Attributes of gammadelta intraepithelial lymphocytes as suggested by their transcriptional profile.

gammadelta T lymphocytes in the intestinal intraepithelial layer (gammadelta IELs) are thought to contribute to immune competence, but their actual function remains poorly understood. Here we used DNA microarrays to study the gene expression profile of gammadelta IELs in a Yersinia infection system to better define their roles. To validate this approach, mesenteric lymph node CD8(+) alphabeta T cells were similarly analyzed. The transcription profiles show that, whereas lymph node CD8(+) alphabeta T cells must be activated to become cytotoxic effectors, gammadelta IELs are constitutively activated and appear to use different signaling cascades. Our data suggest that gammadelta IELs may respond efficiently to a broad range of pathological situations irrespective of their diverse T cell antigen receptor repertoire. gammadelta IELs may modulate local immune responses and participate in intestinal lipid metabolism, cholesterol homeostasis, and physiology. This study provides a strong basis for further investigations of the roles of these cells as well as mucosal immune defense in general.

Type A behavior and coronary atherosclerosis.

OBJECTIVES: To evaluate whether the pathogenesis of type A behavior may involve the premature development of coronary atherosclerosis. BACKGROUND: Type A or coronary-prone behavior is considered a possible risk factor for the development of coronary heart disease. Premature development of coronary atherosclerosis is suspected to play a role. Utilizing electron beam computed tomography, one can accurately determine the degree of coronary artery calcification, which is reflective of coronary atherosclerosis. METHODS: We performed a study of 35 men who had no clinical evidence of coronary heart disease. Twenty exhibited severe type A behavior and 15 exhibited type B behavior. All subjects were given an Electron Beam Computed Tomography scan of their coronary arteries and a treadmill electrocardiogram. RESULTS: Eight of the 20 (40%) type A subjects were found to have coronary calcification compared to none of the type B subjects (P=0.005). The correlation coefficient between type A score and coronary calcium score was 0.39 (P=0.09). CONCLUSIONS: The pathophysiology by which type A behavior increases the risk for coronary heart disease may involve the premature development of coronary atherosclerosis.

B-lymphocyte quiescence, tolerance and activation as viewed by global gene expression profiling on microarrays.

Self-tolerance is achieved by deleting or regulating self-reactive lymphocytes at a series of cellular checkpoints placed at many points along the developmental pathways to plasma cells and effector T cells. At each checkpoint, what are the molecular pathways that determine whether a lymphocyte remains quiescent, begins dividing, differentiates or dies? In splenic B cells, the decision between quiescence, tolerance by anergy, and activation provides a tractable setting to explore these issues by global gene expression profiling on DNA microarrays. Here we discuss the application of microarrays to illuminate a set of cell fate decisions that appear to be determined by summation of numerous small changes in expression of stimulatory and inhibitory genes. Many genes with known or predicted inhibitory functions are highly expressed in naive, quiescent B cells, notably the signal inhibitor SLAP and DNA-binding proteins of the Kruppel family (LKLF, BKLF, GKLF), Tsc-22, GILZ, Id-3, and GADD45. Activation of naive B cells, triggered by acute binding of antigen to the B-cell receptor, involves a rapid decrease in expression of these inhibitory genes. Promitotic genes are induced in parallel, including c myc, LSIRF/IRF4, cyclin D2, Egr-1 and Egr-2, as are the anti-apoptotic gene A1 and genes for the T-cell-attracting chemokines MIP-1alpha and beta. B-cell tolerance through the process of anergy, induced by chronic binding of self antigen, maintains expression of the inhibitory genes found in quiescent B cells and induces an additional set of inhibitory genes. The latter include inhibitors of signaling - CD72, neurogranin, pcp4 - and additional inhibitors of gene expression such as SATB1, MEF2C, TGIF and Nab-2. The effects of tolerance, the immunosuppressive drug FK506 and other modulators of calcium or MAPK signaling allow individual gene responses to be linked to different signal transduction pathways. The global molecular profiles obtained illustrate how quiescence and anergy are actively maintained in circulating B cells, how these states are switched to clonal expansion and how they could be better emulated by pro-tolerogenic drugs.

Genome-wide detection of allelic imbalance using human SNPs and high-density DNA arrays.

Most human cancers are characterized by genomic instability, the accumulation of multiple genetic alterations and allelic imbalance throughout the genome. Loss of heterozygosity (LOH) is a common form of allelic imbalance and the detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes and to characterize tumor stages and progression. Here we describe the use of high-density oligonucleotide arrays for genome-wide scans for LOH and allelic imbalance in human tumors. The arrays contain redundant sets of probes for 600 genetic loci that are distributed across all human chromosomes. The arrays were used to detect allelic imbalance in two types of human tumors, and a subset of the results was confirmed using conventional gel-based methods. We also tested the ability to study heterogeneous cell populations and found that allelic imbalance can be detected in the presence of a substantial background of normal cells. The detection of LOH and other chromosomal changes using large numbers of single nucleotide polymorphism (SNP) markers should enable identification of patterns of allelic imbalance with potential prognostic and diagnostic utility.

