IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens.

Allergy to peanut and tree nuts is characterised by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Although peanut is the most common cause of nut allergy, peanut allergic patients are frequently also sensitive to tree nuts. It is not known if this is due to cross-reactivity between peanut and tree nut allergens. In this study, the major peanut allergen Ara h 2 was cloned from peanut cDNA, expressed in E. coli cells as a His-tag fusion protein and purified using a Ni-NTA column. Immunoblotting, ELISA and basophil activation indicated by CD63 expression all confirmed the IgE reactivity and biological activity of rAra h 2. To determine whether or not this allergen plays a role in IgE cross-reactivity between peanut and tree nuts, inhibition ELISA was performed. Pre-incubation of serum from peanut allergic patients with increasing concentrations of almond or Brazil nut extract inhibited IgE binding to rAra h 2. Purified rAra h 2-specific serum IgE antibodies also bound to proteins present in almond and Brazil nut extracts by immunoblotting. This indicates that the major peanut allergen, Ara h 2, shares common IgE-binding epitopes with almond and Brazil nut allergens, which may contribute to the high incidence of tree nut sensitisation in peanut allergic individuals.

Anaphylaxis to Gelofusine confirmed by in vitro basophil activation test: a case series.

The plasma expander Gelofusine (succinylated gelatin) is a recognised cause of peri-operative anaphylaxis. Current diagnosis of Gelofusine sensitivity is by skin testing, a procedure that itself carries a risk of allergic reaction. We evaluated the reliability of the in vitro basophil activation test as a diagnostic assay for Gelofusine sensitivity in subjects with a clinical history highly suggestive of Gelofusine allergy. Six patients with peri-operative anaphylaxis clinically attributed to Gelofusine were skin tested to confirm sensitivity. Control subjects included three healthy subjects and five subjects allergic to a neuromuscular blocking drug, all negative on Gelofusine skin testing. Whole blood basophil activation to Gelofusine was analysed by flow cytometry for CD63 surface expression. All of the Gelofusine sensitive patients and one of the control allergic subjects showed positive basophil activation to Gelofusine. In this series of subjects, the basophil activation test for Gelofusine allergy had a sensitivity of 100% and a specificity of 87.5%. Our findings suggest that basophil activation testing is a safe and reliable in vitro assay for prediction or confirmation of Gelofusine sensitivity in patients with high clinical suspicion of Gelofusine-induced anaphylaxis.

Functional analysis of cross-reactive immunoglobulin E antibodies: peanut-specific immunoglobulin E sensitizes basophils to tree nut allergens.

BACKGROUND: Peanut and tree nuts are a major cause of food-induced anaphylaxis with an appreciable mortality. Co-sensitization to peanuts and tree nuts is a common clinical observation and may be because of peanut-specific serum IgE antibodies that cross-react with tree nut allergens. It is, however, unclear whether these cross-reactive IgE antibodies are involved in effector-cell activation. OBJECTIVE: To determine if cross-reactivity of peanut-specific IgE antibodies with tree nuts can cause effector cell activation using an in vitro basophil activation assay. METHODS: Two peanut allergic subjects with positive specific IgE for peanut and tree nuts (as measured by CAP-FEIA) were studied. Basophil activation to peanut and tree nuts, as indicated by CD63 expression, was assessed by flow cytometry to confirm co-sensitization to peanut and tree nuts. Inhibition ELISA using sera from the subjects was performed to detect peanut-specific IgE antibodies that cross-reacted with tree nut proteins. To determine whether cross-reactive tree nut allergens can induce effector-cell activation, peanut-specific antibodies were affinity purified from the subject sera and used to resensitize non-peanut/tree nut allergic donor basophils stripped of surface IgE. Basophil activation was then measured following stimulation with peanut and tree nut extracts. RESULTS: The two peanut allergic subjects in this study showed positive basophil activation to the peanut and tree nut extracts. Inhibition ELISA demonstrated that pre-incubation of the peanut allergic subject sera with almond, Brazil nut and hazelnut extracts inhibited IgE binding to peanut extract. IgE-stripped basophils from non-peanut/tree nut allergic subjects resensitized with affinity-purified peanut-specific antibodies from the peanut allergic subject sera became activated following stimulation with peanut, almond and Brazil nut extracts, demonstrating biological activity of cross-reactive IgE antibodies. CONCLUSION: Peanut-specific IgE antibodies that cross-react with tree nut allergens can cause effector-cell activation and may contribute to the manifestation of tree nut allergy in peanut allergic subjects.

Characterization of the T-cell epitopes of a major peanut allergen, Ara h 2.

