The distribution of CY2PC19*2 allele in a Trinidadian population. Is Clopidogrel the right choice?

OBJECTIVE: To determine the distribution of the CYP2C19*2 allele among Trinidadians. DESIGN AND METHODS: This is a cross sectional study among 100 patients attending primary health centers within the North Central Region of Trinidad. A stratified sampling technique was used in which there were three mutually exclusive subgroups: those of South Asian descent (Indo-Trinidadians), African descent (Afro-Trinidadians) and mixed ethnicity. Subsequently, systematic sampling was applied to each stratum to improve the representativeness of the sample. Hence, the 100 subjects recruited for the study were 40 Indo-Trinidadians, 40 Afro-Trinidadians and 20 of mixed descent. Apart from baseline data which included age, gender and ethnicity, DNA was assessed for the CYP2C19*2 allelic variant using a PCR method. RESULTS: There was a high allelic frequency (37%) for CYP2C19*2 which was found to be more common among Indo-Trinidadians (47.5%, 95% CI 32.0-63.0) compared to Afro-Trinidadians (22.5%, 95% CI 9.6-35.4) or people of mixed origin (45%, 95% CI 23.2-66.8). There was a significant difference (p=0.019) between CYP2C19*2 frequencies for Indo-Trinidadians and Afro-Trinidadians. CONCLUSION: We provided evidence that the prevalence of CYP2C19*2 mutation was high in our setting.

Radioimmunotherapy with (111)In/(90)Y-2IT-BAD-m170 for metastatic prostate cancer.

PURPOSE: Over 31,000 Americans die of androgen-independent metastatic prostate cancer each year. New strategies that do not involve hormonal manipulation but instead recognize the biochemical and molecular characteristics of prostate cancer are needed. Radioimmunotherapy (RIT) uses a tumor-specific monoclonal antibody to deliver systemic, targeted radiation to cancer. The objectives of this Phase I study of (111)In-2IT-BAD-m170 (for imaging) and (90)Y-2IT-BAD-m170 (for therapy) were to determine the toxicity and maximum tolerated dose (MTD), the specificity for targeting metastatic prostate cancer, and the efficacy for palliation of pain. EXPERIMENTAL DESIGN: M170 is a mouse monoclonal antibody that targets adenocarcinomas. Patients with adequate renal and liver function, rising prostate-specific antigen, and androgen-independent metastatic prostate cancer were eligible. After estimation of dosimetry and pharmacokinetics with (111)In-2IT-BAD-m170, a single dose of (90)Y-2IT-BAD-m170 (0.185, 0.370, 0.555, or 0.740 GBq/m(2)) was administered to cohorts of three patients. Pain was assessed objectively by questionnaires before and for 8 weeks after RIT; weekly prostate-specific antigen levels were obtained for 2 months after RIT. RESULTS: The MTD of (90)Y-2IT-BAD-m170 was 0.740 GBq/m(2) for patients that had up to 10% of the axial skeleton involved with prostate cancer. Toxicity was almost exclusively confined to reversible myelosuppression. Metastatic prostate cancer was targeted by (111)In-2IT-BAD-m170 in all 17 patients. The mean radiation dose delivered to 39 bone and 18 nodal metastases by (90)Y-2IT-BAD-m170 was 10.5 Gy/GBq (range 2.8-25.1). Thirteen of 17 patients reported pain before (90)Y-2IT-BAD-m170; 7 of these 13 had a partial or complete resolution of pain that lasted an average of 4.3 weeks. CONCLUSIONS: This study determined the MTD of (111)In/(90)Y-2IT-BAD-m170 in patients with metastatic prostate cancer. The drugs were well tolerated, targeted metastases, and temporarily palliated pain.

Dosimetry-based therapy in metastatic breast cancer patients using 90Y monoclonal antibody 170H.82 with autologous stem cell support and cyclosporin A.

