Identification of bovine IgG as a major cross-reactive vertebrate meat allergen

BACKGROUND: Although beef is a main source of protein in Western diets, very little has been published on allergic reactions to beef or the main allergens implicated in these reactions. The aim was to evaluate the IgE antibody response to beef in suspected meat-allergic subjects and assess cross-reactivity of beef with other vertebrate meats. METHODS: Fifty-seven sera from suspected meat-allergic subjects were tested by grid blot for specific IgE antibodies to vertebrate meats (beef, lamb, pork, venison, and chicken), and the patterns of recognition of meat proteins were assessed by immunoblot studies. RESULTS: A 160-kDa band, identified as bovine IgG, was detected in raw beef in 83% (10/12) of beef-allergic subjects but in only 24% of the beef-tolerant subjects. IgE reactivity to a band of similar mol. mass was detected also in lamb and venison, but rarely in pork or chicken. Complete inhibition of the IgE reactivity to the bovine IgG was obtained with lamb, venison, and milk. IgE reactivity to this band also completely disappeared when beef or lamb extracts were separated under reducing conditions, indicating conformational epitopes. CONCLUSIONS: Bovine IgG appears to be a major cross-reacting meat allergen that could predict beef allergy. Further studies with oral IgG challenges should be performed to document the conclusion that in vitro reactivity correlates with clinical hypersensitivity. The role of bovine IgG in other bovine products such as milk, dander, or hair must also be studied, and the hypothesis that it is a cross-reacting allergen with other mammalian products validated.     

Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets

Protein tyrosine phosphatase predominantly determines the status of protein tyrosine kinase-dependent phosphorylation of specific proteins and controls the survival and death of neurons. Previous studies have shown that protein tyrosine phosphatase activity is decreased during hypoxia in cortical membranes of the newborn piglet. We have also shown that nitric oxide (NO) free radicals are generated during hypoxia, and may result in modification of protein tyrosine phosphatase via peroxynitrite-mediated modification. The present study tests the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. Since 3-nitrotyrosine is produced as a consequence of peroxynitrite reactions, we have also examined the effect of 3-nitrotyrosine on protein phophatase activity. Cerebral cortical P(2) membranes were prepared from seven normoxic newborn piglets and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 microM SNP), and a peroxynitrite group (exposed to 100 microM peroxynitrite). Protein tyrosine phosphatase activity was determined spectrophotometrically in the presence or absence of 2 microM bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The protein tyrosine phosphatase activity was 198+/-25 nmol/mg protein/h in the normoxic group, 177+/-30 nmol/mg protein/h in the SNP group (p=NS versus normoxic) and 77+/-20 nmol/mg protein/h in the peroxynitrite group (p<0.001 versus normoxic). The results show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro. Furthermore 3-nitrotyrosine (100 microm), a product of peroxynitrite, decreased the enzyme activity from 926+/-102 to 200+/-77 (p<0.001). We conclude that protein tyrosine phosphatase regulation is mediated by peroxynitrite. We propose that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inactivation in vivo. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity, leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity may be a NO-mediated mechanism of programmed cell death in the hypoxic brain.     

Lack of association between angiotensin converting enzyme polymorphism and sporadic Alzheimer's disease

Epidemiological and pathogenetic evidences suggest a strong association between vascular risk factors and sporadic Alzheimer's disease (sAD). In agreement with the vascular hypothesis of AD, the role of various candidate genes for atherosclerosis has been investigated, leading to conflicting results. In order to clarify the significance of angiotensin-converting enzyme (ACE) gene insertion (I)/deletion (D) polymorphism in a group of patients with sAD, we conducted a case-control study including 149 cases and 149 age and sex matched controls. All subjects were genotyped for ACE and Apolipoprotein E (APOE). There were no significant differences in ACE genotype or allele frequencies between cases and controls, even after stratification for APOE4 carrier status. Our data suggest that the ACE I/D polymorphism is not associated to genetic susceptibility in sAD patients.     

The beta1 adrenergic effects of antibodies against the C-terminal end of the ribosomal P2beta protein of Trypanosoma cruzi associate with a specific pattern of epitope recognition

BALB/c mice immunized with recombinant Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) develop a strong and specific antibody response against its 13 residue-long C-terminal epitope (peptide R13: EEEDDDMGFGLFD) that has a concomitant beta1-adrenergic stimulating activity. However, other animals that undergo similar immunizations seem tolerant to this epitope. To evaluate further the antibody response against the ribosomal P proteins, 25 BALB/c and 25 Swiss mice were immunized with TcP2beta. From the 50 animals, 31 developed a positive anti-R13 response, whereas 19 were non-responsive. From the 31 anti-R13 positive mice, 25 had anti-R13 antibodies that recognized the discontinuous motif ExDDxGF, and their presence correlated with the recording of supraventricular tachycardia. The other six had anti-R13 antibodies but with a normal electrocardiographic recording. These anti-R13 antibodies recognized the motif DDxGF shared by mammals and T. cruzi and proved to be a true anti-P autoantibody because they were similar to those elicited in Swiss, but not in BALB/c mice, by immunization with the C-terminal portion of the mouse ribosomal P protein. Our results show that the recognition of the glutamic acid in position 3 of peptide R13 defines the ability of anti-R13 antibodies to react with the motif AESDE of the second extracellular loop of the beta1-adrenergic receptor, setting the molecular basis for their pathogenic beta1 adrenoceptor stimulating activity.     

