Functional immunoglobulin E cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens.

BACKGROUND: Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. OBJECTIVE: To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. METHODS: Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. RESULTS: In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. CONCLUSIONS: Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.

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.

In Vitro and In Vivo Characterization of a Fully Felinized Therapeutic Anti-Nerve Growth Factor Monoclonal Antibody for the Treatment of Pain in Cats.

BACKGROUND: Limited options are available for the treatment of pain in cats. Monoclonal antibodies (mAbs) that neutralize nerve growth factor (NGF) have demonstrated analgesic capacity in rodent models, people with osteoarthritis, and dogs with degenerative joint disease. HYPOTHESIS/OBJECTIVES: This study describes the design and characterization of a fully felinized anti-NGF monoclonal antibody. In vitro potency, pharmacokinetics, and the ability of the antibody to treat pain in a self-resolving, acute inflammation model were investigated in cats. ANIMALS: Thirty-eight cats at a research colony at Charles River Laboratories, Ireland. METHODS: Felinized anti-NGF mAb, NV-02, was produced using a complementary DNA (cDNA)-based method (PETization). Purified NV-02 was tested for affinity, potency, and immunoreactivity in vitro, then for safety and plasma pharmacokinetic distribution in vivo, and analgesic efficacy in a model of kaolin-induced inflammatory pain. RESULTS: Anti-NGF mAb, NV-02 neutralized NGF with high affinity and potency and did not bind complement. NV-02-administered SC had a plasma half-life of 7-15 days and was well tolerated at dosages up to 28 mg/kg. A dosage of 2 mg/kg NV-02 SC significantly decreased signs of lameness on day 2 (P = .0027), day 3 (P = .016), day 4, (P = .0063), day 5 (P = .0085), day 6 (P = .0014), and day 7 (P = .0034) after induction of inflammation. CONCLUSIONS AND CLINICAL IMPORTANCE: The high affinity, long plasma half-life, safety, and analgesic efficacy of felinized anti-NGF mAb (NV-02) support further investigation of the analgesic potential of this antibody in the cat.

Female-specific sequences isolated from Schistosoma mansoni by representational difference analysis.

Representational difference analysis (RDA), a recently described, polymerase-chain-reaction-based modification of the subtractive hybridization process was employed to isolate female-specific sequences of Schistosoma mansoni. Using HindIII-derived amplicons, an excess of male schistosome DNA was employed to remove sequences common to both male and female adult S. mansoni genomes from female genomic DNA. Following three rounds of RDA, the enriched sequences included two female-specific repetitive elements. One of these exhibited 76% homology to the SM alpha family of retroposons and represents a W chromosome-specific variant of that family. The other sequence represents a novel, female-specific repetitive sequence. These sequences have been designated SM alpha fem-1 and W2, respectively, and both are apparently arrayed as tandem repeats on the W chromosome of S. mansoni. The isolation and characterization of the two female-specific sequences by RDA indicates that this procedure should also find utility in the definition of traits and sequences that differ among other groups of schistosomes.

Variation in the sequence of a mitochondrial NADH dehydrogenase I gene fragment among six natural populations of Schistosoma japonicum from China.

The present study investigated the level of genetic variation among Schistosoma japonicum populations of different geographical origins from mainland China. Polymerase chain reaction-based methods were employed to determine the sequence for a subunit of the mitochondrial NADH dehydrogenase I gene for populations from Zhejiang, Anhui, Jiangxi, Hunan, Hubei and Sichuan. No variation was detectable in the NADH dehydrogenase I sequence within populations from Zhejiang and Hubei, whereas sequence variation of 0.2% was detected within populations from Anhui, Jiangxi, Hunan and Sichuan. Pairwise comparison of the sequences representing the six different populations revealed genetic differences ranging from 0 to 0.6%.

Isolation of Japanese encephalitis virus from mosquitoes (Diptera: Culicidae) collected in the Western Province of Papua New Guinea, 1997-1998.

After Japanese encephalitis (JE) virus emerged in the Torres Strait in Australia in 1995, investigations were initiated into the origin of the incursion. New Guinea was considered the most likely source, given its proximity to islands of the Torres Strait. Almost 400,000 adult mosquitoes were processed for virus isolation from 26 locations in the Western Province of Papua New Guinea (PNG) between February 1996 and February 1998, yielding three isolates of JE virus. Two isolates of Murray Valley encephalitis, 17 isolates of Sindbis, and 1 each of Sepik and Ross River viruses were also obtained. Nucleic acid sequences of the PNG JE isolates were determined in the prM region, and in a region overlapping a part of the fifth nonstructural protein and the 3' untranslated region. The PNG isolates belonged to genotype II, and shared > 99.2% identity with isolates from humans and mosquitoes from the Torres Strait, suggesting that PNG is the source of incursions of JE virus into Australia.

