effective treatment relies on early diagnosis and wide-margin surgical excision.

Minocycline-induced hyperpigmentation.

A 70-year-old man developed hyperpigmentation of his forearms, hands, fingernails, sclerae, ears, and teeth after 9 years of therapy with minocycline for acne rosacea. Minocycline is widely used in the treatment of acne vulgaris and uncommonly produces the side effect of hyperpigmentation. This effect does not appear to be dose-dependent and usually resolves within months to years after discontinuation of therapy. Discoloration of adult teeth, however, is generally permanent.

Passive transfer of autoantibodies from a patient with mutilating epidermolysis bullosa acquisita induces specific alterations in the skin of neonatal mice.

BACKGROUND: Epidermolysis bullosa acquisita is a subepidermal bullous disease characterized by IgG autoantibodies directed against type VII collagen in anchoring fibrils. These autoantibodies are believed to play an important role in the pathogenesis of sub-lamina densa blister formation in this disease. OBSERVATIONS: We describe a patient with epidermolysis bullosa acquisita who has developed mutilating acral involvement with early syndactyly and extensive scarring lesions of the scalp. The patient's serum contains IgG autoantibodies that bind the dermal side of 1-mol/L sodium chloride-separated human skin (at a titer up to 5120), as determined by indirect immunofluorescence microscopy, and type VII collagen, as determined by immunoblot. The severity of this patient's disease and the height of his immune response to type VII collagen prompted us to assess the pathogenicity of his autoantibodies in a murine model. Purified IgG from our patient (or that from a healthy volunteer who served as a control) was administered subcutaneously to BALB/c mice (10 mg/g of body weight) on 2 consecutive days. Light microscopy of normal-appearing skin showed pronounced dermal edema and a dense granulocyte-rich infiltrate in the superficial dermis. Deposits of human IgG, murine C3, and the membrane attack complex of complement were found in the epidermal basement membrane of all experimental mice. Immunogold electron microscopy demonstrated that deposits of human IgG in an experimental subject were localized to anchoring fibrils. Serum samples from mice receiving IgG antibodies from our patient had high titers of circulating antibodies directed against the dermal side of 1-mol/L sodium chloride-separated human skin (titer, 640 to 1280). Light, immunofluorescence, and immunogold electron microscopic studies did not detect such specific alterations in any control mice. CONCLUSIONS: Acquired autoimmunity to type VII collagen in patients with epidermolysis bullosa acquisita may result in a clinical phenotype closely resembling that observed in patients with dystrophic epidermolysis bullosa. Passive transfer of purified IgG autoantibodies from a patient with severe epidermolysis bullosa acquisita to BALB/c mice produces histologic and immunopathologic alterations consistent with those seen in patients with this disease.

Solution properties of Escherichia coli-expressed VH domain of anti-neuraminidase antibody NC41.

The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG), has been expressed in high yield (15-27 mg/L) in Escherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of approximately 4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20 degrees C and concentrations of 5-10 mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its 1H NMR spectrum. Reagents such as CHAPS, n-ocytylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.

Characterization of a basic serine proteinase (pI approximately 9.5) secreted by virulent strains of Dichelobacter nodosus and identification of a distinct, but closely related, proteinase secreted by benign strains.

An extracellular serine proteinase with a PI approximately 9.5 (referred to as 'basic proteinase') was purified to homogeneity, from strains of Dichelobacter nodosus that cause virulent foot-rot, by gel filtration of concentrated culture supernatant on Sephadex G-100 and chromatography on sulphopropyl-Sephadex C-25 at pH 8.6 D. nodosus strains that cause benign foot-rot do not secrete a corresponding basic proteinase with a pI of approximately 9.5. Benign strains secrete a closely related, but distinct, proteinase which has the same molecular mass and N-terminal sequences as the 'virulent' basic proteinase, but a lower pI of approximately 8.6. The basic proteinases from both strains appear to interact with other proteins present in the culture medium, which results in anomalous behavior on gel filtration. Pure D. nodosus 'virulent' basic proteinase has a molecular mass of 36 kDa and showed a low solubility at I < 0.05 precipitating quantitatively from solution as microcrystals. The proteinase shows optimal activity at pH 8.0 and is stable to heating to 55 degrees C for 30 min, but at higher temperatures activity is rapidly lost. Bivalent-metal ions (e.g. Ca2+) are required to maintain the structural integrity and stability of the proteinase; in the presence of EDTA or conditions that cause protein unfolding, the proteinase undergoes rapid and complete autolysis. Cleavage of oxidized insulin A- and B-chain showed that the basic proteinase has a broad specificity, including cleavage at lysine and arginine bonds.