Two sibs with Wiedemann-Rautenstrauch syndrome: possibilities of prenatal diagnosis by ultrasound.

A girl with Wiedemann-Rautenstrauch syndrome was born to a non-consanguineous couple. During the pregnancy, growth retardation particularly in the biparietal and abdominal diameters but not the femoral length was detected through serial ultrasound scans. When the woman became pregnant again, in spite of having been assessed as having a 25% risk of recurrence, the prenatal findings seen in her previous pregnancy led us to suggest sequential echography and a similar pattern of growth retardation was shown. After termination, the male fetus was found to be affected by Wiedemann-Rautenstrauch syndrome. This case shows that ultrasound examination can be a useful tool in the prenatal diagnosis of this rare, autosomal recessive syndrome.     

B-lymphocyte activation with an extract of Nocardia brasiliensis.

An extract from the pathogenic actinomycete Nocardia brasiliensis was mitogenic for murine lymphocytes. This deoxyribonucleic acid-synthetic response of whole spleen cells peaked after 48 h in culture at concentrations of Nocardia extract ranging from 10 to 200 micrograms/ml. The extract appeared to be a mitogen for B lymphocytes since cultures of spleen cells from congenitally athymic nude (nu/nu) mice and of antithymocyte serum plus complement-treated spleen cells from conventional (+/+) mice responded as well as untreated spleen cells from normal +/+ mice. Furthermore, thymocytes did not respond mitogenically to the extract. Mitogenic responses were stimulated in spleen cells from H-2(a), H-2(b), H-2(d), and H-2(k) mice, including lipopolysaccharide-nonresponder C3H/HeJ mice. This Nocardia extract also stimulated polyclonal B-cell activation to the hapten trinitrophenyl, serum protein human gamma globulin, and several mammalian erythrocytes in cultures of cells from both euthymic and nude mice. Additionally, the requirement for helper T cells in the primary in vitro immune response to sheep erythrocytes could be circumvented by the addition of this Nocardia extract. These results indicate that an extract from the pathogen N. brasiliensis can nonspecifically activate murine B lymphocytes and raise the possibility that polyclonal activation of B lymphocytes may contribute to the pathogenesis of nocardiosis.     

Involvement of leukotriene B4 and platelet-activating factor in cytokine priming of human polymorphonuclear leucocytes.

Recombinant human (rh) tumour necrosis factor (TNF) alpha and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) prime human polymorphonuclear leucocytes (PMN) for increased superoxide anion (O2-) generation and for increased platelet-activating factor (PAF) biosynthesis and leukotriene B4 (LTB4) release. Both PAF and LTB4 are candidate mediators for the enhanced O2- generation in cytokine-primed PMN, since exogenous PAF or LTB4 primes PMN. We measured the generation and release of these mediators and examined their potential roles in cytokine priming using the PAF receptor antagonist, WEB 2086, and the inhibitor of 5-lipo-oxygenase, CGS 8515.rhTNF-alpha or rhGM-CSF, alone, increased PAF levels in PMN, but did not cause PAF release or LTB4 synthesis. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated the release of detectable and biologically active amounts of both LTB4 and PAF in primed, but not in non-primed PMN. However, neither blockade of PAF receptors, nor inhibition of LTB4 synthesis influenced the priming of O2- generation by rhTNF-alpha or rhGM-CSF. Simultaneous pretreatment of PMN with WEB 2086 and CGS 8515 also failed to inhibit priming. Our results do not exclude a role for cell-associated PAF in the priming response, but indicate that the release of PAF and LTB4 do not mediate this phenomenon. The ability of cytokines to amplify the production and release of lipids may represent a mechanism to attract and localize the pro-inflammatory actions of stimulated PMN to regions where cytokine levels are also elevated.     

Human T cell responses to peptides of the Mycobacterium leprae 45-kD serine-rich antigen

In order to identify T cell epitopes within the Mycobacterium leprae 45-kD serine-rich antigen, we analysed responses to overlapping 17-mer peptides encompassing the whole antigen in non-exposed UK controls, Pakistani leprosy patients and tuberculosis patients in both the United Kingdom and Pakistan. This antigen has been described as M. leprae-specific, although it has a hypothetical homologue in M. tuberculosis. Human peripheral blood mononuclear cells were stimulated with peptide for 5 days and IFN-gamma measured in supernatants by ELISA. Some peptides were recognized more frequently by T cells from tuberculoid leprosy patients than those from UK controls, suggesting that such T cell epitopes might have diagnostic potential, while other peptides induced greater responses among UK control subjects. Short-term cell lines confirmed that these assays detected specific T cell recognition of these peptides. However, many tuberculosis patients also recognized these potentially specific peptides suggesting that there could be a true homologue present in M. tuberculosis.