Radioimmunoconjugates of 170H.82 (m170), a panadenocarcinoma monoclonal antibody, are effective for imaging primary and metastatic breast cancer. To evaluate m170 as a targeting agent for therapy, we developed (111)In- and 90Y-2-iminothiolane-2-[p-(bromoacetamido)benzyl]-1,4,7,10 tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-m170 immunoconjugates with 99% purity by molecular sieving and immunoreactivity comparable to unmodified antibody. (111)In-m170 pharmacokinetic studies were performed prior to each therapy to determine the maximum dose of 90Y-m170 that could be administered without exceeding a limit of 800 rad to the liver, lungs, or kidneys or 250 rad to the whole body or bone marrow for each of three cycles of treatment. Peripheral blood stem cells (PBSCs) were harvested and cyclosporin A (5 mg/kg twice daily) was administered as strategies to ameliorate myelosuppression and prevent the development of HAMA, respectively. An (111)In imaging/pharmacokinetic study was performed, and the 90Y dose was calculated and administered. The liver was the 90Y dose-limiting organ. The mean and range of calculated doses (in rad/mCi) for the five patients evaluated were as follows: whole body, 2.3 (2.1-2.4); liver, 17.8 (12.7-22.2); lung, 6.4 (4.8-7.2); kidney, 6.9 (6.3-11.5); marrow, 3.6 (1.9-4.4); and tumors (n = 25), 71.5 (14.1-141.5). Of the three patients treated, with doses of 37, 54, and 57 mCi of 90Y, one had a partial response, one had measurable tumor reduction but less than a partial response, and one had stable disease for more than 1 month. PBSCs prevented prolonged myelosuppression. The therapeutic responses, coupled with an absence, thus far, of significant adverse sequelae, suggest that this dosimetry-based approach combined with PBSCs may lead to effective therapy when higher 90Y doses are reached.

Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen.

BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.

Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from rye-grass pollen that demonstrates specific antigenic immunoreactivity.

We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572]. Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix. There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence of the proteinase-inhibitor cystatin homologue from the pollen of Ambrosia artemisiifolia (short ragweed).

The nucleotide sequence of a cDNA (designated IPC1/5) encoding a cystatin (Cyt) proteinase-inhibitor homologue from short ragweed (Ambrosia artemisiifolia) pollen was determined and compared to other plant and animal Cyt. The absence of disulfide bonds in the predicted translation product of the IPC1/5 sequence suggests that it most resembles family-I members of the Cyt superfamily. Significant amino acid (aa) sequence identity was found when comparing the translated sequence of IPC1/5 to rice seed Cyt, human keratocyte Cyt A and human liver Cyt B.

Purification and immunochemical characterization of recombinant and native ragweed allergen Amb a II.

The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Rogers et al. (1991) J. Immun. 147, 2547-2552; Griffith et al. (1991a), Int. Archs Allergy appl. Immun. 96, 296-304]. In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti-Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb aI.1. These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract. These Amb a II isoforms have pI in the 5.5-5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5-5.2 range detected by an anti-Amb a I specific peptide antiserum. The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels. An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis. Recombinant Amb aI.1 and Amb a II have been expressed in E. coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.1. These data suggest that recombinant Amb a I.1 and Amb a II, isolated in a denatured form, differ significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.

Expression and genomic structure of the genes encoding FdI, the major allergen from the domestic cat.

The genes encoding chain 1 (Ch1) and chain 2 (Ch2) of the major allergen of the domestic cat, Felis domesticus I, have been analyzed by genomic cloning and by polymerase chain reaction (PCR). Ch1 has two potential leader sequences, designated A and B. Analysis of a genomic clone encoding Ch1 demonstrated that one structural gene contains sequences corresponding to both leaders, which utilize different Met start codons. PCR analysis showed that genes encoding Ch1 and Ch2 are co-expressed in both the salivary glands and the skin, and that leader sequence A of Ch1 is utilized preferentially in both tissues. Ch2 was shown to have two dominant forms that are differentially expressed in the aforementioned tissues. The long form (Ch2L), composed of 92 amino acids (aa), is preferentially expressed in the salivary glands, while the short form (Ch2S), composed of 90 aa, is preferentially expressed in the skin. There is minor sequence polymorphism in both forms of Ch2. A genomic clone for Ch2 only contained sequences for Ch2S, suggesting that Ch2L is encoded by an exon not contained within this genomic clone.

Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.