Recombinant anti-sialidase single-chain variable fragment antibody. Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex.

The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly4Ser)3, of monoclonal antibody NC10 was expressed in Escherichia coli and purified to homogeneity. This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4 degrees and 20 degrees C. At higher protein concentrations (approximately 5 mg/ml) dimer and higher-molecular-mass multimers were formed and freezing enhanced this aggregation. The dimer was not stable and dissociated to monomer at 20 degrees C with a half-life of approximately 8 days. The higher-molecular-mass multimers and dimer dissociated to monomer in 60% ethylene glycol. Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules bound/sialidase tetramer and that the dimer was bivalent and cross-linked two sialidase tetramers. Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NC10 antigen-binding fragment (Fab) molecule. A complex between tern N9 sialidase and NC10 scFv was crystallized and the structure of the complex was solved at 0.3-nm resolution by X-ray diffraction. Comparison of this scFv/sialidase structure with the parent Fab/sialidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar. There was no discernible electron density for the peptide linker joining the variable heavy (VH) and variable light (VL) chains. A close interaction between two symmetry-related scFv suggests that they may have crystallized as dimers.

Affinity ranking of influenza neuraminidase mutants with monoclonal antibodies using an optical biosensor. Comparison with ELISA and slot blot assays.

A recently developed alternative to the more traditional techniques for studying antigen-antibody interactions has been examined. This method involves the use of an optical biosensor employing surface plasmon resonance detection. In this system one of the reactants is immobilized on the sensor surface and other reactants are passed over the sensor surface sequentially at a constant flow rate. Binding phenomena are detected in real time from changes in the angle at which surface plasmon resonance occurs. This is dependent, among other things, on changes in the refractive index (which is directly proportional to the mass) at or near to the sensor surface. Applications of this biosensor technique for comparing the binding of related neuraminidases, purified from escape mutants of influenza virus NWS/G70C/75 (N9), to two antibody Fab fragments, are described. These results were compared with those obtained from ELISA and slot blot assays on the same neuraminidases interacting with the same two monoclonal antibodies. The biosensor method was shown to be highly specific, permitting rapid screening of binding in such antigen-antibody systems.

Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen.

The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P42(1)2, with unit cell dimensions a = b = 141 A, c = 218 A.

The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor.

Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.

Optical design with Wood lenses 1: infinite conjugate systems.

Spherical aberration in a flat-surfaced radial gradient-index lens (a Wood lens) with a parabolic-index profile can be corrected by altering the profile to include higher-order terms. However, this results in a large amount of third-order coma. An alternative method of aberration correction similar to that used in the catadioptric Schmidt system is presented. A Wood lens with a parabolic-index profile is used to provide all or most of the optical power. Coma is corrected by stop shifting, and spherical aberration is corrected by placing a powerless Wood lens corrector plate at the stop.

Primary structure of Kunitz-type trypsin inhibitor-2a (pI 5.9) from Psophocarpus tetragonolobus (L.) DC seed.

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a, pI 5.9) from Psophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a, pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2, pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1, pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.

A new method for the purification of the influenza A virus neuraminidase.

A rapid new method for the purification of neuraminidase (NA) heads from influenza A virus is described. Virus was pelleted directly from allantoic fluid and was digested with pronase. The cores were removed by centrifugation, redigested and the released NA heads were pooled and concentrated. The NA was separated from all contaminating proteins in a single step on a Superose 12 column. The purified material was suitable for both crystallography and for the production of monospecific antisera.

Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.).

The amino acid sequence of a methionine-rich 2S seed protein from sunflower (Helianthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13-residue hydrophobic pro-sequence which is presumably removed by post-translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C-terminus. Comparison of the sunflower methionine-rich protein sequence with sequences of other seed 2S proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine-rich Brazil nut 2S protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine-rich 2S proteins have diverged as a subclass of the 2S superfamily of proteins which contain only 2-3% methionine. While the related 2S proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC].

The primary sequence of trypsin inhibitor-2 (WBTI-2) from Psophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 from Erythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors from Adenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that the Psophocarpus and Erythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.

Radial gradient-index eyepiece design.

Three- and four-element eyepiece designs are presented each with a different type of radial gradient-index distribution. Both quadratic and modified quadratic index profiles are shown to provide effective control of the field aberrations. In particular, the three-element design with a quadratic index profile demonstrates that the inhomogeneous power contribution can make significant contributions to the overall system performance, especially the astigmatism correction. Using gradient-index components has allowed for increased eye relief and field of view making these designs comparable with five- and six-element ones.