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A A Killeen

Publications and source records attributed to A A Killeen.

18 recordsLinked to original sources
PubMed1997

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a rapid screening method to detect mutations causing Tay-Sachs disease.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay-Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster.

Base Sequence↗
PubMed1997

Quantification of nucleic acids.

Quantification of nucleic acids is an area of growing importance in the clinical laboratory. In addition to routine measurements of total nucleic acid concentrations, quantitative techniques are used to determine the number of microorganisms in samples, the level of expression of genes, or the presence of alterations in gene dosage. This article provides an overview of current methodologies for measurement of total and specific nucleic acid concentrations.

DNA↗
PubMed1995

Sequence analysis of the hepatitis C virus (HCV) core gene suggests the core protein as an appropriate target for HCV vaccine strategies.

Hepatitis C virus (HCV) is a major healthproblem with a prevalence of 1% in the United States population, and a significant percentage of infected patients progress to chronic liver disease and cirrhosis. Interferon therapy has demonstrated that the immune system can be modulated to alter the acute course of the disease, but long-term treatments remain elusive. Prevention of hepatitis C infection is therefore an important strategy to mitigate the impact of this disease. Initial attempts at vaccination have focused on recombinant envelope vaccines, which have shown an ability to protect against very low titre challenges of HCV in chimps. The need for vaccines capable of protecting against higher titre challenges has led to the search for alternative vaccine strategies. The most highly conserved structural protein in the HCV genome is the core protein, and vaccine strategies targeting the core protein have been proposed to increase vaccine efficacy. The variability of HCV core sequences and genotypes in the Ann Arbor patient population are not known, and the present study was undertaken to assess the theoretical feasibility of developing a HCV core vaccine by excluding promiscuous core (C) gene variability as a mechanism of vaccine failure. Results of nucleotide and deduced amino acid sequence analysis from 13 of 14 patients studied reveal a 93% nucleotide and 96.4% amino acid core sequence homology in the C gene regions studied. Genotype analysis revealed four of 14 to be type 1a and nine of 14 to be type 1b with one infection not being sufficiently characterized to determine genotype. These results demonstrate a sufficiently high degree of conservation of HCV core sequences in our patient population to permit design of a vaccine directed against core protein.

Amino Acid Sequence↗
PubMed1994

Quantitative analysis in molecular diagnostics.

Quantitative analysis of DNA products derived from polymerase chain reaction (PCR)-based assays depends on the careful optimization of each of the reaction parameters to achieve highly efficient amplification of target sequences. In practice, however, measurement of the accumulated PCR product is reliable only when analyses are performed at points in the exponential phase of the PCR amplification curve and before the onset of the plateau phase. The recent development of more sensitive DNA product detection systems has permitted the analysis of PCR assays after fewer amplification cycles, where the accumulation of product approaches linearity, while at the same time maintaining superior assay specificity. These methods include the use of high performance liquid chromatography, automated fluorescence detection, electrochemiluminescence, and the ligase chain reaction. Clinical applications of these methods are numerous and include diagnostic testing as well as therapeutic monitoring for neoplastic, infectious, and inherited genetic disease.

Biomarkers, Tumor↗
PubMed1994

Carbamazepine measurement in samples from the emergency room.

The measurement of carbamazepine (CBZ) in samples from the emergency room (ER) raises several issues for drug monitoring. First, the ER frequently requires rapid turnaround time for clinical samples; this need may be more conveniently met by automated immunoassays than by high-performance liquid chromatography (HPLC), the other major analytical technique for measurement of carbamazepine. On the other hand, immunoassays often do not completely measure the pharmacologically active carbamazepine epoxide metabolite and therefore may not indicate the full extent of serum anticonvulsant activity. Second, patients may be admitted to the ER specifically because of seizure activity, which may be an indication of under- or overmedication with carbamazepine and which, if due in large part to high levels of the epoxide metabolite, may not be fully assessed by immunoassay. We examined the results of carbamazepine determination in 102 consecutive samples sent from an ER. Each sample was analyzed by a fluorescence polarization immunoassay (FPIA) and by HPLC. There were good correlations between the FPIA and the HPLC for the parent drug and for the sum of the parent drug plus metabolite (carbamazepine-10,11-epoxide, CBZ-E) with these regression equations: FPIA = 1.13 (HPLC CBZ) + 0.09 (r2 = 0.93) and FPIA = 0.93 (HPLC CBZ+CBZ-E)-0.55 (r2 = 0.89), respectively. There were weak correlations between the FPIA and the epoxide and between the parent drug and the epoxide. Based on the FPIA and HPLC results, we classified each value relative to the therapeutic range, i.e., supratherapeutic, subtherapeutic, or therapeutic.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Catalase, superoxide dismutase and glutathione peroxidase activity in neutrophils of sham-operated and olfactory-bulbectomised rats following chronic treatment with desipramine and lithium chloride.

