Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1.

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175-189 (NA175-189) and nucleoprotein 214-228 (NP214-228), but also respond to a NA175-189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675-689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175-189 or POMT1675-689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1.

Metabolism of OR-1896, a metabolite of levosimendan, in rats and humans.

OR-1896 is a pharmacologically active, long-lived metabolite of levosimendan. In the current study, the metabolism of (14)C-labelled OR-1896 was investigated in six healthy men after intravenous infusion over 10 min and in male rats after an intravenous bolus dose. In human plasma, the only (14)C-compounds detected were (14)C-OR-1896 and its deacetylated form, (14)C-OR-1855, in varying proportions in different subjects. In rat plasma >93% of radioactivity was associated with OR-1896. Radioactivity was mainly excreted to urine in both rats (about 69% of the dose) and humans (about 87% of the dose). OR-1896 was a major urinary compound in both humans and rats. Another major human metabolite was hypothesized as N-conjugated OR-1855. Other human and rat urinary biotransformation products were characterized as N-hydroxylated OR-1896 and N-hydroxylated OR-1855, as well as glucuronide or sulphate conjugates of N-hydroxyl OR-1896. The main difference between rat and human metabolism was a lower amount of OR-1855-related metabolites in the rats. In human faecal homogenates, only OR-1896 and OR-1855 were detected, whereas rat faecal metabolite profile was similar to that in urine.

Efficacy and tolerability of continuous combined hormone replacement therapy in early postmenopausal women.

OBJECTIVE: Continuous combined hormone replacement therapy (ccHRT) based on estradiol valerate (E2V) and medroxyprogesterone acetate (MPA) is effective for relief of menopausal symptoms three years or more after the menopause. This study was undertaken to examine the efficacy and tolerability of ccHRT in early postmenopausal women (last menstrual period 1.3 years before study entry). STUDY DESIGN: This was a 52-week, randomized, double-blind, multinational study of ccHRT comprising three different dose combinations of E2V/MPA in 459 early postmenopausal non-hysterectomized women experiencing 30 or more moderate to severe hot flushes a week and/or vasomotor symptoms requiring treatment. MAIN OUTCOMES MEASURES: The primary endpoint was change in frequency and severity of moderate to severe hot flushes at 12 weeks. Secondary outcome measures included number of bleeding days and evaluation of tolerability. RESULTS: The frequency of hot flushes was reduced by >or=70% after one month (P<0.001 for all doses at week 2 onwards), with little evidence of statistically different dose effects. Severity of flushing was also attenuated by ccHRT. Mean number of bleeding days fell to <1 per 28-day cycle at 52 weeks. Rates of amenorrhoea approached 80-90% at the end of the study, but were significantly lower at several time points with the highest-dose regimen (2 mg E2V + 5 mg MPA) than with the lower-dose options (1 mg E2V + 2.5 mg MPA and 1 mg E2V + 5 mg MPA; P<0.05). Adverse events declined in frequency over time with all regimens but throughout the study were more numerous with the highest-dose regimen than with lower doses (P= 0.0002). CONCLUSIONS: Continuous combined HRT was effective for the relief of climacteric symptoms in early postmenopausal women and was well tolerated.

Patch test reactions to cosmetic allergens in 1995-1997 and 2000-2002 in Finland--a multicentre study.

Contact sensitivity to cosmetics is common, but the sensitizing chemicals vary between countries and study periods. The present survey aimed at revealing the recent trends in patch test sensitivity with cosmetic chemicals in Finland. We report a retrospective multicentre survey of patch test reactions focusing on cosmetic-related substances and comparing the test results in 1995-97 with those in 2000-02. The most striking increases in the frequency of the patch test sensitivity were found with balsam of Peru and propolis from 4.0% to 6.2% (P < 0.001) and from 0.5% to 1.4% (P < 0.001), respectively, whereas the most prominent decreases were found with methylchloro/methylisothiazolinone and chlorhexidine diglugonate from 2.4% to 1.3% (P < 0.001) and from 1.2% to 0.5% (P < 0.001), respectively. The level of patch test sensitivity to methyldibromo glutaronitrile increased, although not significantly, from 1.0% to 1.5%. An increasing tendency was also found with hair dye chemicals 4-aminophenol and toluene-2,5-diamine or toluene-2,5-diamine sulfate from 1.3% to 3.8% and from 1.4% to 5.2%, respectively, while such a tendency was not found among permanent wave chemicals. The sensitivity level of fragrance mix remained the same (6% - 7%). We conclude that surveys revealing the state of sensitivity to cosmetic chemicals should be performed periodically in different countries.

