Post-acute phase and sequelae management of epidermal necrolysis: an international, multidisciplinary DELPHI-based consensus.

BACKGROUND: Long-term sequelae are frequent and often disabling after epidermal necrolysis (Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN)). However, consensus on the modalities of management of these sequelae is lacking. OBJECTIVES: We conducted an international multicentric DELPHI exercise to establish a multidisciplinary expert consensus to standardize recommendations regarding management of SJS/TEN sequelae. METHODS: Participants were sent a survey via the online tool "Survey Monkey" consisting of 54 statements organized into 8 topics: general recommendations, professionals involved, skin, oral mucosa and teeth, eyes, genital area, mental health, and allergy workup. Participants evaluated the level of appropriateness of each statement on a scale of 1 (extremely inappropriate) to 9 (extremely appropriate). Results were analyzed according to the RAND/UCLA Appropriateness Method. RESULTS: Fifty-two healthcare professionals participated. After the first round, a consensus was obtained for 100% of 54 initially proposed statements (disagreement index < 1). Among them, 50 statements were agreed upon as 'appropriate'; four statements were considered 'uncertain', and ultimately finally discarded. CONCLUSIONS: Our DELPHI-based expert consensus should help guide physicians in conducting a prolonged multidisciplinary follow-up of sequelae in SJS-TEN.

Causes of failure of the Kinemax Plus total knee replacement in the first five years.

In order to investigate whether any single batch of polyethylene inserts was responsible for premature failure of total knee replacements (TKR), we performed a retrospective database review of 1133 consecutive primary Kinemax Plus TKR performed by 2 surgical teams between January 1992 and January 2001 at our institution. Cases re-operated in any way within 5 years of the index arthroplasty were identified, case notes studied and radiographs reviewed. The causes contributing to the premature failure of the implants were identified where possible and the polyethylene lot numbers were recorded. Nineteen cases were known to have been revised, 7 cases for deep infection of the prosthesis and 12 for aseptic failure. In 10 cases, an identifiable technical error had contributed to the early failure of the implant. In 4 of these cases, there was inadequate tissue balancing in patients with a significant pre-operative valgus deformity. In 2 cases, the tibial base plate was inserted in a varus mal-alignment. There was one extended tibial component, one internally rotated femoral component, one oversized femoral component and one femoral component with inadequate fixation. Two further cases failed aseptically, one with a fracture of the medial tibial plateau and one idiopathic. The lot numbers of the polyethylene inserts were all different. This study confirms that the premature failure rate of the Kinemax Plus TKR is low. Although a "bad batch" of polyethylene has been suggested as a cause for early failure in TKR, in this series, surgical error was the predominant cause.

Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant, G67.

BACKGROUND: Foot-and-mouth disease viruses (FMDVs) are members of the picornavirus family and cause an economically important disease of cloven-hoofed animals. To understand the structural basis of antigenic variation in FMDV, we have determined the structures of two viruses closely related to strain O1BFS whose structure is known. RESULTS: The two new structure are, like O1BFS, both serotype O viruses. The first, O1 Kaüfbeuren (O1K), is a field isolate dating from an outbreak of FMD in Europe in the 1960s. The second, called G67, is a quadruple mutant of O1K, generated in the laboratory, that bears point mutations conferring resistance to neutralizing by monoclonal antibodies, specific for each of the four major antigenic sites defined previously. The availability of the three related virus structures permits a detailed analysis of the way amino acid substitutions influence antigenicity. Structural changes are seen to be limited, in general, to the substituted side chain. For example, the GH loop of VP1, a highly antigenic and mobile protuberance which becomes ordered only under reducing conditions, was essentially indistinguishable in the three viruses despite the accumulation of up to four changes within its 15-residue sequence. At one of the other antigenic sites, however, changes between the two field strains did perturb both side-chain and main-chain structures in the vicinity. CONCLUSIONS: The conservation of conformation of the GH loop of VP1 adds to the evidence implicating an integrin as the cellular receptor for FMDV, since this loop contains a conserved RGD (Arg-Gly-Asp) sequence structurally similar to the same tripeptide in some other integrin-binding proteins. Structural changes required for the virus to escape neutralization by monoclonal antibodies are generally small. The more extensive type of structural change exhibited by the field isolates probably reflects differing selective pressures operating in vivo and in vitro.