In the olfactory-bulbectomised rat model of depression, neutrophil phagocytosis was significantly decreased and phagocytosis started later in comparison to sham-operated animals. Both desipramine and lithium chloride treatment significantly reversed the depressed neutrophil phagocytosis and shortened the time to commencement of phagocytosis in drug-treated bulbectomised rats. The catalase and glutathione peroxidase (GSH-PX) activities in bulbectomised rats were decreased, while superoxide dismutase (SOD) was significantly increased. Chronic desipramine and lithium chloride treatment slightly improved catalase activity in the bulbectomised rats. Desipramine significantly reversed the reduction in activity of GSH-PX, but failed to reverse the increased activity of SOD. In contrast, lithium chloride significantly reversed SOD activity to normal values, without affecting GSH-PX activity in the bulbectomised rats.

Animals↗
PubMed1993

Extended major histocompatibility complex haplotypes in celiac patients in the west of Ireland.

The highest reported prevalence of celiac disease (gluten-sensitive enteropathy) is found in the West of Ireland. Recent genetic data have suggested that major histocompatibility complex-linked loci may have a dominant genetic effect for disease susceptibility in this population compared with a recessive effect in other groups. To further understand the role of the MHC in celiac disease in the West of Ireland, we analyzed markers for 22 MHC haplotypes from celiac patients and compared them with 18 nontransmitted haplotypes found in the parents of celiac children, and with reported haplotypes from other populations. An extended MHC haplotype including [HLA-B8, DR3, DQw2, Bf*S, C4A*Q0, and C4B*1] accounted for 50% of celiac haplotypes but only 27% of nontransmitted parental haplotypes. Compared with other reported haplotypes in celiacs, patients from the West of Ireland show a higher prevalence of HLA-A1 as a component of this extended haplotype, suggesting that although the core haplotype is similar between Irish patients and others, the celiac population in the West of Ireland differs at other HLA loci. We did not observe any other common haplotypes among our patients unlike the situation in other populations. These differences may underlie the possible dominant effect of HLA-linked loci and the unusually high prevalence of celiac disease in the Irish population. We also found that the serum levels of complement components C3c, C4, and factor B were significantly lower among celiac patients than nonceliacs. The lower serum level of C4 appears to be related to the presence of deletions and null alleles at the C4A and C4B loci in celiacs.

Adult↗
PubMed1992

Prevalence of nonclassical congenital adrenal hyperplasia among women self-referred for electrolytic treatment of hirsutism.

Nonclassical congenital adrenal hyperplasia (NCCAH) is well recognized among women who seek medical attention for hirsutism. However, the prevalence of this disorder among women self-referred for electrolytic treatment of hirsutism is unknown. We hypothesized that the prevalence of NCCAH among women attending an electrolysis clinic might be high. By measuring the morning salivary 17-hydroxyprogesterone (17-OHP) as a screening test for NCCAH in 46 women in the follicular phase of their menstrual cycle, we identified 12 subjects with a high basal salivary 17-OHP. Eleven agreed to have a 60-minute Cosyntropin-stimulation test, as did an additional 6 of 9 women with normal basal salivary 17-OHP, but with a particularly high hirsutism score. One of the women with high basal salivary 17-OHP had a 60-minute Cosyntropin response, which was diagnostic of NCCAH. She was of the Ashkenazi Jewish decent, a group previously reported to have a high prevalence of NCCAH. A second woman with high salivary 17-OPH had a Cosyntropin-stimulation response consistent with heterozygosity for 21-hydroxylase deficiency. None of the Cosyntropin-stimulation responses in those chosen for a high hirsutism score were diagnostic. Thus, 1 of 46 (2.2%) of the women who entered our study had unrecognized NCCAH, a prevalence only about 2-fold greater than that reported in the general population. Therefore, we recommend that electrolysis clinics advise clients from ethnic groups known to have a high frequency of NCCAH of the advisability of having a formal medical evaluation for NCCAH.