Genomic-scale gene expression analysis of lymphocyte growth, tolerance and malignancy.

Immunologists are already comfortable with the need for monitoring many different gene products simultaneously. It is a common challenge to remember what CD-one-hundred-and-something is, and an ever-increasing number of colours are required for identification on the flow cytometer. Gene expression arrays now offer the possibility of extending this approach beyond the cell surface and expanding it dramatically to survey the entire catalogue of gene transcripts in a lymphoid cell.

Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.

Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.

High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays.

A highly reliable and efficient technology has been developed for high-throughput DNA polymorphism screening and large-scale genotyping. Photolithographic synthesis has been used to generate miniaturized, high-density oligonucleotide arrays. Dedicated instrumentation and software have been developed for array hybridization, fluorescent detection, and data acquisition and analysis. Specific oligonucleotide probe arrays have been designed to rapidly screen human STSs, known genes and full-length cDNAs. This has led to the identification of several thousand biallelic single-nucleotide polymorphisms (SNPs). Meanwhile, a rapid and robust method has been developed for genotyping these SNPs using oligonucleotide arrays. Each allele of an SNP marker is represented on the array by a set of perfect match and mismatch probes. Prototype genotyping chips have been produced to detect 400, 600 and 3000 of these SNPs. Based on the preliminary results, using oligonucleotide arrays to genotype several thousand polymorphic loci simultaneously appears feasible.

Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome.

Single-nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, and they provide powerful tools for a variety of medical genetic studies. In a large-scale survey for SNPs, 2.3 megabases of human genomic DNA was examined by a combination of gel-based sequencing and high-density variation-detection DNA chips. A total of 3241 candidate SNPs were identified. A genetic map was constructed showing the location of 2227 of these SNPs. Prototype genotyping chips were developed that allow simultaneous genotyping of 500 SNPs. The results provide a characterization of human diversity at the nucleotide level and demonstrate the feasibility of large-scale identification of human SNPs.

Simultaneous genotyping and species identification using hybridization pattern recognition analysis of generic Mycobacterium DNA arrays.

High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 16S genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure.

Development and evaluation of a computer-based system for dietary management of hyperlipidemia.

OBJECTIVE: To describe the development of a computer-based system for dietary management of hyperlipidemia and to evaluate its efficacy for lowering plasma cholesterol level. DESIGN: Using a stepwise approach, we developed and tested a three-part self-management system in five consecutive clinical studies. Each study assessed plasma cholesterol levels before and after dietary intervention using the system. These studies enabled progressive refinement of (a) a food frequency questionnaire used to assess food intake in the preceding month; (b) computer-generated progress reports, based on questionnaire responses, offering dietary change subgoals and strategies for change; and (c) a dietary workbook providing detailed information on how to achieve goals. SUBJECTS/SETTING: Persons with hyperlipidemia (n=814) were enrolled from worksite and clinical settings in the San Francisco Bay area of California. The attrition rate after randomization was 5%. INTERVENTION: Elements of the dietary intervention evolved in response to the results of five clinical studies. In each study, patients underwent a form of baseline assessment of dietary intake followed by counseling/instruction by various means. Follow-up dietary assessments were provided at specific intervals to facilitate subjects' progress toward their dietary goals. A dietary workbook provided the detailed instruction required to implement the recommendations contained in the periodic progress reports. STATISTICAL ANALYSES PERFORMED: Changes in plasma cholesterol level were measured by paired and unpaired t tests. The relationship between the reported reduction in dietary fat and cholesterol level assessed by food frequency questionnaires and the directly measured change in plasma cholesterol level was measured by multiple linear regression. RESULTS: The three major elements of the final computerized system (food frequency questionnaires, computer-generated progress reports, and dietary workbook) were developed and refined in the course of the five clinical studies. Reductions in total plasma cholesterol level of 5.0% to 6.5% achieved by participants in all five studies were consistent with self-reported reductions in intake of dietary saturated fat and cholesterol. Therefore, the computerized self-management system appears to be an effective tool for reducing plasma cholesterol levels. APPLICATIONS/CONCLUSIONS: A computer-based system for dietary self-management of hyperlipidemia, implemented by mail, was effective in short-term studies. This self-management system can potentially provide health-promoting services to large numbers of people at low cost.

A comprehensive management system for heart failure improves clinical outcomes and reduces medical resource utilization.

