HLA-Dw2: a genetic marker for human immune response to short ragweed pollen allergen Ra5. II. Response after ragweed immunotherapy.

After artificial immunization (immunotherapy) with ragweed antigens, specific immunoglobulin G (IgG) antibody (Ab) response to Ra5 was significantly associated with HLA-Dw2 (P less than 0.0001). From a total of 61 treated patients, all 22 Dw2+ subjects made good IgG Ab responses to Ra5 by year 2 of therapy (21 by year 1), even though 8 of them had no detectable IgG Ab and 9 had no detectable IgE Ab before therapy. The prevalence of IgG Ab response among 39 Dw2- subjects was markedly lower; only 11 (28%) responded well after 1-9 yr of therapy. Both by univariate and multivariate statistical analysis, Dw2 was also found to be strongly associated with the quantity of IgG Ab produced. In particular, both the strength and significance of the association between Dw2 and log[IgG Ab] response to Ra5 increased over a 3-yr period of ragweed therapy (P = 10(-9) by year 3). Multiple regression analysis also revealed a weak association with HLA-B13, which became apparent only after year 2 of therapy. Genetic hypotheses for these findings are discussed. In particular, the possibility of a second Ir gene, Ir-Ra5', separate from HLA-Dw2 and possibly located elsewhere in the genome, is considered.

HLA-Dw2: a genetic marker for human immune response to short ragweed pollen allergen Ra5. I. Response resulting primarily from natural antigenic exposure.

Ultra-pure short ragweed pollen allergen Ra5 (5,000 mol wt) was used to investigate the relationship between human leukocyte antigen (HLA) type and IgE and IgG antibody (Ab) responses to Ra5 in two groups of Caucasian subjects, totaling 447 people. Using highly sensitive radioimmunoassay procedures to measure serum IgE and IgG Ab, qualitative responses to Ra5 in both groups were found to be strongly associated with HLA-Dw2 (P less than 0.0001). For example, 95% of 38 people with IgE Ab vs. 22% of 139 ragweed-allergic persons having no detectable IgE Ab to Ra5 were Dw2+. Quantitative log [IgE Ab] and log[IgG Ab] responses to Ra5 were highly correlated with Dw2 (P = 10(-5) to 10(-14)) in four separate multiple regression analyses, examining the relationship between HLA type (and other variables) and Ab levels in the two study groups. Further studies showed that the primary association of Ra5 response was with Dw2 rather than DR2 and that various combinations of A3, B7, and Dw2 were less strongly associated than Dw2 alone.

Association of the HL-A7 cross-reacting group with a specific reaginic antibody response in allergic man.

A relatively small proportion (17 percent) of individuals highly allergic to ragweed were found to develop marked reaginic (immunoglobulin E-mediated) skin sensitivity to a minor ragweed pollen allergen Ra5 (molecular weight 5200). Sensitivity to Ra5 was significantly associated with the possession of a major histocompatibility antigen of the HL-A7 cross-reacting group. This appears to be the first evidence of a strong association between a specific immune response and a specific group of closely related HL-A antigens in man.

Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations.

Sib-pair analysis of 170 individuals from 11 Amish families revealed evidence for linkage of five markers in chromosome 5q31.1 with a gene controlling total serum immunoglobulin E (IgE) concentration. No linkage was found between these markers and specific IgE antibody concentrations. Analysis of total IgE within a subset of 128 IgE antibody-negative sib pairs confirmed evidence for linkage to 5q31.1, especially to the interleukin-4 gene (IL4). A combination of segregation and maximum likelihood analyses provided further evidence for this linkage. These analyses suggest that IL4 or a nearby gene in 5q31.1 regulates IgE production in a nonantigen-specific (noncognate) fashion.

Monoclonal antibodies to the major Lolium perenne (rye grass) pollen allergen Lol p I (Rye I).

