An unfolded variant of the major peanut allergen Ara h 2 with decreased anaphylactic potential.

BACKGROUND: Peanut allergy causes severe type 1 hypersensitivity reactions and conventional immunotherapy against peanut allergy is associated with a high risk of anaphylaxis. OBJECTIVE: Our current study reports proof of concept experiments on the safety of a stably denatured variant of the major peanut allergen Ara h 2 for immunotherapy. We determined the impact of structure loss of Ara h 2 on its IgE binding and basophil degranulation capacity, T cell reactivity as well as anaphylactic potential. METHODS: The secondary structure of untreated and reduced/alkylated Ara h 2 variants was determined by circular dichroism spectroscopy. We addressed human patient IgE binding to Ara h 2 by ELISA and Western blot experiments. RBL-SX38 cells were used to test the degranulation induced by untreated and reduced/alkylated Ara h 2. We assessed the anaphylactic potential of Ara h 2 variants by challenge of sensitized BALB/c mice. T cell reactivity was investigated using human Ara h 2-specific T cell lines and splenocytes isolated from sensitized mice. RESULTS: Reduction/alkylation of Ara h 2 caused a decrease in IgE binding capacity, basophil degranulation and anaphylactic potential in vivo. However, the human T cell response to reduced/alkylated and untreated Ara h 2 was comparable. Mouse splenocytes showed higher metabolic activity upon stimulation with reduced/alkylated Ara h 2 and released similar IL-4, IL-13 and IFNγ levels upon treatment with either Ara h 2 variant. CONCLUSIONS AND CLINICAL RELEVANCE: Reduced/alkylated Ara h 2 might be a safer alternative than native Ara h 2 for immunotherapeutic treatment of peanut allergic patients.

Monitoring neutrophils and platelets during casein-induced anaphylaxis in an experimental BALB/c mouse model.

BACKGROUND: With respect to the cellular players, mast cells and basophils have been well studied in experimental murine systemic anaphylaxis models, but the role of neutrophils and platelets is not fully understood today. OBJECTIVE: We tested the hypothesis that neutrophils and platelets might participate in an antigen-induced anaphylaxis model. METHODS: BALB/c mice were sensitized intraperitoneally with alum-adsorbed casein. A period of 2 weeks later, mice were challenged with 100 µg casein intravenously and immediate hypersensitivity reactions were assessed by rectal temperature measurements and monitoring the physical activity. Subsequently, leucocytes were counted in the peripheral blood as well as quantified in situ in typical shock organs like lung, liver and spleen, heart and kidney. RESULTS: Mice sensitized with casein showed casein-specific IgG1, IgE, and IgG2a. When sensitized mice were specifically challenged with casein they developed immediate hypersensitivity reactions including drop of temperature and reduced activity. Furthermore, pronounced peripheral neutropenia and reduced platelet counts correlated with the severity of the hypersensitivity reactions. In the histological analyses of collected tissues we observed lung interstitial neutrophilia using Gr-1 staining. These events occurred specifically in mice sensitized and challenged with casein, in contrast to control groups. CONCLUSIONS: On the basis of our data we suggest that in addition to mast cells and basophils, neutrophils and platelets participate in the anaphylactic response in this BALB/c mouse model. Platelet and neutrophils expressing relevant immunoglobulin receptors may therefore have a synergistic effect with allergen specific IgE as well as IgG antibodies in food-induced anaphylaxis. We suggest that management of these cells could be of clinical importance to handle anaphylaxis.

Dose-dependent food allergy induction against ovalbumin under acid-suppression: a murine food allergy model.