The effectiveness of heart failure management in clinical practice is limited by physicians' suboptimal utilization of effective medications, patients' poor adherence to dietary sodium limitation and optimal drug therapy, and the lack of systematic monitoring of patients after hospitalization. The present study evaluated the feasibility and safety of MULTIFIT, a physician-supervised, nurse-mediated, home-based system for heart failure management that implements consensus guidelines for pharmacologic and dietary therapy using a nurse manager to enhance dietary and pharmacologic adherence and to monitor clinical status by frequent telephone contact. Fifty-one patients with the clinical diagnosis of heart failure were followed for 138 +/- 44 days. Daily dietary sodium intake fell by 38%, from 3,393 to 2,088 mg (p = 0.0001); average daily medication doses increased significantly (lisinopril: 17 to 23 mg, p <0.001; hydralazine: 140 to 252 mg, p = 0.01). Functional status and exercise capacity improved significantly (p = 0.01). Compared with the 6 months before enrollment and normalized for variable follow-up, the frequency of general medical and cardiology visits declined by 23% and 31%, respectively (both p <0.03); emergency room visits for heart failure and for all causes declined 67% and 53%, respectively (both p <0.001). Hospitalization rates for heart failure and for all causes declined 87% and 74%, respectively (p = 0.001), compared with the year before enrollment. The MULTIFIT system enhanced the effectiveness of pharmacologic and dietary therapy for heart failure in clinical practice, improving clinical outcomes and reducing medical resource utilization.

Effect of type A behavioral counseling on frequency of episodes of silent myocardial ischemia in coronary patients.

Thirteen of 32 patients with coronary heart disease who also exhibited symptoms and signs of severe time urgency and hostility (the two overt components of type A behavior [TAB]) were found to exhibit multiple episodes of silent myocardial ischemia over a 48-hour period of Holter monitoring as indicated by electrocardiogram ST depressions. Ten of these 13 patients were given 14 months of TAB counseling in an attempt to diminish the intensity of their time urgency and hostility. The remaining three patients served as controls. After counseling, the intensity of the time urgency and hostility of the 10 counseled patients diminished 53% and 59%, respectively, as measured by the videotaped clinical examination. The time urgency and hostility of the three uncounseled control patients did not significantly change. The mean frequency of ischemic episodes in the 10 patients who received TAB counseling significantly declined from an initial 6.6 to 3.1 ischemic episodes per 24 hours. However, the mean frequency of ischemic episodes did not significantly decline in 2 of the 3 uncounseled patients. The third uncounseled patient developed a conduction defect in the 14-month interlude, making an analysis of his repeat 48-hour electrocardiogram for episodes of ST depression impossible.

Relation between insecurity and Type A behavior.

This article presents a new Type A Videotaped Clinical Examination scale that measures insecurity. This scale was validated against an existing insecurity measure in a sample of 204 individuals. The results indicated that this new scale is a valid measure of insecurity. The relation between insecurity and type A behavior was examined in a sample of 3013 people. In this large population insecurity showed a strong positive correlation to type A behavior and to each of the two overt behavioral components, time urgency and free-floating hostility.

A case-management system for coronary risk factor modification after acute myocardial infarction.

OBJECTIVE: To evaluate the efficacy of a physician-directed, nurse-managed, home-based case-management system for coronary risk factor modification. DESIGN: Randomized clinical trial in which patients received a special intervention (n = 293) or usual medical care (n = 292) during the first year after acute myocardial infarction. SETTING: 5 Kaiser Permanente Medical Centers in the San Francisco Bay area. PATIENTS: 585 men and women aged 70 years or younger who were hospitalized for acute myocardial infarction. INTERVENTION: In the hospital, specially trained nurses initiated interventions for smoking cessation, exercise training, and diet-drug therapy for hyperlipidemia. Intervention after discharge was implemented primarily by telephone and mail contact with patients in their homes. All medically eligible patients received exercise training; all smokers received the smoking cessation intervention; and all patients received dietary counseling and, if needed, lipid-lowering drug therapy. OUTCOME: Smoking prevalence and plasma low-density lipoprotein cholesterol (LDL) concentrations were measured 2 months after infarction, and functional capacity was measured 6 months after infarction. RESULTS: In the special intervention and usual care groups, the cotinine-confirmed smoking cessation rates were 70% and 53% (P = 0.03), plasma LDL cholesterol levels were 2.77 +/- 0.69 mmol/L and 3.41 +/- 0.90 mmol/L (107 +/- 30 mg/dL and 132 +/- 30 mg/dL) (P = 0.001), and functional capacities were 9.3 +/- 2.4 METS and 8.4 +/- 2.5 METS (P = 0.001), respectively. CONCLUSION: In a large health maintenance organization, a case-management system was considerably more effective than usual medical care for modification of coronary risk factors after myocardial infarction.

Medical diagnosis of type A behavior.