Thirteen monoclonal antibodies (MAbs) were produced against Lol p I (Rye I), the major Lolium perenne (rye grass) pollen allergen. Spleen cells from A/J and SJL mice immunized with highly purified Lol p I (Lol I) were allowed to fuse with cells from the non-secreting Sp2/0-Ag14 myeloma cell line. Each MAb was analyzed for antigenic specificity by radioimmunoassay (RIA) using 125I-Lol I. The epitope specificities of seven of the MAbs were examined by competitive binding against a labelled standard MAb for the Lol I antigen (Ag). The dissociation constant, Kd, of one MAb (No. 3.2) that was studied most extensively was determined by double Ab RIA to be 3.5 X 10(-6) L/M. This MAb recognized the related 27,000-30,000 Group I glycoproteins found in the pollens of nine other species of grass pollens tested, including weak binding to Bermuda grass Group I (Cyn d I), which by conventional analysis using polyclonal anti-Lol I serum shows no detectable binding. Monoclonal antibody No. 3.2 was coupled covalently to Sepharose 4B and used to prepare highly purified Lol I from a partially purified rye pollen extract. Finally, an RIA was developed which permitted the analysis of the Group I components in rye grass and nine other grass pollen species. The latter assay is likely to prove useful in the standardization of grass pollen extracts according to their Group I contents.

The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein.

The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5. Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1. Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs. When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis. The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis. It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes. CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

Analysis of the three-dimensional antigenic structure of giant ragweed allergen, Amb t 5.

The ragweed allergens Amb t 5 and Amb a 5 are among the smallest inhaled protein allergens known, containing a single, immunodominant T-cell epitope. In this study we analyzed the B-cell epitope structure of Amb t 5. The three-dimensional structures of Amb t 5 and Amb a 5 have been determined by NMR spectroscopy, providing a rare opportunity to analyze three-dimensional antigenic sites. Amb t 5 residues likely to be important for antigenicity were identified by examining the surface area of Amb t 5 accessible to a probe of the size of an antibody molecule. After changing these residues to the corresponding Amb a 5 residues, recombinant proteins were purified and tested for loss of antigenic activity. Inhibition radio-immunoassays, using sera from 8 individuals who had received immunotherapy with giant ragweed extract, allowed the mutations to be divided into three groups: (1) mutations that had little or no effect on antibody binding, (2) mutations that caused a loss of antigenic activity to a different degree in different sera and (3) mutations that drastically reduced antigenic activity in all sera tested. This last set of mutations clustered in the third loop of Amb t 5, suggesting that antibody recognition of Amb t 5, like T-cell recognition, is primarily directed towards a single, immunodominant site.

Stimulation of the high-affinity IgE receptor results in the tyrosine phosphorylation of a 60 kD protein which is associated with the protein-tyrosine kinase, Csk.

The protein tyrosine kinase Csk downregulates the activity of the Src family of kinases and has a negative effect on signal transduction through several Src kinase-associated receptors. Because the Src-family kinase Lyn plays a pivotal role in FcepsilonRI-mediated cellular activation, we examined whether Csk is involved in FcepsilonRI signaling events. Using anti-Csk antibodies and recombinant fusion proteins we detected a single tyrosine-phosphorylated protein of 60 kD (herein referred to as 'p60') that associates with the SH2 domain of Csk after stimulation of the FcepsilonRI. p60 phosphorylation reached a maximum within one minute and remained constant while the receptors were aggregated; disaggregation of the receptors resulted in rapid dephosphorylation of p60. The phosphorylation of p60 was only detected after activation by IgE and antigen and not by stimulation with PMA and/or ionomycin. Phosphorylated p60 was associated entirely with the membrane fraction of the cells. A considerable fraction of Csk was associated with the membrane in both unstimulated and stimulated cells, this fraction did not change upon activation. p60 coprecipitated with Csk from both unstimulated and FcepsilonRI stimulated cells and was phosphorylated by the immunocomplex. Total kinase activity of Csk immunoprecipitates increased upon FcepsilonRI stimulation. p60 did not react with antibodies to a number of known signaling molecules, including the recently cloned, GAP-associated protein, p62dok. Our data demonstrate that Csk associates with a membrane-anchored protein complex that is directly involved in FcepsilonRI signal transduction.