BACKGROUND: Animal models are essential for analyzing the allergenic potential of food proteins and for investigating mechanisms underlying food allergy. Based on previous studies revealing acid-suppression medication as risk factor for food allergy induction, we aimed to establish a mouse model mimicking the natural route of sensitization in patients. METHODS: The effect of acid-suppressing medication on murine gastric pH was assessed by intragastric pH measurements after two injections of a proton pump inhibitor (PPI). To investigate dose-dependency, mice were fed different concentrations of ovalbumin (OVA; 0.2, 0.5, 1.0, 2.5 or 5.0mg) either with or without anti-ulcer medication. Additionally, different routes of exposure (i.p. vs. oral) were compared in a second immunization experiment. Sera were screened for OVA-specific antibody titers (IgG1, IgG2a and IgE) in ELISA and RBL assay. Clinical reactivity was evaluated by measuring rectal temperature after oral challenge and by type I skin tests. RESULTS: Two intravenous injections of PPI significantly elevated the gastric pH from 2.97 to 5.3. Only oral immunization with 0.2mg OVA under anti-acid medication rendered elevated IgG1, IgG2a and IgE titers compared to all other concentrations. Protein feeding alone altered antibody titers only marginally. Even though also i.p. immunizations induced high levels of specific IgE, only oral immunizations under anti-acids induced anaphylactic reactions evidenced by a significant decrease of body temperature. CONCLUSION: Only low-dosage ovalbumin feedings under anti-acid medication resulted in IgE mediated food allergy. Based on this knowledge we have established a suitable food allergy model for further investigations of food adverse reactions.

Studies on skeletal muscle biopsies in endemic skeletal fluorosis.

Neurological manifestations of skeletal fluorosis have been attributed to compressive radiculomyelopathy. Experimental fluorosis has shown evidence of myopathic changes. Data on human muscle pathology is very scanty. This study included 22 patients with established osteofluorosis. 16 of them showed only EMG changes of neurogenic muscle disease. Histochemistry and histopathology of muscle biopsies showed features of muscle atrophy, evidenced by 'type I' atrophy and 'type I' grouping. No myopathic changes were observed. It may be concluded that the primary changes are related to the nerve, with muscle being affected secondarily. There was no evidence of any primary muscle pathology due to fluorosis.

Asymmetric synthesis for process research.

During the past few years, asymmetric synthetic methods have become an indispensable tool in the process research arena for the production of enantiopure materials. The development of a truly efficient, reliable and economically viable asymmetric synthesis is still limited, and searches for such valuable technologies are rapidly growing. This review features an overview of several interesting articles which appeared in the literature from January 1998, through to August 1999, that can greatly impact the area of asymmetric synthesis in process research and development.

Trisomy 18q: 46,XX,-10,+der(10) t(10;18) (p15;q12) pat: a case report.

A 2-month-old female with intrauterine and postnatal growth retardation, multiple congenital anomalies, absent right kidney, congenital heart disease was investigated. Her karyotype revealed, 46,XX,-10,+der(10), t(10;18) (p15;q12) pat. The child died at 2 months 2 weeks. This is the third case of trisomy 18q resulting from translocation of chromosome 10 and 18.

Correlation of fragile sites and bleomycin induced chromosome break points.

Bleomycin (Blm) induced break points in human chromosome preparations were compared with the known fragile sites. A total of 136 breaks were observed from 100 well spread G-banded plates (1.3 bps/cell). These correspond to a total of 57 break prone sites. Of these 57 sites, 24 correspond to the known fragile sites, 5 to sites of protooncogenes and neoplasia, 26 sites correspond to more than one known site of fragility, protooncogene, neoplasia or reciprocal translocation sites, and 2 unknown sites. The findings suggest that fragile sites, either commonly expressed or induced, might be a predisposing factor for chromosome aberrations in human. The expression of fragile sites induced by Blm and their correlation with the known cancer chromosome break points, oncogenes and reciprocal translocation, suggest that the fragile sites are prone to mutagenic action.

Experimental colonic anastomosis in bovines--and angiographic study.