The structure and antigenicity of a type C foot-and-mouth disease virus.

BACKGROUND: Picornaviruses are responsible for a wide range of mammalian diseases and, in common with other RNA viruses, show considerable antigenic variation. Foot-and-mouth disease viruses (FMDVs) constitute one genus of the picornavirus family and are classified into seven serotypes, each of which shows considerable intratypic variation. This antigenic variation leads to continuing difficulties in controlling the disease. To date the structure of only one serotype, O, has been reported. RESULTS: The three-dimensional structure of a serotype C (isolate C-S8c1) FMDV, has been determined crystallographically at 3.5 A resolution. The main chain conformation of the virion is very similar to that of type O1 virus. The immunodominant G-H loop of VP1, the presumed site of cell attachment, is disordered in both types of virus indicating a functional role for flexibility of this region. There are significant changes in the structure of other antigenic loops and in some internal regions involved in protomer-protomer contacts, including the entire amino-terminal portion of VP2, described here for the first time for a picornavirus. Antigenic sites have been identified by genetic and peptide mapping methods, and located on the capsid. The data reveal a major new discontinuous antigenic site (site D) which is located near to the three-fold axis and involves residues of VP1, VP2 and VP3 which lie adjacent to each other on the capsid. CONCLUSION: In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity.

BACKGROUND: Foot-and-mouth disease virus (FMDV) is an extremely infectious and antigenically diverse picornavirus of cloven-hoofed animals. Strains of the A22 subtype have been reported to change antigenically when adapted to different growth conditions. To investigate the structural basis of this phenomenon we have determined the structures of two variants of an A22 virus. RESULTS: The structures of monolayer- and suspension-cell-adapted A22 FMDV have been determined by X-ray crystallography. Picornaviruses comprise four capsid proteins, VP1-4. The major antigenic loop of the capsid protein VP1 is flexible in both variants of the A22 subtype but its overall disposition is distinct from that observed in other FMDV serotypes (O and C). A detailed structural comparison between A22 FMDV and a type O virus suggests that different conformations in a portion of the major antigenic loop of VP1 (the GH loop, which is also central to receptor attachment) result in distinct folds of the adjacent VP3 GH loop. Also, a single mutation (Glu82-->Gly) on the surface of VP2 in the suspension-cell-adapted virus appears to perturb the structure of the VP1 GH loop. CONCLUSION: The GH loop of VP1 is flexible in three serotypes of FMDV, suggesting that flexibility is important in both antigenic variability and structural communication with other regions of the virus capsid. Our results illustrate two instances of the propagation of structural perturbations across the virion surface: the change in the VP3 GH loop caused by the VP1 GH loop and the Glu82-->Gly change in VP2 which we believe perturbs the GH loop of VP1. In the latter case, the amplification of the sequence changes leads to differences, between the monolayer- and suspension-cell-adapted viruses, in host-cell interactions and antigenicity.

Function of minor polypeptides in foot-and-mouth disease virus and poliovirus.

Foot-and-mouth disease virus and poliovirus each contain several minor polypeptides, in addition to the four structural proteins. One of these, the viral RNA polymerase, can also act as a nuclease, hydrolysing the RNA and thus destroying viral infectivity. It is tightly bound to the RNA and may be the packaging signal for assembly of the particle.

Mg- and Ca-actin filaments appear virtually identical in steady-state as determined by dynamic light scattering.

Dynamic light scattering measurements show that although Mg-actin polymerizes more rapidly than Ca-actin (actin at 0.04-0.4 mg/ml polymerized with 0.1 M KCl +/- 2 mM MgCl2 or CaCl2, at room temperature or at 10 degrees C), steady-state filaments exhibit nearly identical intensity autocorrelation functions and similar mean scattered intensities. The dynamic data are used to measure the persistence length of the filaments which is found to be 4.2 microns independent of the bound cation and of the actin concentration.

Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease viruses from serotypes O, A and C have been crystallized. The particular strains studied include O1K, A10(61), A22 Iraq 24/64, A24 Cruzeiro and C-S8c1. In addition, crystals have been grown of G67, a monoclonal antibody neutralization escape mutant derived from O1K, and of virus R100, recovered after the establishment of a persistent infection in baby hamster kidney cells with C-S8c1. Empty particles, capsids which lack the RNA genome, have also been crystallized for subtypes A22 Iraq 24/64 and A10(61). In almost all cases, crystals suitable for high resolution structure determination were obtained from (NH4)2SO4 or mixtures of polyethylene glycol and NH4Cl.

Survey of physician practice behaviors related to diabetes mellitus in the U.S. Physician adherence to consensus recommendations.

OBJECTIVE: To summarize the frequency of physician adherence to consensus recommendations for prevention of diabetic complications. RESEARCH DESIGN AND METHODS: Survey data from a nationwide stratified probability sample of primary-care physicians were analyzed. Adherence to recommendations were reported by physician specialty, age-group, and type of diabetes treated. RESULTS: Adherence was high for eye exams, blood pressure measurements, neurological and circulatory exams, and laboratory procedures using blood. Adherence was low for examination of the teeth and gums, examination of the feet, and laboratory procedures involving the collection of urine. Internists generally had the highest adherence rates and pediatricians the lowest. Reported adherence decreased with physician age. Adherence was higher for the management of individuals with IDDM than for those with NIDDM. CONCLUSIONS: Recommendations for the care of diabetic individuals need to be more widely implemented. Recommendations targeted specifically to pediatricians may be necessary.

Differential expression of putative transbilayer amphipath transporters.

The aminophospholipid translocase transports phosphatidylserine and phosphatidylethanolamine from one side of a bilayer to another. Cloning of the gene encoding the enzyme identified a new subfamily of P-type ATPases, proposed to be amphipath transporters. As reported here, mammals express as many as 17 different genes from this subfamily. Phylogenetic analysis reveals the genes to be grouped into several distinct classes and subclasses. To gain information on the functions represented by these groups, Northern analysis and in situ hybridization were used to examine the pattern of expression of a panel of subfamily members in the mouse. The genes are differentially expressed in the respiratory, digestive, and urogenital systems, endocrine organs, the eye, teeth, and thymus. With one exception, all of the genes are highly expressed in the central nervous system (CNS); however, the pattern of expression within the CNS differs substantially from gene to gene. These results suggest that the genes are expressed in a tissue-specific manner, are not simply redundant, and may represent isoforms that transport a variety of different amphipaths.

Multiple sites of recombination within the RNA genome of foot-and-mouth disease virus.

Recombinant foot-and-mouth disease viruses were isolated from cells infected with a mixture of temperature-sensitive (ts) mutants belonging to different subtype strains. In order to select for recombination events in many different regions of the genome, crosses were performed between various pairs of mutants, with ts mutations in different regions of the genome. ts+ progeny were analysed by electrofocusing virus-induced proteins and RNase T1 fingerprinting of their RNA. All but 5 out of 43 independent isolates, from nine crosses, proved to have recombinant RNA genomes. Maps of these genomes, based on a knowledge of the locations of the unique oligonucleotides, were constructed. Most could be interpreted as being the products of single genetic cross-overs, although three recombinants were formed by two cross-overs each. Cross-overs in at least twelve distinct regions of the genome were identified. This evidence of a large number of recombination sites suggests that RNA recombination in picornaviruses is a general, as opposed to a site-specific, phenomenon.

Isolation and biochemical characterization of intertypic recombinants of foot-and-mouth disease virus.

Recombinants were isolated between two European serotypes (O and A) and between two of the most distantly related serotypes (O from Europe and SAT2 from Africa) using appropriate ts mutants in an infectious centre assay. The recombinants were characterised by electrofocusing of their induced proteins and by RNase-T1 fingerprinting of their RNA. The approximate location of the cross-over event in each recombinant was determined by sequencing the unique distinguishable O or A oligonucleotides and locating them within the known genome sequence. Nine different types of recombinant were identified from the two types of cross (O X A and O X SAT) and all had a single cross-over in the middle or 3' half of the genome, i.e. in the nonstructural coding region. Recombination between the most distantly related viruses (O X SAT2) appeared to occur at a lower frequency than recombination between serotypes of the same group (O X A). A higher incidence of recombinant proteins with unique pI was also observed in the O X SAT2 crosses.