17-alpha-Hydroxyprogesterone↗
PubMed1991

Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene.

The gene encoding steroid 21-hydroxylase activity, P450c21B, is located in the major histocompatibility complex (MHC) class III region, in close proximity to a highly homologous pseudogene, P450c21A. Recombinations between P450c21B and P450c21A have been shown to result in deficiency of 21-hydroxylase activity, the usual cause of congenital adrenal hyperplasia (CAH). A mutant P450c21 gene from a patient with simple virilizing CAH was identified and shown to be consistent with a recombination between P450c21A and P450c21B. Sequence analysis of the mutant gene showed the recombination site to be located between the first exon and the second intron. The mutant gene encodes a leucine instead of the normal proline at codon 31. This mutation resides on a chromosome bearing the HLA-B44 serotype. A comparison of mutations associated with HLA-B44 and that normally found with the HLA-Bw47 serotype suggests that the HLA-B44 mutations are of more ancient origin. The patient's homologous chromosome has a deletion of P450c21B. Endocrinological testing therefore allows for testing of the mutant gene in genetic isolation. Such testing demonstrated that the patient was capable of producing aldosterone and retaining sodium in response to a low-sodium diet, indicating that the mutant gene encodes an enzyme with partial 21-hydroxylase activity.

Adrenal Hyperplasia, Congenital↗
PubMed1989

Serum 5'nucleotidase and alkaline phosphatase--highly predictive liver function tests for the diagnosis of graft-versus-host disease in bone marrow transplant recipients.

The diagnostic efficacy of five serum liver function tests (aspartate and alanine aminotransferase, alkaline phosphatase, 5' nucleotidase, and bilirubin) was investigated in 95 bone marrow transplant recipients in whom acute graft-vs-host disease was graded by the Seattle criteria. The patient population included a control group of 22 autologous transplant recipients (group I), 33 patients with no GVHD (group II), 21 patients with grades 1 and 2 GVHD (group III), 12 patients with grade 3 GVHD (group IV), and 7 patients with grade 4 GVHD (group V). Student t test analysis of the analytes among the five groups of patients showed that 5' nucleotidase and alkaline phosphatase were the best discriminants among all the possible combinations of group pairs. Peak levels of 5' nucleotidase within each group of patients correlated well with those of alkaline phosphatase in all the allogeneic transplant groups (II-V; r = 0.59), but the correlation of these with bilirubin was less frequent. Also, 5' nucleotidase and alkaline phosphatase showed significant discrimination (P less than 0.05) even between groups I and II, suggesting that they are more sensitive than the Seattle criteria in the diagnosis of GVHD. They also showed the best overall discriminatory ability by one-factor analysis of variance (ANOVA; P = 0.0001 as compared with 0.002, 0.009, and 0.04 for aspartate aminotransferase, alanine aminotransferase, and bilirubin, respectively). Receiver-operating curves of the five analytes again revealed that 5' nucleotidase and alkaline phosphatase were by far the best discriminators among the five groups of patients. Bilirubin was relatively insensitive because it was a good discriminator only between the control group and groups IV and V. The hepatocellular enzymes, alanine and aspartate aminotransferase, correlated well (r = 0.80) but discriminated poorly among the four groups of allogeneic transplant recipients (II-V), suggesting that all four groups had some measure of hepatocellular damage that was independent of the severity of GVHD.

5'-Nucleotidase↗
PubMed1988

Diagnosis of classical steroid 21-hydroxylase deficiency using an HLA-B locus-specific DNA-probe.