Association and linkage of atopic dermatitis with chromosome 13q12-14 and 5q31-33 markers.

Atopic dermatitis is a chronic inflammatory skin disease that affects 10-20% of the population. Linkage of atopy, asthma, allergic rhinitis, and total serum IgE levels to several different chromosomal regions have been described extensively, but little is known about the genetic control of atopic dermatitis. We tested for the association and linkage between atopic dermatitis and five chromosomal regions: 5q31-33, 6p21.3, 12q15-24.1, 13q12-31, and 14q11.2/14q32.1-32.3. Marker analysis was performed in two Caucasian populations: (i) 192 unrelated German children with atopic dermatitis and 59 non-atopic children from a German birth cohort study (MAS'90), parental DNA was tested in 77 of 192 children with atopic dermatitis; (ii) 40 Swedish families with at least one family member with atopic dermatitis selected from the International Study of Asthma and Allergy in Children. Evidence for linkage and allelic association for atopic dermatitis was observed for markers on chromosome 13q12-14 and 5q31-33.

Cloning the cDNA encoding the AmbtV allergen from giant ragweed (Ambrosia trifida) pollen.

Ragweed (Ambrosia) pollens contain a number of proteins that cause allergic disease in ragweed-sensitive people. The cloning of the AmbtV cDNA is important, since the 4.4-kDa AmbtV, one of the allergens in giant ragweed (Ambrosia trifida) pollen, serves as a simple model system to study the basic structural requirements for immune recognition of foreign protein allergens. We report the cloning of the AmbtV cDNA by means of the polymerase chain reaction (PCR) using degenerate primers. We generated three sets of overlapping cDNA clones by a combination of PCR and anchored-PCR, and determined the complete nucleotide (nt) sequence. From the nt sequence, the amino acid (aa) sequence of the protein was confirmed and the leader sequence was deduced. This general approach can be used to clone allergen and other cDNAs from complex biological sources provided partial aa sequence information is available. It may be the best available approach in cases where the isolation of clones from a cDNA library is difficult, which proved to be the case for AmbtV.

Cervical myelopathy complicating cerebral angiography. Report of a case and review of the literature.

Transverse cervical myelopathy, at C-6 level, followed injection of Renografin-60 into the right thyrocervical trunk during cerebral angiography. Review of the literature yielded only two cases in which attempted posterior fossa angiography resulted in cervical myelopathy. Two more cases were found. In one, cervical myelopathy occurred during aortography in a patient with coractation of the aorta, and in the other it followed mediastinal angiography. Summation of anoxia, hemorrhage, and cellular toxicity is responsible for spinal cord necrosis following arterial injection of contrast material.

Simplified method for isolation of white cells from whole blood suitable for direct polymerase chain reaction.

A simple method for isolating mononuclear cells from whole blood is described. The procedure utilizes phytohemagglutinin to agglutinate the erythrocytes, separating white cells from whole blood in a very brief handling time. The isolated cells are readily subjected to DNA isolation simply by boiling, and the released DNA can be directly employed for the polymerase chain reaction analysis. The efficiency of this method is similar to other conventional methods, but less costly and less time-consuming. This method is particularly useful in analyzing DNA samples from the peripheral blood cells when the simplicity and low cost of the assay are preferable.

Enhanced diagnostic power of exercise testing for myocardial ischemia by addition of postexercise left ventricular ejection time.