4 different methods of colonic anastomosis, single and double layer inversion and eversion, were performed on cowcalves. Hypervascularization of neovascularized arterioles at the cut ends of colon were visualized on the 7th day by all techniques. The arterioles, later one, tended to reorganise towards normalcy. Inversion technique showed better vascularization at the anastomotic site, and the vascular pattern was nearly normal on the 14th and 28th days from surgery. Eversion technique did not exhibit satisfactory revascularization of the anastomotic site on the 7th day of study. A radiopaque area, davoid of arterioles, was noted on the 14th and 28th days from surgery.

Colonic anastomosis in calves: an experimental study.

Colonic anastomosis was conducted in 12 calves divided equally into four groups. Four suture patterns, viz. single inversion (continuous Connell), double inversion (two rows of continuous Cushing), single eversion (continuous everted mattress), double eversion (single eversion reinforced with simple continuous) were used. In each animal, anastomosis of the same technique was performed at four places. Each technique was evaluated in terms of clinical and gross observations, lumen stenosis and bursting pressure. Observations were made on the 7th, 14th and 28th day after anastomosis. None of the anastomotic techniques showed any untoward incident during the 28 days of postoperative study. Stricture formation at the site of anastomosis was minimal with the single inversion technique whereas it was maximum with the single eversion technique. Pressure for disruption of intestinal segments was higher after inversion than eversion techniques. It was concluded that the one-layer inversion anastomosis technique proved to be the best among the four techniques because (i) of its reduced incidence of adhesions; (ii) it maintains adequate lumen diameter; (iii) it withstands maximum pressure. The double layer inversion technique was conceded to be the second best choice for colonic anastomosis followed by the double eversion and single eversion techniques.

Haemodynamic and blood gas changes in buffaloes (Bubalus bubalis) in the supine position following thiopentone anaesthesia with premedication.

Studies were undertaken on seven adult female buffaloes (Bubalus bubalis) premedicated with triflupromazine hydrochloride (0.3 mg/kg) and chloral hydrate (30 mg/kg). Anaesthesia was induced with thiopentone sodium (10 mg/kg) and animals were kept under intermittent positive pressure ventilation. Measurements were made for 30 min in the lateral position, for 20 min in the supine position and subsequently for 40 min again in the lateral position. Regurgitation during anaesthesia was avoided by evacuation of the rumen by rumenotomy two days prior to the experiment. When animals were in the lateral position there were minimal changes in arterial pressure and heart rate but central venous pressure rose. These changes were related to intermittent positive pressure ventilation. In the supine position, increase in heart rate (28%) was associated with profound hypotension (56%) and decrease in central venous pressure (171%) in these animals. There was a fall in venous oxygen tension and a marked increase (91%) in oxygen extraction ratio. Restoration to the lateral position ameliorated all these changes rapidly. It was suggested that a 'low pressure-low flow' circulatory state exists in anaesthetised adult buffaloes in the supine position.

Antibodies directed against guanylic acid and guanosine are nucleolus specific probes.

Purified antibodies against guanylic acid and guanosine binding to RNA at guanosine residues were used to probe human lymphocyte preparations by indirect immunofluorescence. Neither antibody gave any banding pattern with metaphase chromosomes but both showed binding to specific sites in the interphase nuclei. Evidence presented indicates that these sites are guanosine residues on rDNA transcripts at the nucleolar organizer regions.

Patho-anatomy of herniation of the reticulum through the diaphragm in the bovine.

Dissection of embalmed and untreated water buffalo carcasses (n=10) revealed that hernias had occurred at the musculotendinous junction of the diaphragm, ventral to the foramen venae cavae and slightly lateral to the median plane. The diameter of the hernial ring varied from 7 cm to 20 cm. Herniation was more common in the right thoracic cavity with the reticulum firmly adherent to the hernia ring. Adhesions between the herniated portion of the reticulum and pleura, lung, pericardium or thoracic wall were present, while in a few cases thick fibrous tracts concealing metallic bodies were found. In two cases, involvement of esophageal groove with malalignment of cardia and reticulo-omasal opening was observed. Displacement and compression of the heart was observed in four animals.