The HLA-B and steroid 21-hydroxylase loci are known to be closely linked. Restriction fragment length polymorphisms seen after digestion of genomic DNA with MspI and TaqI with the HLA-B locus-specific DNA-probe, pHLA-1.1, were examined in 7 nuclear families with classical steroid 21-hydroxylase deficiency. In each family 2 polymorphic hybridizing bands (corresponding to the 2 HLA-B genes) were seen. In all families, TaqI-generated polymorphisms allowed for identification of children previously shown on clinical and biochemical criteria to be affected by 21-hydroxylase deficiency from their unaffected sibs. The results were in complete agreement with the clinical diagnoses. Among the unaffected children, carriers could be distinguished from non-carriers in all cases by TaqI polymorphisms. MspI-generated polymorphisms allowed for full identification of genotypes in 5 families. In one family, MspI-generated polymorphisms could be used to identify affected from unaffected children, but could not distinguish between carriers and non-carriers. In another family, no identification of genotypes was possible by MspI-generated polymorphisms alone. The HLA-B locus-specific DNA-probe, pHLA-1.1, can be used for diagnosis and genotyping of individuals from families with 21-hydroxylase deficiency. This technique can be used as an alternative to HLA-serotyping, or in situations where HLA-serotyping is technically difficult, for example in chorionic villus samples.

Adrenal Hyperplasia, Congenital↗
PubMed1988

Conjugated hyperbilirubinemia and renal function in bone marrow transplant recipients.

Although it has long been recognized that hyperbilirubinemia is enhanced in patients with impaired renal function, a clear correlation between the levels of bilirubin and creatinine in serum has never been established. We have studied this relationship in 13 bone marrow transplant recipients who had episodes of combined conjugated hyperbilirubinemia and renal impairment. Most of the patients had graft-vs-host disease and various degrees of hepatic cholestasis as evidenced by histologic examination results and abnormal liver function tests. Serial serum specimens obtained during this period showed a good correlation between the two analytes, with a mean (+/- SD) correlation coefficient of .86 +/- .10. The slopes of the regression lines, represented as bilirubin-creatinine ratios, varied widely (0.2 to 3.6), reflecting the wide variation in the degree of hyperbilirubinemia attributable to renal impairment.

Adolescent↗
PubMed1987

Kallikrein inhibition and C1-esterase inhibitor levels in patients with the lupus inhibitor.

It has been suggested that kallikrein inhibition may predispose patients with the lupus inhibitor to thrombosis by interfering with the Factor XII-mediated activation of plasminogen. To further investigate this suggestion, the authors measured kallikrein inhibition in 19 patients with the lupus inhibitor. They found that kallikrein inhibition was greater than 100% of that of a normal plasma pool in all patients and greater than 125% in 11 of 19. Kallikrein inhibition was significantly correlated with C1-esterase inhibitor (C1S-INH) concentration, which they measured by rocket immunoelectrophoresis (r = +0.55, P less than 0.05). In three patients the C1S-INH was more than 30% greater than the kallikrein inhibition. Crossed immunoelectrophoresis for C1S-INH in these patients' plasma revealed an electrophoretic mobility identical with that of the normal plasma pool. The authors suggest that C1S-INH-mediated kallikrein inhibition, in conjunction with other coagulation abnormalities, predisposes patients with the lupus inhibitor to thrombosis.

Adolescent↗
PubMed1987

Fetal supraventricular tachycardia treated with high-dose quinidine: toxicity associated with marked elevation of the metabolite, 3(S)-3-hydroxyquinidine.

We observed a patient at 33 weeks' gestation who was administered exceptionally large doses of quinidine to treat a fetal supraventricular tachycardia. The patient had clinical evidence of quinidine toxicity at low to mid-therapeutic levels of quinidine, but markedly elevated levels of the metabolite 3(S)-3-hydroxyquinidine (3-hydroxyquinidine). This pattern is consistent with a "fast metabolism" of quinidine. There is substantial evidence that 3-hydroxyquinidine has pharmacologic activity. We conclude that elevated levels of 3-hydroxyquinidine can be associated with a clinically significant quinidine toxicity. This can occur in the presence of a nontoxic serum quinidine level. We also measured the levels of quinidine and 3-hydroxyquinidine in the amniotic fluid and cord blood. The levels in the cord blood were 31 and 27%, respectively, of those in a simultaneously drawn maternal blood sample.

Adult↗