BACKGROUND: The development of safe and effective immunotherapy for peanut allergy has been complicated by the high anaphylactic potential of native peanut extracts. We sought to map the T-cell epitopes of the major peanut allergen, Ara h 2 in order to develop T-cell targeted vaccines. METHODS: A panel of eight peanut-specific CD4+ T-cell lines (TCL) was derived from eight peanut-allergic subjects and proliferative and cytokine responses to stimulation with a set of overlapping 20-mer peptides representing the entire sequence of Ara h 2 determined. Proliferation was assessed in 72 h assays via tritiated thymidine incorporation, while interleukin (IL)-5 and interferon (IFN)-gamma production were assessed via sandwich enzyme-linked immunosorbent assay (ELISA) of cell culture supernatants. RESULTS: Eight of the 17 Ara h 2 peptides were recognized by one or more subjects, with the two peptides showing highest reactivity [Ara h 2 (19-38) and Ara h 2 (73-92)] being recognized by three subjects each. Adjoining peptides Ara h 2 (28-47) and Ara h 2 (100-119) induced proliferative responses in two subjects. Each of these peptides was associated with a Th2-type cytokine response. CONCLUSION: Two highly immunogenic T-cell reactive regions of Ara h 2 have been identified, Ara h 2 (19-47) and Ara h 2 (73-119), providing scope for the development of safe forms of immunotherapy for peanut allergy.

Immunological analysis of allergenic cross-reactivity between peanut and tree nuts.

BACKGROUND: Peanut and tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross-reactive allergens remains unknown. OBJECTIVE: The aim of this study was to investigate the presence of allergenic cross-reactivity between peanut and tree nuts. METHODS: Western blotting and ELISA were performed using sera from subjects with or without peanut and tree nut allergy to assess immunoglobulin E (IgE) reactivity to peanut and tree nut extracts. Inhibition ELISA studies were conducted to assess the presence of allergenic cross-reactivity between peanut and tree nuts. RESULTS: Western blot and ELISA results showed IgE reactivity to peanut, almond, Brazil nut, hazelnut and cashew nut for peanut- and tree nut-allergic subject sera. Raw and roasted peanut and tree nut extracts showed similar IgE reactivities. Inhibition ELISA showed that pre-incubation of sera with almond, Brazil nut or hazelnut extracts resulted in a decrease in IgE binding to peanut extract, indicating allergenic cross-reactivity. Pre-incubation of sera with cashew nut extract did not cause any inhibition. CONCLUSION: These results show that multiple peanut and tree nut sensitivities observed in allergic subjects may be due to cross-reactive B cell epitopes present in different peanut and tree nut allergens. The plant taxonomic classification of peanut and tree nuts does not appear to predict allergenic cross-reactivity.

Microsatellite instability and mismatch-repair protein expression in hereditary and sporadic colorectal carcinogenesis.

Aberrant crypt foci (ACF) are microscopic clusters of altered colonic crypts considered premalignant lesions in the large bowel. Genomic instability at short tandem repeats in the DNA, referred to as microsatellite instability (MSI) is the hallmark of hereditary nonpolyposis colorectal carcinoma (HNPCC) caused by mutations in DNA mismatch-repair genes, mostly hMLH1 and hMSH2. In this study, we evaluated for MSI ACF (n = 16), adenomas (n = 18), carcinomas (n =22), and lymph node metastases (n = 3) from 17 patients with colorectal cancer positive for MSI. Ten patients were members of HNPCC families; 7 patients had no family history of cancer. MSI was found in 7 of 7 (100%) ACF and 11 of 12 (91%) adenomas from patients with HNPCC. MSI was not related to histology and size of ACF. A progressive increase in instability as estimated by the number of shifted bands was observed along the ACF-adenoma-carcinoma sequence. In contrast, two of nine (22%) ACF and none of six adenomas from patients with MSI sporadic carcinoma were unstable at microsatellite loci. hMLH1 or hMSH2 protein expression was altered only in MSI-positive premalignant lesions (ACF and/or adenomas), but not in all MSI-positive lesions in patients with HNPCC. These observations provide evidence of the premalignant nature of ACF in HNPCC and suggest that MSI is a very early event both in HNPCC and in sporadic colorectal carcinogenesis, although in the latter it seems infrequent.

Problems in the identification of hereditary nonpolyposis colorectal cancer in two families with late development of full-blown clinical spectrum.

The recognition of Hereditary Nonpolyposis Colorectal Cancer (HNPCC) remains difficult despite the most recent advancements of molecular biology and technology. We describe two families with early onset of cancer but no suspicion of hereditary tumors; during follow-up, both families developed a tumor spectrum highly suggestive of HNPCC, thus emphasizing the importance of family history for a proper identification of hereditary tumors or cancer aggregation. Microsatellite instability was negative in tumors from both families and, as expected, no germline mutations of the major DNA mismatch repair genes (MSH2 and MLH1) could be detected. Suspicion of the disease at the time of proband's lesion might have led to prevention, or early diagnosis, of at least three malignant tumors. We conclude that a possible genetic origin should always be suspected in individuals with early-onset neoplasms of the large bowel and probably of other organs such as the endometrium, small bowel, and urothelium, even when the initial pedigree does not show marked aggregation of cancers or vertical transmission.

