Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients.

We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation.

Transcriptional regulation of parathyroid hormone-related protein promoter P2 by NF-kappaB in adult T-cell leukemia/lymphoma.

Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of patients with adult T-cell leukemia/lymphoma (ATLL) due to human T-cell lymphotropic virus type-1 (HTLV-1) infection. We previously showed that ATLL cells constitutively express high levels of PTHrP via activation of promoters P2 and P3, resulting in HHM. In this study, we characterized a nuclear factor-kappaB (NF-kappaB) binding site in the P2 promoter of human PTHrP. Using electrophoretic mobility shift assays, we detected a specific complex in Tax-expressing human T cells composed of p50/c-Rel, and two distinct complexes in ATLL cells consisting of p50/p50 homodimers and a second unidentified protein(s). Chromatin immunoprecipitation assays confirmed in vivo binding of p50 and c-Rel on the PTHrP P2 promoter. Using transient co-transfection with NF-kappaB expression plasmids and PTHrP P2 luciferase reporter-plasmid, we showed that NF-kappaB p50/p50 alone and p50/c-Rel or p50/Bcl-3 cooperatively upregulated the PTHrP P2 promoter. Furthermore, inhibition of NF-kappaB activity by Bay 11-7082 reduced PTHrP P2 promoter-initiated transcripts in HTLV-1-infected T cells. In summary, the data demonstrated that transcriptional regulation of PTHrP in ATLL cells can be controlled by NF-kappaB activation and also suggest a Tax-independent mechanism of activation of PTHrP in ATLL.

Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts.

The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression.

Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping.

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.

Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.

Identification of leptin-induced transcripts in the mouse hypothalamus.

Long-form leptin receptor (OB-R(L)) is a signal-transducing member of the cytokine receptor superfamily that is essential for mediating the effects of leptin on mammalian body weight homeostasis. At present, the range of transcriptional targets responsive to OB-R(L) activation, and consequently, the likely mediators of leptin action, remain undefined. In this report, we have used cDNA subtractive hybridization to identify transcripts induced by leptin in immortalized hypothalamic neurons expressing OB-R(L). Differential expression of the identified transcripts in these cells was confirmed by both array technology and Northern blotting. In situ hybridization studies indicate that these transcripts are expressed in the mouse central nervous system, including nuclei of the hypothalamus that coexpress OB-R(L). Comparative in situ analysis of slices of hypothalami generated from control and leptin-injected ob/ob mice demonstrates that a subset of the identified transcripts is induced in vivo after leptin injection. The potential role of the proteins encoded by these transcripts in mediating the effects of leptin on body weight and energy homeostasis are discussed.

Recombinant Fel d.I: Expression, purification, IgE binding and reaction with cat-allergic human T cells.

This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the domestic cat (Felis domesticus). An inframe linker encoding polyhistidine has been added to the 5' ends of the Fel d I chains 1 and 2 cDNAs to facilitate purification using Ni2+ ion affinity chromatography. This method provides high yields in a single step of rchain 1 and rchain 2 of Fel d I with a > 90% level of purity. Polymerase chain reaction (PCR) methods were used to introduce a thrombin cleavage site (LVPR decreases GS) at the N-terminus of both chains. Thrombin cleavage of rchain 1 and rchain 2 followed by HPLC purification of the cleavage products allowed the isolation of each recombinant chain with only two additional residuals (GS) at the N-terminus of the native sequence. Amino acid sequencing analysis of the N-terminus and mass spectrometry of these polypeptides demonstrated that they are highly pure and full-length. Direct ELISA assays showed that IgE from cat-allergic patients binds to both rchain 1 and rchain 2 of Fel d I, demonstrating that both these chains contribute to the allergenicity of this heterodimeric protein. An examination of the reactivity of T cells derived from cat-allergic patients revealed that both polypeptide chains contribute to the T cell response to this allergen. Consequently, it is concluded that the immunological response to Fel d I is composed of a reaction at both the B and T cell level to each of the two chains that constitute the native allergen.

Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes.

The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

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.

Complications in patient with triple penile prosthesis.

A case report of a man with three poorly functional prosthetic implants in his penis is presented. Complications resulting from the insertion of penile implants are reviewed and suggestions are made on evaluating a patient who had previous prosthetic surgery.

Cognitive-behavioral treatment for alcohol dependence: a review of evidence for its hypothesized mechanisms of action.

OBJECTIVE: This review examined support for the hypothesis that cognitive-behavioral treatment (CBT) for alcohol dependence works through increasing cognitive and behavioral coping skills. METHOD: Ten studies were identified that examined the hypothesized mechanisms of action of CBT. These studies involved random assignment (or its near equivalent) of participants to CBT and at least one comparison condition. RESULTS: Although numerous analyses of the possible causal links have been conducted to evaluate whether CBT works through increasing coping, the results indicate little support for the hypothesized mechanisms of action of CBT. CONCLUSIONS: Research has not yet established why CBT is an effective treatment for alcohol dependence. Negative findings may reflect methodological flaws of prior studies. Alternatively, findings may indicate one or more conceptual assumptions underlying CBT require revision.

