Factors associated with frequency of 12-Step attendance by drug abuse clients.

Comparison was made of treatment clients attending Narcotics Anonymous and/or Alcoholics Anonymous meetings less than weekly (n = 41) with treatment clients attending meetings at least three times a week (n = 30). The frequent attenders (attending an average of 30.6 meetings monthly) differed from non- and infrequent attenders (attending an average of 0.4 meetings monthly) in terms of histories of greater lifetime drug use, more arrests and treatment experiences, and an earlier age of first use of alcohol. Although the frequent attender was also older, age was not found to influence the differences found between groups. Measures of religiosity, use of community services, and support from others for recovery and psychological functioning, other than ratings of the helpfulness of 12-Step, were not differentiated among groups. The findings suggest that 12-Step groups are more likely to be selected by clients with more severe histories of drug use and criminal activity, i.e., those most in need of the support to behavior change those groups provide. The role of treatment programs in facilitating the use of 12-Step groups is discussed.

Factors associated with frequent and infrequent HIV testing.

Drug user treatment clients with 5 or more HIV tests (frequent testees N=43) and 0-2 HIV tests (infrequent testees-N = 56) were compared on demographic characteristics, risk behaviors, perceived risk of HIV infection to self, involvement with family members, and psychological functioning. Extreme groups of HIV testees did not differ on any variables other than an index of perceived vulnerability to HIV infection (e.g., " You think that you really could get AIDS"). That measure of felt vulnerability was not correlated significantly with needle or sexual risk behaviors, family involvement, psychological functioning or other measures of perceived risk. It was reasoned that, in a community in which both dangers and protective behaviors are widely understood, frequent testees experience a generalized and heightened concern unrelated to specific behaviors or characteristics.

Characterization of the rnc-97 mutation of RNAaseIII: a glycine to glutamate substitution increases the requirement for magnesium ions.

The rnc-97 mutation of the Escherichia coli double-stranded-RNA-specific ribonuclease III (RNAaseIII) was previously isolated by virtue of the lethal expression of RNAaseIII in Saccharomyces cerevisiae. Here we show that rnc-97 is a single point mutation causing the substitution of glycine 97 by glutamic acid. The mutation eliminates the lethal phenotype of RNAaseIII expression in yeast and reduces fourfold the effect of RNAaseIII expression on bacteriophage gy1 propagation in E. coli. Mutant RNAaseIII-G97E and wild-type RNAaseIII were purified according to published procedures. The apparent molecular masses of the two enzymes on SDS polyacrylamide gels are the same but they differ in pI (6.85 for RNAaseIII-G97E and 7.3 for RNAaseIII). Whereas the two enzymes (under standard assay conditions) do not show a great difference in activity towards double-stranded RNA and defined single-stranded RNAaseIII substrates, they differ dramatically (20-fold or more) under conditions of Mg2+ limitation. The hypothesis that limitation of Mg2+ ions in vivo is responsible for the phenotypes of the rnc-97 mutation in S. cerevisiae and E. coli is discussed.

The M20 IL-1 inhibitor. I. Purification by preparative isoelectric focusing in free solution.

An interleukin-1 (IL-1) inhibitor produced by the M20 myelomonocytic cell line has been shown to be active in various in vitro and in vivo IL-1 induced parameters. This inhibitor has been purified from the conditioned medium by gel filtration through a Sephacryl S-300 column or dye ligand chromatography on Affi-Gel blue column, followed by isoelectric focusing in free solution in the pH range 3-5 using the Rotofor cell. When gel filtration by FPLC with the Superose 12 column was used as the final step, the combined sequence of purification procedures resulted in a 1600-fold purification of the IL-1 inhibitor. The purified IL-1 inhibitor has a molecular weight of approximately 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. By SDS-PAGE analysis the inhibitor preparation thus obtained showed the presence of two protein bands, while a few closely spaced protein bands were seen by analytical isoelectric focusing in polyacrylamide gels (pH 3-6). Some of these bands in PAGIF might correspond to different degrees of glycosylation of the inhibitory protein. Although the M20 IL-1 inhibitor has not yet been purified to homogeneity, it should be stressed that the procedures used, allowed us to remove the great majority of the proteins present in the medium in which the M20 cells were cultured, and to recover in satisfactory yield the inhibitor which we consider likely to be present in the conditioned medium in subnanomolar concentrations.

