Production of a Ric c3 hypo-allergen with no IgE binding or anaphylactogenic activity.

Several studies have been carried out to expand the use of Ricinus communis L. castor bean (Ricinus communis L castor bean.). This oilseed finds appropriate conditions for its development in Brazil, with more than 700 applications. The main allergens of this plant are Ric c1 and Ric c3, that cross-react with various aeroallergens and food allergens such as peanuts, soybeans, corn, and wheat. This study aimed to determine the effect of mutations in Ric c3 amino acid residues known to affect IgE binding and allergy challenges. Based on the Ric c3 structure, B-cell epitopes, and amino acid involved in IgE binding, we produce recombinant mutant protein, mrRic c3, secreted from E. coli. Strategic glutamic acid residues in IgE-biding regions were changed by Leucine. The allergenicity of mrRic c3 was evaluated by determination of IgE, IgG1, and total IgG in immunized Balb/c mice and by degranulation assays of mast cells isolated from Wistar rats. The mrRic c3 presented a percentage of mast cell degranulation close to that seen in the negative control, and the immunization of mice with mrRic c3 presented lower levels of IgE and IgG1 than the group treated with the protein without mutations. The mutant mrRic c3 had an altered structure and reduced ability to stimulate pro-inflammatory responses and bind IgE but retained its ability to induce blocking antibodies. Thus, producing a hypoallergenic mutant allergen (mrRic c3) may be essential in developing new AIT strategies.

Complex transcriptional regulation of the Saccharomyces cerevisiae CYB2 gene encoding cytochrome b2: CYP1(HAP1) activator binds to the CYB2 upstream activation site UAS1-B2.

Expression of the Saccharomyces cerevisiae gene encoding cytochrome b2 (EC 1.2.2.3), CYB2, was investigated by direct analysis of mRNA transcripts and by measurement of the expression of lacZ fused to the CYB2 control regions. These studies indicated that regulation of the CYB2 gene is subject to several metabolic controls at the transcriptional level: inhibition due to glucose fermentation, induction by lactate, and inhibition in anaerobiosis or in absence of heme biosynthesis. Furthermore, we have shown that the CYB2 promoter contains one cis negative regulatory region and two heme-dependent positive regions, one of which is controlled by the transcriptional regulator CYP1 (HAP1) which is involved in the modulation of the expression of several oxygen-regulated genes. The CYP1 (HAP1)-binding sequence was located by gel retardation and DNase I footprinting experiments and compared with the binding sequences previously characterized in detail (UAS1CYC1, UAS'CYP3 (CYC7), and UASCTT1).

Femoral hernia repair. Kugel retroparietal approach versus plug alloplasty: a prospective study.

Plug insertion for primary femoral hernia repair may cause p.o. discomfort. The Kugel technique may avoid this problem. Patients' satisfaction to the Kugel and the plug techniques is compared in the present study. Demographics, surgical, outcome and analgesic consumption data of 26 patients treated for with the plug technique (P group) are compared with 24 operated with the Kugel patch (K group). Patients' p.o. discomfort to the two procedures was measured with quantitative (VAS score) and a qualitative (the short form of McGill pain questionnaire, SF-MPQ) methods, and compared. P group presented higher early p.o. pain (P<0.001), higher analgesic consumption and a significative delay in the return to physical activity (P<0.001). SF-MPQ scores at p.o. day 8, day 30 and month 6 were significantly lower for K group (P<0.001, P<0.001, P<0.005). The Kugel technique for femoral hernia treatment seems to cause less p.o. discomfort to patients than the plug technique.

Kugel hernia repair: open "mini-invasive" technique. Personal experience on 620 patients.

A large monoinstitutional series adopting the Kugel retroparietal technique for inguinal hernia surgery is analysed. Our aim is to assess the "mini-invasiveness" of this technique. Six hundred and twenty patients (pts) affected by monolateral inguinal hernia were treated with a preperitoneal alloplasty with a posterior approach (Kugel hernia repair, KHR) between January 2002 and September 2004. The surgical incision extension was 3.5 cm on average (range 2-4.5). The mean operation time was 33 min (range 20-45). Spinal anaesthesia and ambulatory procedure were applied in 595 cases (96%). Postoperative complications affected 20 pts (3%). The postoperative pain was well controlled. No chronic neuropathic pain was registered at follow-up. Patients resumed work after an average of 9 days (range 7-12) from operation. Recurrence rate was 0.8%. Conclusions. The Kugel hernia repair satisfies the standards to be awarded as a "mini-invasive" technique.

Expression of a lipocalin in Pichia pastoris: secretion, purification and binding activity of a recombinant mouse major urinary protein.

The proteins of the mouse major urinary protein complex (MUP), members of the lipocalin family, bind volatile pheromones and interact with the vomeronasal neuroepithelium of the olfactory system. We report the expression of a MUP protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. Mature recombinant MUP (rMUP) is secreted at a concentration of 270 mg/l in minimal medium and it is isolated from the culture supernatant by one step ion-exchange chromatography in a nearly pure form. Binding activity, tested with an odorant molecule which displays high affinity for native MUP, indicates that rMUP has a behavior similar to the native one. This finding suggests that the protein, and in particular its hydrophobic binding pocket, is properly folded.

Immunogenicity of yeast-derived hepatitis B vaccine in Pakistani adults.

A study of the immunogenicity and safety of 20 micrograms recombinant DNA yeast derived hepatitis B vaccine was conducted in 153 Pakistani adults. All participants were in good physical condition, had negative hepatitis B serological markers (HBsAg, anti-HBs, anti-HBc) and normal ALT. Anti-HBs developed in 33%, 81% and 98% of subjects one month after the first, second and third dose respectively. Minor systemic and local side-effects were observed in 39% of individuals. We conclude that yeast derived hepatitis B vaccine is safe, effective and immunogenic in Pakistani adults.

Sub-specificities of hepatitis Bs antigen.

A preliminary survey of HBsAg subtypes shows that ayw is the most common subtype in Pakistan. adw. was detected in one blood donor and ayr in a Baluchi female with acute viral hepatitis.

Rosette forming T lymphocytes in healthy subjects and patients with liver disease.

The number of rosette forming T cells was significantly reduced in patients with acute viral hepatitis, cirrhosis of the liver and liver cancer. The frequency of T cells in various hepatic disorders correlated well with the impairment of delayed hypersensitivity response to DNCB.

Immunoglobulins in healthy subjects and blood donors.

Levels of immunoglobulins in serum were determined in 41 healthy subjects and 128 professional blood donors. In healthy subjects mean levels +/- S.D. were: IgG 1260 +/- 492.9 mg/dl, IgM 62.4 +/- 18.2 mg/dl, and IgA 183.4 +/- 70.1 mg/dl. Of 128 blood donors 112 were hepatitis Bs antigen (HBsAg) negative and 16 were positive. A significant (P is less than 0.001) elevation of all three immunoglobulins was observed in HBsAg positive group suggesting increased antibody formation due to persistance of antigen.