The complete primary structure of Fel dI (International Union of Immunological Societies nomenclature), the major allergen produced by the domestic cat, Felis domesticus, was determined by protein sequence analysis and cDNA cloning. Protein sequencing of Fel dI from an immunoaffinity-purified extract of house dust revealed that the allergen is composed of two polypeptide chains. Degenerate oligonucleotides derived from the protein sequence were used in polymerase chain reaction amplification of cat salivary gland cDNA to demonstrate that the two chains are encoded by different genes. Chain 1 of Fel dI shares amino acid homology with rabbit uteroglobin, while chain 2 is a glycoprotein with N-linked oligosaccharides.

Complete sequence of the allergen Amb alpha II. Recombinant expression and reactivity with T cells from ragweed allergic patients.

This study defines the complete primary structure of Amb alpha II, an important allergen produced by short ragweed (Ambrosia artemisiifolia). The deduced amino acid sequence derived from the cDNA indicates that Amb alpha II shares approximately 65% sequence identity with the Amb alpha I multigene family of allergens. Full-length cDNA encoding Amb alpha I.1 and Amb alpha II have been expressed in E. coli and purified. An in-frame linker encoding polyhistidine has been added to the 5' end of the cDNA to facilitate purification using Ni2+ ion affinity chromatography, yielding greater than 90% pure recombinant protein in a single step. T cells from patients allergic to ragweed proliferate in response to pollen extract as well as purified recombinant Amb alpha I.1 and Amb alpha II. T cell lines established using either Amb alpha I.1 or II as the stimulating Ag exhibit a high level of cross-reactivity to both proteins. This result is entirely consistent with the extensive primary sequence identity shared by these two proteins. These data suggest that allergic humans recognize shared T cell epitopes on these two related molecules.

Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

Wegener's granulomatosis in two siblings: a family study.

Two brothers with Wegener's granulomatosis are presented. HLA status and associated genes in the brothers and the immediate family are explored. No specific association could be found with tissue types within the family.

Sequence polymorphism of Amb a I and Amb a II, the major allergens in Ambrosia artemisiifolia (short ragweed).

Two of the major allergens in the pollen of short ragweed are Amb a I and Amb a II (formerly antigen E and antigen K, respectively). The genes for Amb a I and Amb a II have recently been cloned, and it was shown that Amb a I is a family of proteins with at least three distinct polymorphic family members. This study addresses the number of individual Amb a I and Amb a II family members, the polymorphism in each family member and the expression of these genes in ragweed plants from different geographical locations. This work led to the cloning and characterization of a fourth Amb a I family member, designated Amb a I.4.

Cloning of Amb a I (antigen E), the major allergen family of short ragweed pollen.

To determine the structure of Amb a I (previously called antigen E), the major allergen from short ragweed, cDNA from pollen was cloned into lambda gt11 and lambda gt10. One of the three distinct clones isolated from the lambda gt11 library by screening with anti-denatured Amb a I antibodies was used to screen both libraries for other Amb a I sequences. Multiple clones were isolated and sequenced and proved to be highly homologous but nonidentical. The clones could be divided into three groups based on sequence similarity, and in accordance with the International Union of Immunological Societies-approved nomenclature (Marsh, D. G., Goodfriend, L., King, T. P., Lowenstein, H., and Platts-Mills, T. A. E. (1986) Bull. WHO 64, 767-770) they have been designated Amb a I.1, Amb a I.2, and Amb a I.3. Clones within a group have greater than 99% identity, and similarity among groups is 85-90% at the nucleotide level. The amino acid sequence of four peptides (isolated from antigen E obtained from the Research Resources Branch of the National Institutes of Health) containing 132 amino acids was identical to one of the clones (Amb a I.1). The presence of multiple naturally occurring isoelectric forms of Amb a I was demonstrated by two-dimensional gel electrophoresis and Western blotting. Southern blot analysis demonstrates the presence of multiple Amb a I-related sequences in the ragweed genome. Amb a I is therefore not a single molecule but rather a family of closely related proteins.

Functional and molecular characterization of I-A kappa beta mutants is consistent with the predicted three dimensional structure of class II MHC molecules.