The structure of an immunodominant loop on foot and mouth disease virus, serotype O1, determined under reducing conditions.

Residues 136-159 of VPI of foot and mouth disease virus (FMDV) comprise the G-H loop of the protein and form a prominent feature on the surface of virus particles. This sequence contains an immunodominant neutralizing epitope, which can be mimicked with synthetic peptides, and includes an Arg, Gly, Asp motif which has been implicated in the binding of the virus to cellular receptors. Crystallographic analysis of native virus particles failed to resolve the structure of this region due to its disordered state. However, reduction of a disulphide bond between cysteine residues 134 of VP1 and 130 of VP2 caused the G-H loop to collapse onto the surface of the virus particle and allowed its conformation to be determined.

Baculovirus expressed 2C of foot-and-mouth disease virus has the potential for differentiating convalescent from vaccinated animals.

Determining whether animals have been infected with foot-and-mouth disease virus or vaccinated is important because infected animals frequently become carriers of the virus, shed it intermittently and thus may be the source of new outbreaks of the disease. We had shown previously that the sera of convalescent animals contain antibodies to 2C, a highly conserved non-structural protein, whereas the sera of vaccinated animals do not. This is explained by observation that 2C is retained on the membranes of cells used for growing the virus for vaccine production. In contrast, the non-structural protein 3D, which is released into the medium, is not removed by centrifugation or filtration during vaccine production and therefore stimulates an immune response in both vaccinated and convalescent cattle. In this study we produced 2C and 3D in insect cells infected with recombinant baculoviruses. As demonstrated by serology and electron microscopy, 2C is also retained on the membranes of the insect cells. Both expressed proteins react with sera of convalescent animals, indicating that they are conformationally similar, but the 2C does not react with sera from vaccinated animals. The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test.

In vitro photodynamic inactivation of herpes simplex virus with sapphyrins: 22 pi-electron porphyrin-like macrocycles.

The photodynamic inactivation of HSV-1, a virus having a membranous envelope, with both a decaalkyl sapphyrin and its dicarboxy-substituted analog was studied. The decaalkyl sapphyrin was as efficient in the inactivation of HSV-1 on a per macrocycle basis as DHE, whereas the efficiency of the dicarboxy-substituted sapphyrin was approximately two orders of magnitude less. Fluorescence studies of sapphyrin's binding to liposomes and VSV suggested that the decaalkylsapphyrin bound monomerically to cholesterol-rich regions of the viral envelope, whereas its charged analog localized in a more polar environment.

Regular pattern of preventive dental services--a measure of access.

Having a regular pattern of care should be an indicator of access to and periodic use of preventive and health maintenance services. The analyses reported in this study are intended to provide a better understanding of the factors related to having a regular pattern of preventive dental care. The data were collected in 1981 as part of a U.S. household survey, 'A Study of Dental Health Related and Process Outcomes Associated with Prepaid Dental Care', the most comprehensive cross-sectional data base available in dentistry. Descriptive analyses of a constructed variable, representing perceived and realized access and a preventive orientation, indicate that 53% of the population had a regular pattern of preventive care. Those with a regular pattern of care were more likely to be white, younger, have dental insurance, have no cost barriers, have more than 12 years of education, be dentate, have no perceived symptoms, and no fear of pain. Logistic regressions indicated that there was an increased probability of having a regular pattern of preventive care if individuals had no economic access problems, had positive attitudes, had higher income, reported few oral symptoms, and were non-Black. Overall, the descriptive models used suggested that individuals with resources in the form of finances and education, and a sense of self-efficacy as expressed in attitudes toward oral health, had the greatest probability of having a regular pattern of preventive care.

Comparison of Mycoplasma gallisepticum subunit and whole organism vaccines containing different adjuvants by western immunoblotting.

Chickens were vaccinated with subunit (adhesin protein) or whole organisms of Mycoplasma gallisepticum (MG) adjuvanted with multilamellar positively charged liposomes or oil-emulsion. Sera were collected before and following the first (13 weeks of age) and second (17 weeks of age) vaccination. The chicken sera were used in western immunoblotting against whole MG polypeptides. Vaccination with the subunit (MG-adhesin) bacterin containing positively charged liposomes resulted in antibody response specific to adhesin band (75 kD) at 3 weeks post the first and second vaccination; however, crossreactions of the same antibodies occurred to MG proteins of 85 kD (3 weeks after the first vaccination) and 56 kD (3 weeks after the second vaccination). Vaccination with whole MG proteins containing positively charged liposomes resulted in significant immunopotentiation of antibodies against low molecular weight polypeptides of MG (less than 48.0 kD). The addition of Salmonella typhimurium cell wall proteins mitogens (STP) to the different bacterins suppressed the antibody responses to some MG polypeptides.