High-level production of human type I collagen in the yeast Pichia pastoris.

Four human genes, two of them encoding the proalpha1 and proalpha2 chains of type I procollagen and two of them the two types of subunit of prolyl 4-hydroxylase (4-PH), were integrated into the genome of Pichia pastoris. The proalpha1 and proalpha2 chains expressed formed type I procollagen molecules with the correct 2:1 chain ratio, and the 4-PH subunits formed an active enzyme tetramer that fully hydroxylated the proalpha chains. Chains lacking their N but not C propeptides formed pCcollagen molecules with the 2:1 chain ratio and, surprisingly, the expression levels of pCcollagen were 1.5-3-fold relative to those of procollagen. Both types of molecule could be converted by pepsin treatment to collagen molecules that formed native-type fibrils in vitro. The expression levels obtained for the pCcollagen using only single copies of each of the four genes and a 2 l fermenter ranged up to 0.5 g/l, indicating that it should be possible to optimize this system for high-level production of recombinant human type I collagen for numerous medical applications.

A multicenter study of patch test reactions with dental screening series.

BACKGROUND: Dental products contain many allergens, and may cause problems both for patients undergoing dental treatment and for dental personnel because of occupational exposure. Individual patch test clinics may not study sufficient numbers of patients to collect reliable data on uncommon allergens. OBJECTIVE: To collect information on dental allergens based on a multicenter study. MATERIALS AND METHODS: The Finnish Contact Dermatitis Group tested more than 4,000 patients (for most allergens, 2,300 to 2,600 patients) with dental screening series. Conventional patch testing was performed. The total number and percentage of irritant (scored as irritant [IR] or doubtful [?]) and allergic (scored as +, ++, or +++) patch test reactions, respectively, were calculated, as well as the highest and lowest percentage of allergic patch test reactions recorded by the different patch test clinics. A reaction index (RI) was calculated, giving information on the irritancy of the patch test substances. RESULTS: The most frequent allergic patch test reactions were caused by nickel (14.6%), ammoniated mercury (13%), mercury (10.3%), gold (7.7%), benzoic acid (4.3%), palladium (4.2%) and cobalt (4.1%). 2-hydroxyethyl methacrylate (2.8%) provoked most of the reactions caused by (meth)acrylates. Menthol, peppermint oil, ammonium tetrachloroplatinate, and amalgam alloying metals provoked no (neither allergic nor irritant) patch test reactions. CONCLUSION: Patch testing with allergens in the dental screening series, including (meth)acrylates and mercury, needs to be performed to detect contact allergy to dental products.

Expression of recombinant human type I-III collagens in the yeast pichia pastoris.

An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and-III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.

Preoperative stereotactic hookwire localization of nonpalpable breast lesions with and without the use of a further stereotactic check film.

A series of 394 women underwent 399 stereotactically guided preoperative hook-wire localization of non-palpable breast lesions. 250 lesions were localized without checking the position of the needle tip with a further stereo film. Later the position of the localization needle was checked by means of a further stereo film in 149 lesions. If the needle tip was beside the lesion (failed x- or y-coordinates), we took a new stereo film. Errors in needle depth (z-coordinate) produced particularly subtle signs. Therefore a polystyrene phantom and a metallic object was used to ascertain how to recognize needle depth error on the stereotactic film. In 51 lesions readjustment of the needle depth was necessary. Measured by our criteria, successful and acceptable localization procedures with or without a check stereo-image film showed no significant differences. Unsuccessful localizations without the check film were significantly higher (16.0%) than with the check film, without readjusting the needle depth (7.1%) or after readjusting the needle depth (9.8%). These unsuccessful localizations were mostly architectural distortions or microcalcifications. Our results suggest that success of the localization procedure is better by using the stereo check film as without it, even in lesions which are difficult to resolve and target on films. However, readjustment of the needle depth did not always lead to successful localization.

Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris.