To improve both the sensitivity and specificity of the multistage treadmill test, postexercise systolic time intervals were prospectively studied in 73 patients with angina-like chest pain and normal resting ST-T segments. The decision to perform coronary angiography was made independent of the exercise test. Twenty-three patients had normal coronary arteries and 50 had more than 50 percent reduction of luminal diameter of one or more major coronary arteries. The systolic time intervals were measured before and 2,4,6,8 and 10 minutes after exercise. Of all the systolic time intervals, the 4 minute postexercise left ventricular ejection time proved most discriminating between normal subjects and those with coronary artery disease. The deviations of this interval from the normal regression with heart rate both before and after exercise were used to calculate the net delta left ventricular ejection time after exercise. A net increase of more than 31 msec represented 2 standard deviations above normal. Twenty-three (46 percent) of the patients with coronary artery disease had an abnormal net delta ejection time after exercise. Twenty-five (50 percent) had a positive electrocardiographic response with a 9 percent false positive rate. Thirteen (26 percent) had only a positive postexercise net delta ejection time so that a total of 76 percent of patients with coronary artery disease were identified. Thus, measurement of the postexercise net delta ejection time a simple and useful adjunct to multistage treadmill testing.

Effect of heparin in anticoagulant doses on the electrocardiogram and cardiac enzymes in patients with acute myocardial infarction. A clinical pilot study.

The effect of heparin in clinical anticoagulant doses on S-T segment and cardiac enzymes was studied in 18 patients with acute myocardial infarction by electrocardiogram and enzyme evaluation 1 hour and 24 hours after initial heparin infusion. Intestinal mucosa heparin was given by infusion, 10,000 units after the admission electrocardiogram, and 5,000 units every 6 hours. Data in the nine control and nine treated patients were statistically similar on admission. The electrocardiograph findings were improved, but not significantly, 1 hour after administration of heparin. At 24 hours of heparin therapy, the S-T deviations were reduced 64% (from 139 +/- 2.1 [standard error of the mean] to 50.5 +/- 1.2 mm); in control patients S-T deviations were reduced 21% (from 109 +/- 1.8 to 86 +/- 0.9 mm (t=2.9, P less than 0.019). At 24% hours electrocardiographic leads with 2 mm or more deviation were reduced 86% in heparin-treated patients and 28% in control subjects. Cardiac enzymes were comparably elevated at 24 and 48 hours in both groups, with no clear trend. It is concluded that heparin in anticoagulant doses reduces the 12 lead electrocardiographic pattern of injury without discernibly modifying cardiac enzymes. The question of heparin efficacy in acute myocardial ischemic injury, reopened by findings with large dose heparin in therapy in dogs and anticoagulant dose in this study, awaits further expanded investigation.

Human immune responsiveness to Lolium perenne pollen allergen Lol p III (rye III) is associated with HLA-DR3 and DR5.

A well-characterized allergen of Lolium perenne (perennial rye grass) pollen, Lol p III, has been used as a model antigen to study the genetic control of the human immune response. Associations between HLA type and IgE or IgG antibody (Ab) responsiveness to Lol p III were studied in two groups of skin-test-positive Caucasoid adults (N = 135 and 67). We found by nonparametric and parametric analyses that immune responsiveness to Lol p III was significantly associated with HLA-DR3 and DR5. No association was found between any DQ type and immune responsiveness to Lol p III. Geometric mean IgE or IgG Ab levels to Lol p III were not different between B8+, DR3+ subjects and B8-, DR3+ subjects, showing that HLA-B8 had no influence on the association. Lol p III IgG Ab data obtained on subjects after grass antigen immunotherapy showed that 100% of DR3 subjects and 100% of DR5 subjects were Ab+. A comparison of all the available protein sequences of DRB gene products showed that the first hypervariable region of DR3 and DR5 (and DRw6), and no other region, contains the sequence Glu9-Tyr-Ser-Thr-Ser13. Our observations are consistent with the possibility that immune responsiveness to the allergen Lol p III is associated with this amino acid sequence in the first hypervariable region of the DR beta 1 polypeptide chain.

Association of responsiveness to the major pollen allergen of Parietaria officinalis with HLA-DRB1* alleles: a multicenter study.