Hereditary colorectal cancer in the general population: from cancer registration to molecular diagnosis.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common inherited disorders predisposing to cancer. The genes responsible for the disease have recently been cloned and characterised; their mutations induce a generalised genomic instability which is particularly evident at microsatellite loci (replication error (RER)+ phenotype). AIMS: To investigate how to select individuals and families in the general population who should be screened for constitutional mutations predisposing to colorectal cancer. PATIENTS/METHODS: Between 1984 and 1995, 1899 colorectal malignancies in 1831 patients were registered, and in 1721 of these (94%), family trees could be obtained. Patients and families were classified into five categories according to a more or less likely genetic basis: HNPCC; "suspected" HNPCC; juvenile cases; aspecific cancer aggregation; sporadic cases. In 18 families with HNPCC as well as in 18 with suspected HNPCC, microsatellite instability in tumour tissues and constitutional mutations of two DNA mismatch repair genes (MSH2 and MLH1) could be evaluated. RER status was studied with five markers (BAT40, D2S123, D18S57, D17S787, and BAT26) in paraffin embedded tissues. Germline mutations of MSH2 or MLH1 genes were assessed on DNA and RNA extracted from lymphomonocytic cells, using reverse transcription polymerase chain reaction, single strand conformation polymorphism analysis, and direct DNA sequencing. RESULTS: HNPCC represented 2.6% and suspected HNPCC 4.6% of all registered colorectal neoplasms. Eleven out of 18 HNPCC families (61%) showed microsatellite instability as opposed to four (of 18) suspected HNPCC (22%; p<0.02). Three germline mutations (two in MSH2 and one in MLH1 gene) were found in three different large HNPCC families, whereas no mutations were detected in suspected HNPCC. CONCLUSIONS: In this study of cancer genetic epidemiology, data from a tumour registry were analysed and this ultimately led to the identification and selection of families that should be tested for mutator gene mutations. With the use of a population based approach, the incidence of mutations was appreciably lower than previously reported and limited to families with full blown HNPCC. It is possible that in most families with a clinical spectrum of HNPCC (or suspected HNPCC) other DNA mismatch repair genes are involved in the pathogenesis of the disease.

Scanning electron microscopy of aberrant crypt foci in human colorectal mucosa.

BACKGROUND: Aberrant crypt foci (ACF) are clusters of morphologically altered crypts which can be observed by light or stereomicroscopy on the mucosal surface of the colon after staining with methylene-blue. They probably represent one of the earliest events in human colorectal carcinogenesis. The main purpose of the present study was to observe the surface features of aberrant and normal colonic crypts in humans using scanning electron microscopy (SEM) in order to find and measure differences between aberrant and normal. MATERIALS AND METHODS: Fifteen mucosal specimens containing ACF and 8 with normal mucosa taken from patients operated on for colon cancer were observed under a scanning electron microscope. RESULTS: By SEM ACF were easily observed on the mucosal surface, because they showed a well defined border and were elevated on the mucosal surface. Under higher magnification luminal openings of aberrant crypts had a larger overall average diameter than normal (37.6 microns +/- 13.5, mean +/- SD, vs 15.9 microns +/- 4.9, P = 0.001), though when crypt multiplicity of ACF (number of crypts per ACF) was higher, the diameter of luminal openings tended to be smaller and similar to those of normal crypts, with weak negative correlation between crypt multiplicity of ACF and mean diameter of aberrant luminal openings (r = 0.27). Finally, the mucosal surface among aberrant crypts was flattened because of a loss of microvilli. in conclusion, scanning electron microscopy allows a better definition of the topological features of aberrant crypt foci than light or stereomicroscopy.

Potential for cross-contamination from use of a needleless injector.