Do treatment process factors mediate the relationship between type A-type B and outcome in 12-Step oriented substance abuse treatment?

AIMS: One underutilized strategy for enhancing treatment research is to examine intervening factors that link client characteristics to endpoint outcomes. This study tested the hypothesis that Type B substance abusers would demonstrate difficulties engaging in the treatment process, and that these problems would mediate their poorer outcomes. DESIGN: Longitudinal naturalistic study. SETTING: Two intensive 12-Step substance abuse treatment programs. PARTICIPANTS: A sample of 115 men and women seeking treatment. MEASUREMENT: Empirical clustering techniques were used to divide the sample, and the link between type, process factors, and 12-month outcomes was examined. FINDINGS: Hypotheses were not supported. Type Bs did not demonstrate difficulties with the treatment process, but had greater problems sustaining gains posttreatment. Contrary to prediction, Type Bs were better matched to self-help affiliation than Type As. CONCLUSIONS: Findings argue for a more optimistic perspective on treating Type Bs, and for the utility of Type A-Type B in informing treatment research.

The generalizability of the dependence syndrome across substances: an examination of some properties of the proposed DSM-IV dependence criteria.

DSM-III-R and proposed DSM-IV schemes for the diagnosis of psychoactive substance use disorders are based largely on the dependence syndrome concept. However, there is an absence of empirical support for the generalizability of the dependence syndrome across substances. This study examines how consistently proposed DSM-IV dependence criteria function to measure dependence across seven substances: alcohol, cannabis, cocaine, stimulants, hallucinogens, sedatives and opiates. Using structured research diagnostic interviews, dependence diagnoses were determined for 295 American subjects in treatment for alcohol/drug problems. Several factor analytic techniques were used to assess whether criteria formed single dimensions and how consistently individual criteria measured dependence across substances. The ability and consistency of criteria to measure a continuum of severity across substances were also assessed. Only subjects who used the substance at least six times were entered in the analyses. Overall, results provide strong support for the DSM approach for alcohol, cannabis, cocaine, stimulants, sedatives and opiates, but not for hallucinogens. Results indicate that a single strong factor adequately described the criteria for these six substances. All criteria loaded strongly and uniformly on single factors indicating that all were good measures of dependence. Criteria provided a dimensional measure of severity based on several indices for these substances. In addition, four criteria provided relatively stable indicators of high or low severity across these substances. Results did not support the use of dependence criteria for hallucinogens as these criteria did not form a single factor. Results suggest that very few hallucinogen users experience an inability to cut down or control use, a key indicator of loss of control.

Lifetime DSM-IV diagnosis of alcohol, cannabis, cocaine and opiate dependence: six-month reliability in a multi-site clinical sample.

Psychiatric research increasingly emphasizes the diagnosis of symptoms and syndromes on a longitudinal basis. This study tests the reliability of lifetime DSM-IV diagnoses of alcohol, cannabis, cocaine and opiate dependence. The CIDI-SAM was administered at intervals not less than six months apart to a multi-site sample of 201 clinical respondents. The reliability of lifetime diagnosis of the syndromes, of the criteria which constitute the syndromes, and of the ages of onset reported for the criteria and for the dependence syndromes as a whole, were studied and the effects of patient characteristics suspected to degrade reliability were examined. There was generally good agreement, statistically, at both the syndrome and criterion level between the two interviews. Lifetime diagnoses for three of the drugs--alcohol, cannabis and opiates--were made at or near levels of agreement generally considered excellent under less strict testing conditions, and cocaine dependence was only marginally below this level. Most criteria showed good reliability and all delivered about equal results when averaged across the four substances, although a relationship between reliability and centrality of the symptom to the individual drug abuse pattern was found. Age of onset was almost uniformly highly reliable. Most patient characteristics bore no detectable relationship to reliability, although patients with multiple drug use patterns may warrant more careful probing by interviewers. Overall, these data indicate that lifetime symptoms and diagnoses can be queried reliably, although they must be reported with less confidence than current state diagnoses.

Measuring diagnostic agreement.

Diagnostic agreement tests the reliability and concordance of diagnostic systems. The introduction of measures of agreement with reputations for baserate independence (e.g., Yule's Y and Q), and new studies occasioned by the publication of the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV; American Psychiatric Association, 1994) and the International Classification of Diseases--10 (ICD-10; World Health Organization, 1992) make it necessary to study the relationship of illness baserates to measures of agreement. Testing diagnostic concordance for diagnoses of drug dependence from the third edition of the DSM (American Psychiatric Association, 1980) versus DSM-IV diagnoses of drug dependence under 3 baserate conditions, it was found that Yule's Y and Q proved as vulnerable to differences in baserates as kappa or percent agreement and that specificity covaried with baserate rather than being fixed, as most theoretical discussions assume. The uncritical use of Y and Q, therefore, is likely to lead to optimistic interpretations of agreement. Kappa should be preferred for most purposes, although an adjustment to the computational formulas for Y and Q is presented that can diminish their positive bias.