Class II MHC (Ia) molecules have been shown to be critical as restriction elements in the T helper/inducer cell recognition of antigen. Efforts to determine the role of allelic variation in MHC restricted antigen presentation have included the use of serologically selected mutants to correlate structural variations in Class II molecules with changes in the antigen presenting function of Ia bearing cells. Such studies have revealed that serologically selected mutations tend to occur in a single immunodominant region and that even a single amino acid substitution can alter T cell recognition of Ia molecules. We report here the characterization of two more serologically selected Class II A beta chain mutations. Each is due to a single base change which alters a single amino acid. One of these mutations is in the third hypervariable region (amino acid 64--glutamine to arginine) and alters the antigen presenting function. The second mutation at amino acid 48, though a relatively non-conservative change (arginine to cysteine), has no effect on APC phenotype. Such a result would be predicted based on comparisons made with the proposed three dimensional crystallographic structure of Class I molecules and models proposed for Class II molecules based on Class I structure. The amino acid change at position 48 is in a portion of the molecule that is most likely unavailable to bind antigen or interact with T cell receptor whereas the mutation at amino acid 64 is on an exposed face of the alpha helix, a region which could affect interaction with either antigen and/or the T cell receptor.

Titanium grommets: a trial to assess function and extrusion rates.

Titanium grommets have been promoted as having the advantage of a slower extrusion rate than other types of ventilation tube. A prospective trial was therefore designed to compare the function and extrusion rates of these grommets with those of the widely used Shepard design of Teflon grommet in a single group of patients. Thirty-one children had one type of grommet inserted in one ear and the other type in the opposite ear. After eight months there were significantly more Titanium grommets still functioning (p less than 0.05) but after 12 and after 16 months there was no significant difference in the extrusion rates of the two types of grommet. There was a higher incidence of infection with granulation tissue formation around the Titanium grommet. Accordingly it is concluded that the extra expense of the Titanium grommet is not justified, particularly as the long-term effects of these grommets on the tympanic membrane are not known.

An intronic 10-base-pair deletion in a class II A beta gene affects RNA processing.

Several biologically important examples of posttranscriptionally regulated genes have recently been described (T. Gerster, D. Picard, and W. Schaffner, Cell 45:45-52, 1986; R. Reeves, T.S. Elton, M.S. Nissen, D. Lehn, and K.R. Johnson, Proc. Natl. Acad. Sci. USA 84:6531-6535, 1987; H.A. Young, L. Varesio, and P. Hwu, Mol. Cell. Biol. 6:2253-2256, 1986). Little is known, however, regarding sequences that mediate posttranscriptional RNA stability. Characterization in our laboratory of a mutant murine B lymphoma, M12.C3, revealed a posttranscriptional defect affecting the synthesis of a major histocompatibility complex class II gene (A beta d) whose product normally controls both the specificity and magnitude of the immune response. Molecular studies revealed that the mutation responsible for diminished A beta d gene expression was an intronic deletion of 10 base pairs (bp) located 99 bp 5' of the third exon. This deletion lies in a region not known to be critical for accurate and efficient splicing. Furthermore, sequence analysis of amplified A beta-specific cDNA demonstrated that the small number of A beta d transcripts produced in the mutant cells was correctly spliced. It appears that the mechanism by which this intronic 10-bp deletion acts to decrease RNA stability is unlikely to be at the level of RNA splicing.

Antigen presentation abrogated in cells expressing truncated Ia molecules.

Oligonucleotide site-directed mutagenesis was used to introduce a premature stop codon in wildtype A beta k and A alpha k cDNA clones to create truncated A beta k and A alpha k molecules lacking the cytoplasmic domain. Transfected B lymphoma cells expressing an I-Ak molecule with a truncated beta-chain or with truncated alpha- and beta-chains showed profound defects in two Ia-related functions: Ia-restricted Ag presentation and intracytoplasmic signaling. The ability of these transfected cell lines to activate autoreactive T hybrids was markedly impaired whereas loss of Ag presentation to nominal Ag-specific T hybrids was more subtle. Ia-mediated transmembrane signaling as measured by PKC translocation from cytosol to nucleus after stimulation with anti-Ak antibody was greatly affected by truncation of the A beta and A alpha cytoplasmic domains. These results indicate an important role for the highly conserved cytoplasmic domain in Ia-mediated responses.