Automated detection of single nucleotide polymorphism in beta-2 adrenergic receptor gene using LCx(R).

Beta-2 adrenergic receptor (B2AR) agonists are the most widely prescribed rescue agents used in the treatment of asthma. Recent studies have indicated a relationship between a polymorphism at codon 16 of the B2AR gene, and the response to recurrent beta-agonist therapy. The B2AR polymorphism of interest involves a single nucleotide change from A to G, resulting in an amino acid change from Arginine (Arg) to Glycine (Gly). Clinical efforts to further investigate this relationship require an accurate, reliable and inexpensive method for detecting the polymorphism. In this study, we report an LCx(R) assay for the detection of a single nucleotide polymorphism at codon 16 of the beta-2 adrenergic receptor. This assay is capable of detecting patients harboring any of the three possible genotypes at this locus, namely, homozygous wild type, homozygous variant or heterozygous individuals with a single genomic DNA sample of 25-500 ng. It requires minimum hands-on time with automated detection. The assay would be suitable for use in research labs for screening of a large number of samples. We believe that this type of assay will facilitate research and clinical investigations in elucidating the association of SNPs with disease states, diagnosis, prognosis and treatment.

Automated detection of the factor V Leiden mutation using the LCx microparticle enzyme immunoassay.

The factor V Leiden mutation, a G-->A transition at position 1691 in exon 10 of the gene that codes for factor V, produces an Arg506Gln substitution and is the most common genetic risk factor for venous thrombosis. We have developed a rapid, sensitive, and specific method to detect the factor V Leiden mutation in genomic DNA from whole blood by PCR amplification and microparticle enzyme immunoassay detection using the Abbott LCx instrument. We compared this automated method with the standard procedure using restriction endonuclease digestion of PCR products followed by gel electrophoresis in blinded experiments. In 130 patients (from Veterans Affairs medical centers) with deep venous thromboses, including 24 heterozygotes with the factor V Leiden mutation, there was complete agreement between the two methods. The assay was also able to distinguish heterozygotes from homozygotes. This method, which carries a low potential for cross-contamination of samples, should be a useful routine test for the factor V Leiden mutation in clinical laboratories with sufficient demand for molecular diagnostic assays using the LCx instrument.

Acute jaundice in Vientiane, Lao People's Democratic Republic.

Analysis of serum samples from patients with acute jaundice by means of enzyme-linked immunosorbent assay and polymerase chain reaction testing provided the first profile of this condition in Vientiane, Lao PDR, in 1995 and 1996. In a case-control, hospital-based study, evidence of acute infections due to hepatitis A and B viruses was found in 14% and 10% of cases, respectively. Hepatitis E virus, however, did not appear to contribute to clinically recognized acute jaundice. Similarly, antibody to hepatitis C virus was recognized in almost equal proportions of cases (8%) and controls (6%), thus representing probable background infections. The detection of hepatitis G virus marks the first report of this virus in Lao PDR. The large proportion (21%) of new leptospiral infections in cases without acute hepatitis A or B was notable. This finding suggests significant regional underreporting of leptospirosis as a cause of acute jaundice. The limited laboratory diagnostic capabilities for confirming a differential diagnosis of leptospirosis contribute to the lack of attention paid to this important health problem.

Detection of GB virus C by the RT-PCR LCx system.

The recent publication of representative genomic sequences of GBV-C has permitted the selection of PCR primers for detection of GBV-C in clinical samples by PCR techniques. Traditional amplification methodologies which couple reverse transcription polymerase chain reaction (RT-PCR) and Southern blot detection are slow, cumbersome, and can be technique dependent. This has hampered studies to determine the clinical significance of GBV-C. We report the selection of highly conserved PCR primers and a probe useful for semi-automated RT-PCR using the Abbott LCx system. This adaptation of the LCx system expands its capabilities to include the detection of RNA by RT-PCR, in addition to DNA detection by ligase chain reaction (LCR).

Antibody to GBV-C second envelope glycoprotein (anti-GBV-C E2): is it a marker for immunity?