The M20 IL-1 inhibitor. II. Biological characterization.

Interleukin-1 (IL-1) is an important mediator in inflammation and immunological processes. The findings of native IL-1 inhibitors suggest a negative feedback mechanism to down-regulate IL-1 mediated acute inflammation. IL-1 inhibitors were also found elevated in disease states associated with high IL-1 levels. We have previously described one such IL-1 inhibitor derived from the human M20 myelomonocytic cell line. In this paper we present several biological and biochemical characteristics of the M20 IL-1 inhibitor. Various in vitro activities of the inhibitor are described and its IL-1 specificity in these assays is demonstrated. Purification of the inhibitor was performed by DEAE-high performance liquid chromatography, isoelectric focusing, gel filtration and dye ligand chromatography column. This protein factor has a MW of 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. The inhibitor has no cross-reactivity against a panel of known cytokines (IL-1 alpha, IL-1 beta, IL-2, sIL-2R, IL-6, tumor necrosis factor (TNF), interferon-gamma (IFN-gamma)) and is distinct from the IL-1 receptor antagonist (IL-1ra). The purified IL-1 inhibitor was destroyed by trypsin, 2-mercaptoethanol, sodium dodecyl sulfate and extremes in pH and in temperature. Only IL-1 induced (but not the IL-2, IL-6 or TNF induced) thymocyte proliferation and PGE2 production by fibroblasts were inhibited by the inhibitor, thus showing specificity to IL-1 in these assays.

Detection of albumin binding abnormalities in sera of patients with familial dysalbuminaemic hyperthyroxinaemia using isoelectric focusing.

Definitive diagnosis of familial dysalbuminaemic hyperthyroxinaemia (FDH) requires finding a high concentration of [125I]T4 bound to albumin. We used isoelectric focusing (IEF) in agarose gels to study the sera of three members of a family with FDH and compared the distribution of [125I]T4 obtained by IEF with that obtained by conventional agarose gel electrophoresis (AGE). Both IEF and AGE confirmed the diagnoses of FDH. In case #1, the % [125I]T4 bound to albumin was 15.2 and 20.0, with IEF and AGE, respectively, in case #2 23.6 and 26.5, and in case #3, 22.1 and 23.0, compared to normal controls of 5.9 and 7.4, respectively. IEF has not previously been used to diagnose FDH, to our knowledge. IEF has the advantage of eliminating TBG interference with albumin migration, which can potentially complicate the diagnosis of FDH when the AGE method is used. To date, reverse flow electrophoresis, a more cumbersome method with poorer resolution than IEF, has been utilized to eliminate TBG interference. IEF, in agarose gels, is a relatively simple and accurate method to diagnose FDH, and avoids artifacts which may be encountered with AGE.

The specific IL-1 inhibitor from the human M20 cell line is distinct from the IL-1 receptor antagonist.

Several inhibitors of IL-1 have been described. Four appear to be the same: one purified from urine of patients with monocytic leukemia, another from IgG-stimulated monocytes, a third from PMA-induced U937 cels, and a fourth from keratinocytes. Because these IL-1 inhibitors compete with bona fide IL-1 for occupancy of IL-1 receptors, they are now called the IL-1 receptor antagonist (IL-1ra) or IL-1 receptor antagonist protein (IRAP). We have described another IL-1-specific inhibitor produced by the human myelomonocytic leukemia cell line, M20. This inhibitor specifically blocks IL-1-induced effects both in vitro and in vivo. In the present study, we compared the M20 IL-1 inhibitor with IL-1ra using neutralization in an IL-1 bioassay and immunoblotting with an anti-IL-1ra antibody that recognizes natural IL-1ra. Neutralization experiments, immunoblotting, and western blotting obtained after transfer from SDS-PAGE revealed that anti-IL-1ra does not recognize the M20 IL-1 inhibitor. In addition, the isoelectric point and molecular weight of the M20 IL-1 inhibitor were different from those of the IL-1ra. From these data, we conclude that the M20 IL-1 inhibitor is antigenically unrelated to the IL-1ra but is a distinct and specific IL-1 inhibitor.