It was recently reported that co-expression of the proalpha1(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only approximately 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human proalpha1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical proalpha1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments. The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule.

Coexpression with collagen markedly increases the half-life of the recombinant human prolyl 4-hydroxylase tetramer in the yeast Pichia pastoris.

Recent coexpression studies of the subunits of human prolyl 4-hydroxylase (4-PH) in the yeast Pichia pastoris have indicated that only a minor fraction of them were present in the alpha2beta2 tetramer, while coexpression with type III procollagen markedly increased their assembly level. We report here that the half-life of the recombinant 4-PH tetramer in Pichia when studied by pulse-chase experiments was only 50 min. Coexpression with the pro alpha1(III) chains increased this half-life to 12.5 h. Coexpression with the pro alpha1(I) chains, which were produced at half the level of the pro alpha1(III) chains, gave a half-life of 6.5 h. Coexpression with collagen thus markedly increases the half-life of the 4-PH tetramer, and the half-life may be related to the level of collagen expression.

Dose dependency of antibody response in infants and children to pneumococcal polysaccharides conjugated to tetanus toxoid.

Three injections of tetravalent pneumococcal polysaccharide-tetanus toxoid conjugate vaccine (PncT) were given to infants at 2, 4 and 6 months of age simultaneously with diphtheria-tetanus-pertussis and Haemophilus influenzae type b-tetanus toxoid conjugate vaccines. Three doses (1, 3 or 10 microg) of polysaccharides were used. Children were boosted with unconjugated polysaccharide vaccine at 14 months of age. No dose dependency was seen after primary immunization. However, booster response to three vaccine serotypes was highest in the group primed with the lowest dose of conjugate vaccine. As the magnitude of the response to booster may be related to the number of polysaccharide-specific memory B cells, we hypothesize that the 10 microg dose of the tetravalent conjugate vaccine is too high for optimal induction of immunologic memory.

Mutacin production by Streptococcus mutans may promote transmission of bacteria from mother to child.

The production of bacteriocin-like inhibitory substances, mutacins, by mutans streptococci varies among isolates. To find if the degree of mutacin activity of an isolate was related to its transmission between mother and her child, 19 mothers and their 18-month- to 3-year-old children were sampled for their oral mutans streptococci. In addition, the stability of mutacin activity was studied with isolates from the mothers and with isolates from five unrelated 5-year-old children in 5- to 7-year follow-up studies. A total of 145 oral mutans streptococcal isolates were serotyped by immunodiffusion, ribotyped, and mutacin typed by the stab culture technique. Mutacin was produced by 88% of the strains against more than 1 of the 14 indicator strains, representing mutans streptococci, Streptococcus sanguis, Streptococcus salivarius, Streptococcus oralis, Streptococcus gordonii, and Streptococcus pyogenes. Streptococcus mutans isolates showed more inhibitory activity than did Streptococcus sobrinus isolates. Identical ribotypes had similar mutacin activity profiles within a subject, initially and in the follow-up studies, in all but two cases. The mothers harbored a total of 37 different mutans streptococcal ribotypes. Six children were negative for mutans streptococci. Transmission was probable in 9 of 20 mother-child pairs on the basis of the presence of identical strains, as determined by ribotyping and bacteriocin (mutacin) typing. S. mutans strains shared between a mother and her child showed a broader spectrum of inhibitory activity than did nontransmitted strains. In conclusion, the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonization by S. mutans.

Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase.

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.

Pentavalent pneumococcal oligosaccharide conjugate vaccine PncCRM is well-tolerated and able to induce an antibody response in infants.