The clinical significance of GB virus C (GBV-C E2) antibody is under investigation. The prevalence rates of GBV-C RNA and antibody to GBV-C E2 glycoprotein were determined in a population of 123 Egyptian anti-hepatitis C virus (HCV)-positive patients with chronic liver disease (CLD) who had not been treated previously with interferon. Sera were tested for GBV-C RNA by the LCx assay (Abbott Laboratories, North Chicago, IL), and for GBV-C E2 antibody by enzyme immunoassay. GBV-C RNA was present in 11.4% of patients. GBV-C E2 antibody was detected in 55.9% of GBV-C RNA-negative patients and in 2.2% of GBV-C RNA-positive patients (P = 0.006). GBV-C RNA was associated significantly with a history of schistosomiasis (relative risk [RR] = 5.83, 95% confidence interval [CI] 1.99-17.14, P < 0.005) but not with parenteral risk factors. The presence of GBV-C E2 antibody was not associated with age, gender, parenteral risk factors, schistosomal infection, or HCV viremia. The HCV genotype and level of viremia were similar in GBV-C anti-E2-positive and negative patients. There was a trend toward more severe histological disease with anti-E2 seropositivity (RR = 1.45, 95% CI 0.89-2.45, P = 0.11), an association which was independent of the evidence of schistosomiasis. It is concluded that GBV-C infection is common among HCV-infected Egyptian patients with CLD due to HCV infection. A significant negative correlation between the GBV-C viremia and GBV-C E2 antibody suggests that an antibody response is associated with viral clearance. This antibody response presumably occurs spontaneously, as none of the patients had received antiviral therapy. The unexpected association between GBV-C RNA and schistosomiasis suggests that nonparenteral or occult parenteral routes of GBV-C infection are likely to be important.

Comparative hemodynamic effects of OPC-18790 and dobutamine in patients with advanced heart failure.

OPC-18790 (Otsuka America Pharmaceutical, Rockville, MD), a novel positive inotropic agent, produces titratable hemodynamic benefits in patients with advanced heart failure. In such patients, OPC-18790 has been shown to acutely increase the cardiac index, while reducing systemic vascular resistance and left ventricular filling pressure, without an associated increase in heart rate. This study was performed to compare the acute hemodynamic effects of OPC-18790 and the beta-adrenergic receptor agonist, dobutamine, in patients with advanced heart failure. OPC-18790 and dobutamine were compared on successive days in 13 patients with worsening New York Heart Association class III or IV heart failure. The mean (+/- SEM) left ventricular ejection fraction was 15 +/- 2% (range, 6-29%). Pretreatment hemodynamics were: heart rate, 96 +/- 2 beats/min; mean arterial pressure, 77 +/- 3 mmHg; cardiac index, 1.80 +/- 0.10 L/min/m2; pulmonary capillary wedge pressure, 27 +/- 1 mmHg; mean pulmonary arterial pressure, 41 +/- 2 mmHg; and systemic vascular resistance, 1,732 +/- 152 dynes.s/cm5. At infusion rates yielding comparable increases in the cardiac index (5 micrograms/kg/min for 2 hours for each drug), OPC-18790 produced significantly more favorable effects on heart rate (-2 +/- 3% vs 11 +/- 4%; P = .01), pulmonary capillary wedge pressure (-32 +/- 4% vs -17 +/- 8%; P = .04), mean pulmonary arterial pressure (-14 +/- 3% vs 6 +/- 11%; P = .06), stroke volume index (48 +/- 8% vs 29 +/- 7%; P = .02), stroke work index (70 +/- 11 vs 42% +/- 12%; P = .03), and rate pressure product (2 +/- 4% vs 14 +/- 4%; P = .05). The hemodynamic profile for OPC-18790 differs from dobutamine, with OPC-18790 exhibiting no increase in heart rate, greater preload reduction, and an increase in cardiac performance at a lower estimated metabolic cost.

HTLV-III serology in hemophilia: relationship with immunologic abnormalities.

We investigated the relationship of the presence of antibodies to HTLV-III and immunologic abnormalities in patients with hemophilia. Serum antibodies to HTLV-III were analyzed by ELISA assay, immunoprecipitation of labeled cell extracts, and immunoprecipitation of purified HTLV-III p24. Thirty-four (61%) of the total group (n = 56) had antibody to HTLV-III; 34 (76%) of 45 patients given commercial factor VIII preparations were seropositive, compared with none of 11 patients treated exclusively with cryoprecipitate obtained from volunteer blood donors. Of patients who were seropositive for HTLV-III antibody, 94% had abnormal T4/T8 ratios, and 33% of those whose serum was antibody negative had abnormal T4/T8 ratios; five patients, each antibody positive, have lymphadenopathy syndrome. Sequential studies in a subset of patients indicate that there is a changing pattern of antibody production to HTLV-III antigens after seroconversion.