Preliminary data on efficacy of Mycoplasma gallisepticum vaccines containing different adjuvants in laying hens.

Chickens were vaccinated subcutaneously twice, at 13 and 17 weeks of age. The vaccines used were the whole organisms of Mycoplasma gallisepticum (MG) adjuvanted with multilamellar positively charged (MPC) liposomes or oil-emulsion. Other chickens received the same bacterins but supplemented with Salmonella typhimurium cell wall protein mitogen (STP) (50 micrograms/dose). At 21 weeks of age, each bird was challenged in the right and left caudal thoracic air sacs. The challenge dose/chicken was 1.3 x 10(5) CFU of MG (R-strain). A significant immunoglobulin (Ig) response specific to MG was observed in sera of chickens collected 3 weeks after the first and second vaccination with MG adjuvanted with MPC liposomes or oil-emulsion. The same two treatments had highly significant MG-titers in eggs collected during the first and second month post challenge. Both groups had highly significant protection (P less than 0.05) against MG transmission in eggs layed during the first month post challenge. Vaccination with MG organisms adjuvanted to MPC liposomes or oil-emulsion resulted in higher egg production, during the first month following challenge, in comparison to the unvaccinated-challenged birds; the same two groups had higher egg production in the second month following challenge compared to unvaccinated-challenged birds, but not significantly different (P greater than 0.05). The addition of STP to bacterins containing MG organisms adjuvanted to MPC liposomes or oil-emulsion, resulted in a significant reduction (P less than 0.05) of the Ig-specific to MG in sera and in a significant drop in egg production (P less than 0.05) during the first month following challenge.

Immunopathology of periodontal disease. I. Immunologic profiles in periodontitis and juvenile periodontitis.

Eleven patients with periodontitis, ten patients with juvenile periodontitis and ten control subjects were studied to compare systemic and local immunologic responses and various other blood parameters. A more intensified immunologic response was seen in juvenile periodontitis as compared to periodontitis or controls as evidenced by: (1) greater number of plasma cells and lymphocytes in biopsy specimens of involved gingiva stained by Harris hematoxylin and eosin; (2) significant decrease in serum C4 levels and slightly elevated serum IgG levels as determined by radial immunodiffusion; (3) marked increase of positive fluorescing cells in biopsy specimens stained with antihuman immunoglobulin conjugate and an increase in complement deposition in the same tissues as determined by immunofluorescence. No significant differences among the two groups of patients and control subjects were observed with respect to complete blood counts, coagulation studies, or blood glucose levels. These findings suggest that the immunologic responses in periodontitis and juvenile periodontitis are qualitatively similar, but that the intensity of the response is greater in juvenile periodontitis.

Immunopathology of periodontal disease. II. Immunofluorescent studies on the localized immune response in periodontitis and juvenile periodontitis.

Local immunologic responses were studied in eleven patients with periodontitis, ten patients with juvenile periodontitis, and ten control subjects by performing immunofluorescence on gingival biopsies. F(ab')2 monospecific conjugates to human IgG, IgM, IgA, IgD, and kappa and lambda light chains were used as well as monospecific conjugates to human IgE and properdin. Biopsies from patients with juvenile periodontitis displayed a more intensified immunologic response as evidenced by increases of (I) positive cells stained with a F(ab')2 antihuman immunoglobulin conjugate; (2) IgG, IgM, and kappa and lambda containing cells; (3) extracellular fluorescence of IgG, IgM, IgA, and kappa and lambda light chains; and (4) properdin deposition. Neither IgD nor IgE conjugates reacted significantly with biopsies from the three patient groups. These findings support our previous conclusion that immunologic responses in periodontitis and juvenile periodontitis appear qualitatively similar, but that patients with juvenile periodontitis exhibit a more intense immune response.