Parietaria, a plant belonging to the family of Urticaceae, is a major source of allergenic pollen in Europe. In the context of a multinational study, we investigated whether in allergic subjects antibody response towards Par o 1, the major allergen from P. officinalis, was associated with defined HLA-DRB1* alleles. The study population consisted of 234 allergic patients: 65 from Bulgaria, 30 from Israel, 99 from Italy, and 40 from Spain. In the Italian study group, the prevalence of ST positivity to Parietaria was 77%. In Parietaria ST-positive subjects, the prevalences of IgG and IgE serum Ab towards Par o 1 were 91% and 75%, respectively. HLA-DRB1*1101 and/or 1104 were significantly positively associated with the presence of IgG Ab and with high levels of IgE Ab towards this allergen (p = 0.0007 and p = 0.012, respectively). In the Spanish study group, the positive association of DR1100 with responsiveness to Par o 1 was confirmed (p = 0.02, RR = 4, and p = 0.002, RR = 7, for IgG and IgE Ab, respectively). None of the Bulgarian patients had IgE Ab to Par o 1, whereas IgG Ab response was observed in 7 out of 65 subjects and was positively associated with DRB1*1101 and/or 1104 (p = 0.025). In the Israeli study group, responsiveness to Par o 1 was not associated with specific HLA-DRB1* alleles. In conclusion, this study shows that in allergic patients from three European populations antibody response to the major allergen from the pollen of Parietaria is associated with HLA-DRB1*1101 and/or 1104. Our data suggest that this association is stronger in subjects monosensitized to Parietaria.

Inheritance of immunoglobulin E: genetic model fitting.

Total serum immunoglobulin E (IgE) concentration was measured on 316 members of five pedigrees selected through breast cancer probands. Sex- and age-adjusted natural logarithm-transformed IgE level was not found to differ between individuals with breast cancer or with cancer of any site and other relatives. Likelihoods of the polygenic and of one-locus, two-allele major gene and mixed models were computed. The analysis provided evidence for the presence of a polygenic component in the determination of IgE; it did not show evidence of a major gene effect.

A genetic-epidemiologic study of human immune responsiveness to allergens in an industrial population: I. Epidemiology of reported allergy and skin-test positivity.

Four hundred six subjects, comprising a 10% random sample of all employees, and a sample of "self-reported" allergic employees of a light industrial plant participated in an epidemiologic study of allergy. Puncture skin testing with a wide variety of crude allergens revealed a significantly higher prevalence of IgE-mediated sensitivity in males than females (29% males and 7% females in a random group; 60% males and 30% females in a self-reported allergic group); however, reported prevalence rates for "allergy" and different allergic symptoms were generally not different between males and females. Interestingly, reported asthma was greater in skin-test-positive subjects than in skin-test-negative subjects. We also noted a decrease in skin-test positivity with increasing age in self-reported allergic subjects. This was significant in the case of several crude allergens but not in the case of positivity to at least one allergen. We also found evidence that people born in and who have been resident in "Zone I) (MD, PA, Del, NJ, or DC) for most of their lives exhibit a greater prevalence of skin-test positivity than people who were born in and have lived for much of their lives in the northeastern United States (east of the Mississippi River and to the north of South Carolina) other than in Zone I.

The inheritance of immunoglobulin E: genetic linkage analysis.

Linkage analyses between 21 genetic markers including HLA-A, B, and the postulated locus for determining total serum IgE levels were done to try to clarify the inheritance of total IgE levels and to map the locus. A total of 316 individuals from five Mormon kindreds were studied, and data from an additional 204 Amish individuals from 11 families were analyzed for possible HLA linkage. Segregation analyses of both data sets did not give clear definition of the mode of inheritance of total IgE levels, but purely environmental models were rejected. Linkage analyses gave significant evidence against HLA linkage with the codominant, recessive, or dominant model of inheritance for total IgE levels. No significant evidence for linkage with any of the genetic markers was obtained. Since total serum IgE levels are correlated with allergies, understanding the genetics of total IgE levels is important to understanding the genetics of allergic disease in man.