Partial characterization of a C5a-inhibitor in peritoneal fluid.

We have recently described a 40-kDa protein in peritoneal fluid that neutralized the chemotactic activity of the C fraction C5a. It was deficient in peritoneal fluids of patients suffering from familial Mediterranean fever. Further characterization of the inhibitor with the use of 125I-rC5a binding to dibutyryl cAMP-induced U937 cells revealed dependence on the peritoneal fluid concentration, on the time of incubation and on temperature and pH. Fractionation of 125I-C5a on Sephadex G-50 column, before and after incubation with peritoneal fluid, revealed similar fractionation patterns despite loss of biologic activity of the treated C5a (but not its binding to polyclonal anti-C5a antibody). Analysis of rC5a by SDS-PAGE before and after treatment with partially purified C5a inhibitor, revealed slight modification of the inhibitor-treated C5a. Using various protease inhibitors (i.e., PMSF) suggested that the C5a inhibitor is a serine protease. It neutralized C5a by means of limited proteolysis which did not change C5a immunologic properties and changed only slightly its m.w. but abolished its receptor binding and chemotactic functions. It is suggested that the C5a inhibitor plays a role in the regulation of inflammation in serosal tissues and that its deficiency in familial Mediterranean fever may explain the attacks of sterile inflammation characteristic of this disease.

Identification and purification of a functional amine transporter from bovine chromaffin granules.

The amine transporter from bovine chromaffin granules has been purified in a functional state. Two isoforms with different pI values have been separated and shown to be active. One with an unusually acidic pI (approximately 3.5) has been shown to be a glycoprotein with an apparent Mr of 80,000. The purified polypeptide catalyzes transport of serotonin upon reconstitution with an apparent Km of 2 microM and a Vmax of 140 nmol/mg/min, 150-200-fold higher than the one determined in the native system. Transport is inhibited by reserpine and tetrabenazine, ligands which bind to two distinct sites on the transporter. These findings suggest that the binding sites for both drugs reside on a single polypeptide. The reconstituted purified transporter binds [3H]reserpine with a biphasic kinetic behavior, KD values of 0.3 and 30 nM and Bmax of 310 and 4200 pmol/mg protein, respectively. In addition, binding of [3H]reserpine is accelerated upon imposition of a pH gradient across the proteoliposome. From these findings it is evident that a single polypeptide catalyzes the various functions of the transporter.

Effects of radical scavengers on the development of experimental diabetes.

The possible value of radical scavengers in the prevention of beta cell destruction was studied in two animal models of type I diabetes. Eight compounds were tested alone or in combinations. In the low-dose streptozotocin model treatment started 24 hr after the last injection of the beta cell toxin, in the BB rat daily administration was started at 50 days of age (i.e., 20-70 days before diabetes onset). No effect was seen in the low-dose streptozotocin model for 3-aminobenzamide, N-acetyl-DL-homocysteine thiolactone, ebselen, and butylated hydroxyanisole whereas partial suppression of hyperglycaemia was seen in mice receiving cysteamine. In BB rats diabetes development was delayed and partially suppressed by administration of ebselen plus vitamin E plus MaxEPA (fish lipid concentrate), but not when ebselen was replaced by nicotinamide. We conclude that the disease process is not readily modifiable by treatment with exogenous radical scavengers.

The natural anti-alpha-galactosyl IgG on human normal senescent red blood cells.