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.

Mammographically detected microcalcifications due to pseudoxanthoma elasticum.

Mammographic detection and analysis of microcalcification is a key component in the detection of early breast cancer. A casting configuration or granular morphology has a strong association with malignancy. These are relatively easily recognised, but with the proliferation of mammographic screening, other varieties of calcification are being encountered which may pose diagnostic dilemmas. This case report describes the findings in a patient with Pseudoxanthoma Elasticum (PXE) who presented in this way. To our knowledge, this has not previously been documented. Such calcification is not usually visible on conventional radiographs.

A retrotransposon of the non-long terminal repeat class from the human blood fluke Schistosoma mansoni. Similarities to the chicken-repeat-1-like elements of vertebrates.

The genomes of representative species of fishes, amphibians, and reptiles contain non-long-terminal-repeat (non-LTR) retrotransposons showing strong sequence identity to the chicken repeat 1 (CR1) non-LTR retrotransposon from birds. These nonavian retroelements have been termed CR1-like elements. We have isolated sequences of a non-LTR retrotransposon from the human blood fluke Schistosoma mansoni. These schistosome sequences, which we have termed the SR1 family of non-LTR retrotransposons, contain regions of deduced amino acids characteristic of the CR1-like elements. SR1 elements possess atypical 3' termini consisting of the tandem repeat (AACCATTTG)2 which are similar in structure to the imperfect tandem repeat of the 3' termini of CR1. There are at least 200 copies of SR1 interspersed through the genome of S. mansoni. The structural and amino acid sequence similarities of SR1 with members of the CR1-like elements suggest that the SR1 family belongs to the CR1-like category of non-LTR retrotransposons. Although other non-LTR retrotransposons have been described in invertebrates, this is the first CR1-like element reported from a nonvertebrate taxon, suggesting that the phylogenetic distribution of CR1-like retrotransposons is not restricted to vertebrates.

Host-like sequences in the schistosome genome.

A series of recent papers has indicated that widespread genomic rearrangements take place in the genome of schistosomes during the life cycle of the parasite. These results have been controversial since genomic rearrangements are not common in eukaryotes, probably because excessive genome plasticity would carry a heavy evolutionary price. Here, Karen Clough, Alec Drew and Paul Brindley present data that ostensibly support the concept of widespread genomic rearrangements, but for which they suggest a different interpretation. They conclude that artefactual contamination of schistosome genome preparations with host DNA can probably explain the Southern hybridization results which led to the original hypothesis of developmental, genomic rearrangements.

SR2 elements, non-long terminal repeat retrotransposons of the RTE-1 lineage from the human blood fluke Schistosoma mansoni.

As in other eukaryotes, a substantial portion of the genome of the human blood flukes belonging to the genus Schistosoma appears to be composed of mobile genetic elements and other repetitive sequences. The constitutent elements and their relative organization are not well understood, although retroposons (the SM alpha elements) and a family of non-long terminal repeat (LTR) retrotransposons (the SR1 elements) have been reported from the genome of Schistosoma mansoni. Here, we report the presence of a second family of non-LTR retrotransposons from S. mansoni which we have termed the SR2 elements. SR2 elements are members of a recently described lineage of non-LTR retrotransposons typified by the RTE-1 non-LTR retrotransposon of Caenorhabditis elegans. We determined the sequence for approximately 3.9 kb of a consensus full-length SR2 element, which included a long 5' untranslated region (UTR), potential first and second open reading frames (ORFs) of 78 and 1,018 amino acid residues, respectively, a short 3' UTR, and an A-rich 3' terminus. SR2 elements were bound by target site duplications. The putative first and second ORFs did not overlap. The second ORF was homologous to retroviral pol and encoded an apurinic/apyrimidinic endonuclease and a reverse transcriptase. A number of extremely short SR2 elements of less than 0.5 kb, reminiscent of SINEs, were also characterized. These consisted solely of the 5' and 3' UTRs of full-length SR2 elements, having both ORFs deleted. Analysis indicated that these SINE-like SR2 elements were produced by replication of a SINE-like SR2 element, rather than by repeated deletions within larger SR2 elements.

A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.