Primed lymphocyte testing specificity of alloreactive lymphocyte clones for HLA-B locus determinants.

We have generated two alloreactive T-lymphocyte clones that recognize the products of the HLA-B locus. The primed lymphocyte testing (PLT) reactivity of clone HJ21 was monospecific for the HLA-B antigen Bw49, a subtype specificity of HLA-Bw21. The PLT specificity of clone RD3 was strongly associated with HLA-Bw21. Several monoclonal antibodies against class I HLA molecules inhibited the PLT responses of these clones whereas class II monoclonal antibodies had no effect. These observations provide experimental evidence that class I products of the HLA-B locus may directly be involved in lymphocyte alloactivation.

Detection of non-HLA-DR determinants by alloreactive lymphocyte clones.

Using the soft-agar colony assay, we have generated three MT3-associated clones: HJ1, HJ13, and HJ39, from an MLR combination of two unrelated individuals. Another clone, HJ37, appeared to recognize a novel HLA-D determinant. PLT inhibition studies with monoclonal anti-Ia-like antibodies (Mab) were conducted on clones HJ1, HJ39, and HJ37. Five different anti-DR Mab had no significant inhibitory effect on these clones. On the other hand, two Mab SG171 and Q5/13 which appear to react with DR and MT3 (I-A like) molecules strongly inhibited the two MT3-specific PLT clones. While SG171 and Q5/13 had little effect on HJ37, it was observed that a polymorphic Mab 17.15 had a strong inhibitory effect. These results, in concordance with biochemical data on Ia molecules precipitated by these Mab, suggest that these alloreactive clones may recognize non-DR PLT determinants. They also provide further indirect support that MT3 molecules represent the human homologue of murine I-A molecules.

Detection of HLA-DR associated PLT determinants by alloreactive lymphocyte clones.

This study deals with alloreactive T-cell clones which recognize cellular determinants associated with HLA-DR antigens. Two clones, CB55 and DS56, exhibited a PLT specificity that was perfectly associated with DR5. On the other hand, clones CB7, DS1 and HS1 showed PLT reactivity with approximately one-half of the DR5 positive cells and none of the DR5 negative cells, whereas clone MD4 largely reacted with the other half of DR5 positive cells. Another MLR culture generated two alloreactive clones DS6 and DS9 with PLT specificity for DR2. However, these clones did not respond to DR2 cells, which were also positive for the DR2-associated HLA-B7 and B18 antigens. Monoclonal antibody (mAb) inhibition studies showed heterogenous patterns, whereby monomorphic non-DR mAbs inhibited the DR2-associated PLT clones while the DR5-associated PLT clones were inhibited by different groups of anti-DR and non-DR mAbs. These observations suggest the existence of several lymphocyte-activating determinants associated with HLA-DR antigens. This diversity may be an important consideration in studies of the role of HLA-DR in immune mechanisms and transplant compatibility.

Association of PLT specificity of alloreactive lymphocyte clones with HLA-DR, MB and MT determinants.

The HLA-D region encodes for several serologically defined systems, including DR, MB, and MT. The antigens of MB and MT are strongly associated with two or more DR specificities. The purpose of this study was to determine the role of MB and MT antigens in lymphocyte alloactivation. A soft agar colony assay was used to generate alloreactive lymphocyte clones primed in mixed leukocyte culture against a stimulator who typed as HLA-DR4,-;MB3,-;MT3,-. In secondary primed lymphocyte typing (PLT) assays, several clones were identified with PLT specificities strongly associated with DR4, MB3, or MT3. The data suggest that HLA-D controls different lymphocyte-activating determinants associated with the serologically defined DR, MB, or MT antigens.

Molecular basis for the temperature-dependent insolubility of cryoglobulins. X. The amino acid sequence of the heavy chain variable region of McE.