BACKGROUND: The emergence of resistant pneumococci makes the treatment of pneumococcal diseases difficult. The currently available polysaccharide vaccines have very limited efficacy in young children. The immunogenicity can be improved by covalent coupling to protein carriers as has been shown with Haemophilus influenzae type b. METHODS: Thirty healthy infants were immunized with a pneumococcal conjugate vaccine at 2, 4 and 6 months of age. Oligosaccharides were derived from capsular polysaccharides of types 6B, 14, 18C, 19F and 23F and conjugated to the nontoxic mutant diphtheria toxin CRM197. The final vaccine was a mixture of these conjugates, containing 10 micrograms of each oligosaccharide. The infants received simultaneously H. influenzae type b oligosaccharide-CRM197 conjugate vaccine. Serum samples were taken before each dose and 1 month after the third dose. Control material was composed of 25 serum samples taken from children of the same age without pneumococcal vaccination. Enzyme-linked immunosorbent assay was used to measure serum IgG anti-pneumococcal polysaccharide concentrations and radioimmunoassay for the serum Ig anti-H. influenzae type b concentrations. RESULTS: PncCRM vaccine was well-tolerated. Pneumococcal type 18C induced a significant antibody increase after the first dose, whereas the other five oligosaccharides, including H. influenzae type b oligosaccharides, induced an increase after the second or third dose. The specific IgG concentrations at 7 months of age were significantly higher among the vaccinated infants than in the controls for all the five pneumococcal types. CONCLUSIONS: Pneumococcal oligosaccharide-CRM197 conjugate vaccine is able to induce an IgG serum response in infants and anti-pneumococcal antibody concentrations were significantly higher than in controls of same age.

Metabolite profiles of two [14C]-labelled catechol O-methyltransferase inhibitors, nitecapone and entacapone, in rat and mouse urine and rat bile.

The metabolites of two inhibitors of catechol O-methyltransferase, nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)2,4-pentanedione] and entacapone [(E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide++ +], excreted in urine and bile by rats and in urine by mice, were compared and quantified by using HPLC with radiochemical detection after administration of [14C]-labelled compounds. With the exception of 3-O-methylated nitecapone, no major metabolites were found in rat bile that were not found in rat urine. For both compounds the major biotransformations were the same in the mouse and the rat. However, a bisulfite adduct of nitecapone was found in rat urine only, and reduction of the C = C and C = O groups of the nitecapone side chain was more extensive in the mouse. After entacapone administration, the products of amide N-dealkylation were more abundant in rat urine than in mouse urine. Most of the dose was excreted in urine and bile as O-conjugates. Most abundant were the O-glucuronides, while smaller amounts of O-sulfates and O-methylated metabolites were found in both species. One non-glucuronide glycoside of entacapone was found in urine of both rats and mice.

Preoperative hook-wire localization of nonpalpable breast lesions by use of standard and stereotactic technique.

A series of 151 women underwent 153 preoperative hook-wire localization of nonpalpable breast lesions, all of them without reinserting the needle. Measured by our criteria, the success for localization procedure was stereotactically 73% and conventionally 47%. Measured by criteria of surgeon acceptability and evidence of successful removal of the lesion, the success rate for localization was stereotactically 84% and conventionally 97%. Failure to remove the suspicious lesion in our series was more common after stereotactical than conventional localization with larger specimens. At present our experience and results of stereotactic wire localization are improving. We believe that the stereotactic wire localization without previous checking of the needle position is accurate and shortens the time required for the whole procedure.

Serum cortisol and outcome of ischemic brain infarction.

The predictive value of serum cortisol level on the prognosis in acute brain infarction of the carotid circulation territory was studied in 101 patients younger than 70 years. The levels of 7 a.m. and 7 p.m. serum cortisol were measured initially and at 1 week. All patients underwent a computed cerebral tomography (CT) within 2 days of the onset of symptoms, and a second CT 3 weeks or 3 month later. Serum cortisol values predicted the stroke outcome. Both the 7 a.m. and the 7 p.m. values in the initial and 1-week samples correlated positively with the severity of hemiparesis on the corresponding days. The 7 p.m. values predicted better than the 7 a.m. values the functional outcome and case fatality during the 3 month follow-up. Initially and at 1 week, the median 7 p.m. serum cortisol values were statistically significantly higher in those with frontally extending infarcts than in those with non-frontal infarcts. Both 7 a.m. fasting blood glucose and glycosylated hemoglobin (HbA1c) measurements were taken within 3 days of the onset in 95 cases. The patients were diagnosed to have prestroke normoglycemia (n = 73) and hyperglycemia (n = 22) on the basis of the HbA1c level. A highly significant (P = 0.0001) correlation was demonstrated between the initial 7 p.m. cortisol and 7 a.m. fasting blood glucose values in those with prestroke normoglycemia, suggesting that hyperglycemia during the acute phase of stroke is a stress response.