A novel method for the isolation of schistosome eggs and miracidia from livers of mice infected with Schistosoma japonicum or S. mansoni is described. The method employed collagenase B to degrade the interstitial matrix of mouse liver tissue, after which the schistosome eggs were separated from the liver cells by 2 single-step density centrifugations through Percoll. Using this procedure sufficient quantities of miracidia were obtained to generate a cDNA library. Southern blot analysis demonstrated that miracidia isolated by this method were free from contaminating host DNA.

Screening for diabetic retinopathy.

OBJECTIVES: To identify factors associated with a high risk of diabetic retinopathy in order to rank patients for urgency of examination by fundal photography. DESIGN: Retrospective case survey. PATIENTS: Eight hundred and eighty-eight consecutive diabetic patients who were routinely referred for mydriatic fundal photography to the Queen Elizabeth Hospital, Adelaide, between August 1987 and May 1992. OUTCOME MEASURES: The prevalence of non-proliferative and proliferative diabetic retinopathy and patient biochemical and demographic characteristics and urinary albumin excretion rate. RESULTS: The prevalences of nonproliferative and proliferative diabetic retinopathy were 18.1% and 2.4%, respectively. Multiple logistic regression analysis established that treatment with insulin or oral hypoglycaemic drugs was associated with the highest risk of diabetic retinopathy (odds ratio [OR], 14.7; 95% confidence interval [CI], 3.4-63.2), while duration of diabetes > or = 7 years (OR, 3.6; CI, 1.9-6.8), age 50-66 years (OR, 2.1; 95% CI, 1.1-4.0) and albumin excretion rate > or = 21 micrograms/min (OR, 2.2; 95% CI, 1.1-4.5) were also significant risk factors. Non-significant variables were hypertension, obesity and sex. CONCLUSIONS: Diabetic patients may be ranked for urgency of retinal photographic screening based on mode of treatment and duration of diabetes, thereby facilitating examination of patients at highest risk of asymptomatic diabetic retinopathy and increasing the efficiency of the screening program.

Tandemly repeated genomic sequence demonstrates inter- and intra-strain genetic variation in Schistosoma japonicum.

Genetic variability within and among four geographical strains of Schistosoma japonicum was examined using a novel repetitive element. The element, termed Sirh1.0, was isolated from genomic DNA of a Philippine strain of S. japonicum using a combination of restriction fragment PCR and band-stab PCR. Sjrh1.0 is a tandemly repeated element, the sequence of which appears to be species-specific, in that it hybridized to DNA from S. japonicum but not to DNA from S. mansoni. Its sequence does not match previously deposited sequences in GenBank. When employed as a probe in Southern hybridization analysis, radiolabelled Sjrh1.0 revealed sex-specific and strain-specific differences in genomic DNA of individual worms. We also found individual genetic variation within geographical isolates of the Asian schistosome.

The hevein domain of the major latex-glove allergen Hev b 6.01 contains dominant T cell reactive sites.

BACKGROUND: Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex-glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C-terminal fragment (14 kDa). Hevein is an abundant protein in latex-glove extracts. As the immune response to allergens is initiated by activation of allergen-specific CD4(+) T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy. OBJECTIVE: To identify dominant T cell epitopes of Hev b 6.01 in latex-allergic glove users. METHODS: Ten latex-allergic frequent glove users and six non-latex-allergic atopic control subjects were selected, based on clinical symptoms and positive latex-specific serum IgE. Serum IgE reactivity to glove extract and recombinant Hev b 6.01 (rHev b 6.01) were analysed by ELISA. Latex-specific short-term oligoclonal T cell lines were generated from peripheral blood of latex-allergic subjects. These lines were tested for proliferative responses to overlapping 20-mer peptides of the Hev b 6.01 molecule. CD4(+) T cell intracellular cytokines, IL-4 and IFN-gamma were assessed following stimulation with immobilized anti-CD3 in the presence of IL-2. RESULTS: All ten of the latex-allergic patients showed serum IgE binding to glove extract while eight of these also showed IgE binding to rHev b 6.01 by ELISA. Western blotting confirmed reactivity with rHev b 6.01 at around 20 kDa. T cell proliferation assays showed that latex-specific T cell lines from all subjects responded to one or more peptides, with greatest frequency of reactivity to peptides Hev b 6.01 p(10-29) and Hev b 6.01 p(19-38) in the hevein domain. An allergic-type cytokine profile with considerable IL-4 in addition to IFN-gamma was evident from intracellular cytokine staining. CONCLUSION: Hevein is an important T cell as well as B cell immunogen and contains dominant T cell reactive sites.

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.