The objective of this investigation was to develop a medically oriented examination (including a search for physical signs in addition to elicitation of symptoms) for the accurate diagnosis of type A and type B behaviors. Comprising the study were 99 post-myocardial infarction patients, 15 clinically well persons in whom clinical coronary heart disease subsequently developed, and 23 healthy type B subjects. All participants were subjected to a videotaped clinical examination during which, in addition to eliciting responses to questions, 14 possible physical or psychomotor signs (many of which are newly discovered) of type A behavior were also observed. Each physical sign and symptom was given an arbitrarily weighted score (according to its observed frequency of occurrence in previously studied and authenticated type A behavior). These total scores were then statistically analyzed to obtain a critical "diagnostic score" for the presence of type A behavior. The medically oriented videotaped clinical examination detected the presence of type A behavior in 97 of 99 (98%) successively examined postinfarction patients and in 14 of 15 (93%) subjects who were clinically well at the time of their videotaped clinical examination but who subsequently had clinical coronary heart disease. Conversely type A behavior was diagnosed by videotaped clinical examination in only 1 of 23 (4%) healthy men who previously had been found to exhibit type B behavior by prior diagnostic procedures.

Cholesterol-related counseling by registered dietitians in northern California.

BACKGROUND: An estimated 40 million Americans have serum cholesterol levels that warrant medically supervised dietary intervention. Although registered dietitians are expected to play an important role in treating these patients, current treatment practices in the community are largely unknown. METHODS: A questionnaire concerning treatment practices was mailed to all 377 registered dietitians listed in the directories of the American Dietetic Association for two large California districts. Number of patients seen and length and content of dietary counseling were ascertained for three types of patients: (a) hypercholesterolemic outpatients without heart disease, (b) hypercholesterolemic outpatients who have heart disease, and (c) inpatients with myocardial infarction. RESULTS: A return rate of 59% (n = 252) was obtained for the questionnaire. A total of 44% of the registered dietitians counseled hypercholesterolemic patients in any of the categories surveyed. About 30% of the respondents counseled hypercholesterolemic outpatients without heart disease. They saw an average of 4.9 such patients a week, spent an average of 53 min in an initial session, and usually did not see the patient again in follow-up. Fewer than 10% of patients had as many as four sessions. About 27% of the respondents saw hypercholesterolemic outpatients with heart disease, averaging 3.5 such contacts per week. The reported practices were similar to those provided to noncardiac outpatients. About 22% of registered dietitians worked with hospitalized myocardial infarction patients. They spent an average of a total of 41 min over 2.5 visits with each patient. CONCLUSION: Currently, outpatient registered dietitian counseling for hypercholesterolemia appears to be limited in both the number of patients reached and the duration of the counseling. Further research into the impact of, barriers to, and efficacy of alternative delivery methods of dietary counseling is needed.

Algorithms for identifying local molecular sequence features.

Efficient algorithms are described for identifying local molecular sequence features including repeats, dyad symmetry pairings and aligned matches between sequences, while allowing for errors. Specific applications are given to the genomic sequences of the Epstein-Barr virus, Varicella-Zoster virus and the bacteriophages lambda and T7.

Efficient algorithms for molecular sequence analysis.

Efficient (linear time) algorithms are described for identifying global molecular sequence features allowing for errors including repeats, matches between sequences, dyad symmetry pairings, and other sequence patterns. A multiple sequence alignment algorithm is also described. Specific applications are given to hepatitis B viruses and the J5-C (J, joining; C, constant) region of the immunoglobulin kappa gene.

Multiple-alphabet amino acid sequence comparisons of the immunoglobulin kappa-chain constant domain.

We compare the amino acid sequences of the constant domains of the immunoglobulin kappa chain of human, mouse, and rabbit by using four classification schemes ("alphabets") of the 20 amino acids based on their chemical, functional, charge, and structural properties. The comparison reveals three regions of pronounced similarity across the three species, independent of allotype. Two of these regions (residues 65-73 and 99-103) entail a high degree of identity at the DNA level and are distinguished from the rest of the constant domain in codon usage and in the dinucleotide sequence at abutting sites of adjacent codons. Residues 22-29 are highly conserved among the three species in the chemical and functional alphabets but do not show any three-sequence significant amino acid block identities. These results are discussed in terms of transcript processing, effector functions, and structural interactions within the constant domain and with the heavy chain.

Comparative statistics for DNA and protein sequences: single sequence analysis.

Four categories of data representations are used to help interpret structures and similarities of nucleic acid and protein sequences. Statistical significance of the observed relationships revealed by these representations are assessed by a hierarchy of permutation procedures and by comparisons with theoretical random models. Applications are presented for various DNA sequences including papovaviruses, Epstein-Barr virus, mitochondrial genomes, and several globin and immunoglobulin genes.