Affiliation with Alcoholics Anonymous after treatment: a study of its therapeutic effects and mechanisms of action.

Relatively little is known about how substance abuse treatment facilitates positive outcomes. This study examined the therapeutic effects and mechanisms of action of affiliation with Alcoholics Anonymous (AA) after treatment. Patients (N = 100) in intensive 12-step substance abuse treatment were assessed during treatment and at 1- and 6-month follow-ups. Results indicated that increased affiliation with AA predicted better outcomes. The effects of AA affiliation were mediated by a set of common change factors. Affiliation with AA after treatment was related to maintenance of self-efficacy and motivation, as well as to increased active coping efforts. These processes, in turn, were significant predictors of outcome. Findings help to illustrate the value of embedding a test of explanatory models in an evaluation study.

Testing the effectiveness of cognitive-behavioral treatment for substance abuse in a community setting: within treatment and posttreatment findings.

This study evaluated the short-term effectiveness of cognitive-behavioral treatment (CBT) for substance abuse delivered in a community setting. At entry into outpatient community substance abuse treatment, participants (N = 252) were randomly assigned to 3 conditions: high-standardization CBT, low-standardization CBT, and treatment as usual. Treatment consisted of 12 weekly individual therapy sessions. There was a significant decrease in substance use from baseline, with participants reporting being abstinent on 90% of within-treatment days and 85% of days during the 6 months posttreatment. However, there were no significant differences in outcomes across conditions. Findings do not support the hypothesis that disseminating CBT to community settings will improve outcomes and suggest that standard substance abuse counseling may be more effective than previously thought.

On criterion weighting in the DSM-IV.

Differential weighting of illness signs and symptoms has surfaced recurrently in psychiatric nosology. Six alternately weighted algorithms for diagnosing alcohol dependence in accordance with the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV; American Psychiatric Association, 1994), based on statistical, unit, rational and random criterion weighting systems, were used to predict an array of concurrent validators and 6-month drinking outcomes in a regional clinical sample of 365 participants. Comparable predictive efficiency across all algorithms, including the randomly weighted versus statistical best-fit model, was observed. Further analyses and geometric modeling suggested that this was due to the extremely high internal consistency of the DSM-IV criteria. An alternative strategy that favors factorially complex, less homogeneous criteria was used to develop an experimental DSM-IV algorithm from an array of 39 candidate criteria. This algorithm had extremely low internal consistency, high difficulty, and complex factor loadings. Differential weighting of its criteria produced a good range of efficiencies, predictive power for rational models exceeding the random weight model, and a best-fit algorithm with substantial surplus predictive power. These results illustrate an emerging conflict in nosology between 2 opposing trends: a press for the promulgation of criterion arrays with high internal consistency and a clear desire to assign some criteria extra weight for prognosis or decision making. Both cannot be had in the same algorithm. An alternative approach emphasizing diagnostic criteria with complex structures can satisfy the multiple demands of brevity, validity, and weighting performance.

The comorbidity of alcoholism and personality disorders in a clinical population: prevalence rates and relation to alcohol typology variables.

This study assessed prevalence rates and overlap among Diagnostic and Statistical Manual of Mental Disorders (3rd ed., revised; DSM-III-R; American Psychiatric Association, 1987) personality disorders in a multisite sample of 366 substance abusers in treatment. In addition, the relation of antisocial personality disorder (APD), borderline personality disorder (BPD), and paranoid personality disorder (PPD) to alcohol typology variables was examined. Structured diagnostic interviews and other measures were administered to participants at least 14 days after entry into treatment. Results indicated high prevalence rates for APD and non-APD disorders. There was extensive overlap between Axis I disorders and personality disorders, and among personality disorders themselves. APD, BPD, and PPD were linked to more severe symptomatology of alcoholism and other clinical problems. However, only APD and BPD satisfied subtyping criteria, after controlling for other comorbidity. Implications for classifying alcoholics by comorbid disorders are discussed.

DSM-III, DSM-IV and ICD-10 as severity scales for drug dependence.

The construct of illness severity serves many scientific and clinical functions. This study tested the performance as severity scales of three systems for diagnosing drug dependence--DSM-III, DSM-IV and ICD-10--in a multisite regional sample of 370 clinical subjects. Both lifetime and current severity of four drug problems--alcohol, cannabis, cocaine and opiate dependence--was studied in three stages: (a) item difficulty and internal consistency analysis; (b) probabilistic modeling of distribution behavior; and (c) concurrent validation against a set of independent measures. All three systems, for most drugs correlated with most test variables, had good to excellent concurrent validity. Unexpectedly, DSM-III showed in some instances better item behavior, composite score behavior and concurrent validity than the other systems, though DSM-IV and ICD-10 are based on slimmer generic algorithms, and may represent a good balance between simplicity and concurrent validity. Results suggest that the design of future diagnostic algorithms start at the item level and strive for moderate levels of both internal consistency and difficulty. Composite score distributions can then be modeled in field research, and necessary item corrections can be made before the algorithm is widely promulgated.