A highly sensitive antiglobulin test based on rosette formation due to the interaction between IgG bearing red blood cells (RBC) and Fc receptors on K562 cells, was used to study the immunoglobulin molecules present on human senescent RBC. Normal human RBC were separated into young and senescent subpopulations on the basis of age-dependent differences in density by centrifugation on a discontinuous density Percoll gradient, and by flotation on phthalate ester mixtures. The senescent but not the young RBC were found to bear membrane bound IgG. Most of the bound IgG molecules could be specifically eluted by galactose in its alpha-anomeric form. Antigalactosyl (anti-Gal) IgG antibodies with similar reactivity were found to be present in high titres in every one of the 400 normal human sera tested. The natural anti-Gal antibodies isolated from normal sera by affinity chromatography could bind to IgG depleted senescent RBC but not to young RBC. Erythrophagocytosis experiments indicated that the anti-Gal bound to the senescent RBC induced their destruction by macrophages. It is suggested that the natural anti-Gal antibodies interact with cryptic alpha-galactosyl residues which are exposed in the course of the RBC senescence and mediate the removal of these RBC from circulation by cells of the reticuloendothelial system.

A unique natural human IgG antibody with anti-alpha-galactosyl specificity.

A new natural anti-alpha-galactosyl IgG antibody (anti-Gal) was found to be present in high titer in the serum of every normal individual studied. The antibody was isolated by affinity chromatography on a melibiose-Sepharose column. The reactivity of the antibody was assessed by its interaction with alpha-galactosyl residues on rabbit erythrocytes (RabRBC). The specificity was determined by inhibition experiments with various carbohydrates. The anti-Gal interacts with alpha-galactosyl residues, possibly on glycolipids of human RBC (HuRBC), after removal of membrane proteins by treatment with pronase. In addition, the anti-Gal bind specifically to normal and pathologically senescent HuRBC, suggesting a physiological role for this natural antibody in the aging of RBC. The ubiquitous presence of anti-Gal in high titers throughout life implies a constant antigenic stimulation. In addition to the theoretical interest in the antibody, the study of the anti-Gal reactivity seems to bear immunodiagnostic significance. Decrease in the antibody titer was found to reflect humoral immunodeficiency disorders.

Continuous production of monoclonal rheumatoid factor by EBV-transformed lymphocytes.

Human B lymphocytes are immortalized by Epstein-Barr virus (EBV, ref. 1). The virus can be used to establish lymphoblastoid cell lines that produce and actively secrete specific antibodies. The original method, which we have used for various antigens is based on selection of the specific surface antigen receptor-positive lymphocytes from the peripheral blood lymphocytes of a donor who was previously sensitized to the corresponding antigen. Furthermore, by cloning the polyclonal anti-NNP cell line we have produced human monoclonal antibodies for the first time in vitro. About 5-20 microgram ml-1 stably produced specific antibody is obtained in the supernatant of the cell lines. This approach can be used for the in vitro production of monoclonal human autoimmune antibodies by EBV-immortalized lymphocytes from patients with autoimmune diseases. We demonstrate the continuous production in vitro of a monoclonal IgM and anti-IgG antibody (rheumatoid factor, r.f.) by a lymphoblastoid cell line established from a patient with rheumatoid arthritis.

Immune response to sheep red blood cells in mice pretreated with mycobacterial fractions.

Ten micrograms of trehalose-6,6' -dimycolate (cord factor) injected into the footpads of mice increased the antibody response to sheep red blood cells (SRBC) subsequently injected into the same sites. There is a relationship between the antibody response and the cellular reaction induced locally and in the draining lymph nodes by cord factor, as judged by a much weaker response when antigen is injected into the contralateral footpads. The time intervals between injection of cord factor and antigen were from 5 to 20 days. A similar increased antibody response to SRBC was evident after preliminary administration of Freund's complete adjuvant or living BCG bacilli into the footpads. There was no increased antibody response in mice pretreated with living BCG or Freund's adjuvant to SRBC injected into the contralateral footpads. Administration of wax D from human strains Peurois and Test was without any effect. Administration of SRBC emulsified in incomplete Freund's adjuvant containing 5 mug of cord factor induced a very strong antibody response in the mice as compared to that after injection of the same amount of antigen in incomplete Freund's adjuvant containing wax D or mycobacteria. The results are discussed.