The amino acid sequence of the VH region of McE, a monoclonal IgM cryoimmunoglobulin, has been determined employing automated sequencing methodology. Digestion of the intact Fab mu component, derived by trypsin cleavage of the parent protein at elevated temperature with CNBr, followed by complete reduction and alkylation, yielded the intact light chain as well as the 2 CNBr fragments that constituted the VH. N-terminal sequencing of the larger unblocked CNBr fragment, along with a component fragment derived by cleavage by BNPS-Skatole, established the structure of the VH from position 88 through the V leads to C switch region. Citraconylation of the smaller, blocked fragment effected sufficient solubilization for enzymatic deblocking and direct sequencing of the N-terminal 20 residues of the VH. Complete trypsin digestion of the N-terminal CNBr fragment yielded 9 peptides that could be isolated by preparative cation exchange chromatography and gel filtration. The complete sequence of these peptides along with 4 chymotryptic peptides completed the primary structure of the VH region. The primary structure of McE appears to resemble that of He, previously identified as belonging to the VH II subgroup. The presence of characteristic CDR and FR regions as well as the identification of a probable site of glycosylation suggest that the cryoimmunoglobulin closely resembles noncryoglobulins in terms of overall structural composition. The cryoglobulin property may arise through alterations in individual residues or unfavorable arrangements of CDR and FR segments.

Molecular basis for the temperature-dependent insolubility of cryoglobulins. XII. Anomalous mobility of monoclonal cryoimmunoglobulin heavy chains accompanying polyacrylamide gel electrophoresis in sodium dodecyl sulfate.

When subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), the fully reduced and alkylated heavy chains isolated from three monoclonal IgG1 cryoimmunoglobulins exhibited various degrees of retardation in mobility when compared to noncryoglobulin references. The anomalous electrophoretic mobility was not correlated with the thermal magnitude of cryoprecipitation of the individual proteins. High sensitivity analytical gel filtration in 5 M guanidine-HCl failed to distinguish heavy chains of the cryoimmunoglobulins from noncryoglobulin references, suggesting that the proteins possess equivalent molecular weights. Other possible causes for the anomalous mobility such as atypical amino acid and carbohydrate composition, charge and quantitative SDS binding do not appear to be likely. It remains possible that the shape and/or charge of the SDS-protein complexes are unique. Examination of the gel electrophoretic mobility in SDS of fully reduced and alkylated Fab components suggests that the Fd portion of these proteins may be abnormal. The gel electrophoresis anomaly is the only atypical structural feature thus detected which is shared by these three monoclonal cryoimmunoglobulins.

Immunoglobulin diversity in the phylogenetically primitive shark, Heterodontus francisci. Suggested lack of structural variation between light chains isolated from different animals.

A two-step procedure employing gel filtration and anion exchange chromatography has been utilized to isolate LMW immunoglobulin from the horned shark, Heterodontus francisci. Light chains obtained by complete reduction and alkylation of the parent protein have been compared by several analytical techniques. Amino acid composition data implies a limited degree of variation in the light chains isolated from individual animals. Polyacrylamide gel electrophoresis of the CNBr digests of the light chains reveal indistinguishable banding profiles of the major peptides. Isoelectric focusing indicates limited heterogeneity in the light chain spectrotype and identity in the pI of the majority of bands detectable by staining. The suggested degree of structural similarity in the light chains of this phylogenetically primitive shark is discussed in terms of the evolutionary position of the species and current theories concerning the origins of structural diversity in immunoglobulins.

Physicochemical characterization of six monoclonal cryoimmunoglobulins: possible basis for cold-dependent insolubility.

The physical and chemical properties of five human and one canine monoclonal cryoimmunoglobulin have been compared. By many criteria, the proteins cannot be distinguished from the noncryoglobulin reference proteins analyzed in parallel; however, certain hydrodynamic and spectroscopic properties of the proteins indicate that cryoimmunoglobulins differ in tertiary structure relative to their cold-soluble counterparts. These differences seem to favor low-temperature-induced association between cryoglobulin molecules as an immediate consequence of increased intermolecular ionic or van der Waals forces. No evidence was found for the formation of cold-dependent antigen-antibody complexes or the ubiquitous presence of low-temperature-dependent conformation changes as a component of cryoprecipitation. Rather, the anomalous solution behavior of monoclonal cryoimmunoglobulins can be considered a direct result of the individual solubility properties of these proteins.