BACKGROUND: Medical devices that are used on patients in fields containing potentially infectious body fluids can become contaminated and transmit infectious agents to other sites on the patient or to other patients if the devices are not properly cleaned and decontaminated after use on each patient treatment site. One such device is the needleless or jet injector, which is widely used in medicine and dentistry to deliver local anesthetic in procedures such as bone marrow aspirations, lumbar punctures, and cutaneous and intraoral injections. This study was conducted to determine whether cross-contamination can occur on in vitro reuse of a needleless injector and whether a manufacturer's recommended method of injector decontamination (ie, immersion sterilization) is effective in the prevention of cross-contamination. METHODS: The study was performed with new autoclaved injectors, fluorescein dye, and Streptococcus crista (the bacteria commonly found in saliva) in the field of use to determine whether these devices can become contaminated during use and carry over the contamination to other sites during immediate reuse. RESULTS: Fluorescein dye and bacteria tests with the needleless injectors showed that contamination or carryover does occur. It appeared to reduced to a minimum when a autoclaved, sterile rubber cap used over the head of the device during injection was replaced between each use, although replacement of the rubber cap alone did not prevent carryover. Immersion of the head of the injector in a 2% glutaraldehyde solution for 30 minutes followed by a sterile water rinse and the replacement of the rubber cap with a sterile cap between uses was shown to curtail bacterial growth and prevent cross-contamination on immediate reuse of the device. CONCLUSION: This study demonstrated that needleless injectors become contaminated during in vitro use and direct contact with contaminated surfaces and that needless injectors carry over the contamination to subsequent sites of release. The replacement of the injector's rubber cap with a new one after initial discharge or the removal of an exposed rubber cap and immersion of the head of the injector in 2% glutaraldehyde followed by a rinse of the head in sterile water, as recommended by one injector manufacturer, can minimize or eliminate the carryover.

Lack of PMS2 gene-truncating mutations in patients with hereditary colorectal cancer.

Hereditary non-polyposis colorectal cancer (HNPCC) is a genetically heterogeneous disease for which PMS2 gene, a member of the human PMS gene family, is believed to have a marginal role. To better define the contribution of PMS2 to hereditary colorectal cancer, we investigated this gene in 22 unrelated Italian patients that, despite a positive family history and/or early onset and development of tumors with microsatellite instability (MSI), did not carry constitutional mutations of MLH1 and MSH2 genes. No mutations with clear-cut pathogenetic significance were detected in the coding regions of PMS2 gene, but only 8 polymorphisms (7 common and 1 rare, 3 silent and 5 missense) and 3 unique molecular variants (2 missense substitutions and one 3-nucleotide deletion) were seen. Lack of PMS2 truncating mutations in our study does not disagree with its supposed marginal involvement in hereditary colorectal cancer, but at the same time points out the need to investigate the phenotypic molecular and clinical characteristics more specifically associated with PMS2 mutations.

Characterisation of Trypanosoma cruzi populations by DNA polymorphism of the cruzipain gene detected by single-stranded DNA conformation polymorphism (SSCP) and direct sequencing.

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas' disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.

Genetic structure of Trypanosoma cruzi in Central America and its comparison with South American strains.

Genetic characterization by isozyme analysis was performed on 68 isolates of Trypanosoma cruzi; 57 from Guatemala in Central America and 11 from South American countries. Ten zymodemes (isozyme patterns) were identified by examining zymograms of 12 enzymes (13 loci). These zymodemes were classified to 3 major distinctive groups: (1) major Guatemalan, (2) minor Guatemalan and (3) unique South American, by the genetic distances and the phylogenetic dendrogram drawn by UPGMA. Based on the results obtained, genetic structures and phylogenetic relations of T. cruzi in Guatemala and South America are discussed. Clonal reproduction seemed to be consistent with the observation of deviation from Hardy-Weinberg equilibrium in several loci.

K-ras and p53 mutations in human colorectal aberrant crypt foci.

Aberrant crypt foci (ACF) are putative precursor lesions of colon cancer, recently identified on the methylene blue-stained mucosal surface of human colon. No mutations in K-ras or p53 genes were found by non-radioactive single-strand conformation polymorphism analysis in 14 ACF collected from five patients. Using the more sensitive method of allele-specific polymerase chain reaction (PCR) for K-ras, 8 of 14 ACF were found to contain K-ras mutations, suggesting that mutated cells are present in minute clones in ACF. No dysplasia was observed in any of the ACF containing a mutated clone. The presence of K-ras mutations in ACF suggests that these lesions occur at a very early stage in human colorectal carcinogenesis.

Bile lipid composition and bile acid pool size in diabetes.

Since the prevalence of gallstones is higher in diabetics than in controls and since cholelithiasis is often associated with supersaturated bile, we measured bile lipid composition and bile acid pool size in 8 patients with juvenile diabetes, 16 with maturity-onset diabetes, and 10 control subjects. Bile lipid composition was expressed as "saturation index." In the maturity-onset diabetics the saturation index (1.60 +/- 0.45 SDM) was significantly higher (P less than 0.005) than that in the controls (0.82 +/- 0.20) and in patients with juvenile diabetes (0.75 +/- 0.24). The absolute values for biliary bile acid concentration were significantly lower (P less than 0.01) in the maturity-onset diabetics than in the other two groups. There were no differences in either the proportion of the individual biliary bile acids or the size of the bile acid pool between the three groups. The results suggest that the incidence of cholelithiasis in diabetes is associated with the secretion of a supersaturated bile only in the maturity-onset subgroup.