Need for an external proficiency testing program for cytokines, chemokines, and plasma markers of immune activation.

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Ibeta, soluble TNF receptor II, soluble IL-2 receptor alpha, beta(2)-microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.

Improved brain delivery and in vitro activity of zidovudine through the use of a redox chemical delivery system.

OBJECTIVE: Improved therapy for AIDS dementia and related encephalopathies may be achieved through enhanced delivery of effective antiretroviral agents to the central nervous system (CNS). DESIGN: A novel chemical delivery system (CDS) was used, which utilized redox trapping of drugs in the brain. This study was aimed at defining the pharmacokinetics of a zidovudine (ZDV)-CDS as well as establishing its in vitro antiviral efficacy against HIV in both lymphocytes and in a neural cell line. RESULTS: ZDV-CDS administered parenterally to rats produced significantly higher brain levels of ZDV [area under the curve (AUC), 425 micrograms x min/g] than equimolar ZDV (AUC, 13.5 micrograms x min/g). Native ZDV uptake was minimal after 1 h when analyzed in CEM lymphocytes and in SKNMC neuroblastoma cell line. By contrast, marked uptake of ZDV-CDS was followed by biochemical conversion of ZDV-CDS to its main metabolites (ZDV-CDS quaternary salt, ZDV-Q+, and native ZDV). These improved uptake profiles were associated with greater in vitro virucidal effect. ZDV-CDS at 0.5 microM was 80% more effective than ZDV in suppressing p24 production in a lymphocyte culture infected with 6000 median tissue culture infective doses (TCID50) of the HIV N1T strain and 50% more effective at 0.05 microM. Furthermore, syncytia formation was completely suppressed at a ZDV-CDS dose of 0.5 microM (600 TCID50) but native ZDV at the same dose was ineffective. Finally, while ZDV (at 0.5 microM) is not active in reducing viral replication in an SKNMC neural cell line, the ZDV-CDS complex significantly suppressed p24 synthesis. CONCLUSION: The ZDV-CDS complex is capable of delivering higher ZDV doses to lymphocytes and neural cells, with improved antiretroviral activity.

Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease.

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced, > 2.5-fold, the levels of TNF-alpha. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to-cell interactions between HIV-infected brain macrophages and astrocytes.

PC12 cells differentiate into chromaffin cell-like phenotype in coculture with adrenal medullary endothelial cells.

Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

Neurotrophic activity of monomeric glucophosphoisomerase was blocked by human immunodeficiency virus (HIV-1) and peptides from HIV-1 envelope glycoprotein.

Glucophosphoisomerase (GPI), a glycolytic enzyme, was recently described to share 90% sequence homology with neuroleukin, a recently discovered growth factor which promotes motor neuron regeneration in vivo, survival of peripheral and central neurons in vitro, and affects B cell immunoglobulin synthesis. Interestingly, neuroleukin activity was described to be antagonized by the human immunodeficiency virus (HIV-1) envelope glycoprotein (gp120), with which neuroleukin was found to share partial sequence homology. In this study, reduced GPI demonstrated similar activity to neuroleukin in a novel bioassay using human and rat neuroblastoma cell lines. In the presence of reduced GPI, these cells were found to differentiate, in terms of enhanced neurite extension at a reduced proliferation rate. These results demonstrate the existence of a novel growth factor activity of an evolutionary ancient enzyme. The nonreduced commercial form of GPI, probably the dimer, was found to be inactive in this bioassay. Using the neuroblastoma cells model system, we further investigated the significance of the region of homology to HIV-1 envelope glycoprotein (gp120) as the putative binding site of GPI to its receptor on neuronal cells.

Specific adhesion between pheochromocytoma (PC12) cells and adrenal medullary endothelial cells in co-culture.

Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.

Target dependent and independent stages in regeneration.

Goldfish retinae deprived of their target by tectal ablation showed characteristics of regeneration, such as sprouting activity in culture and changes in protein synthesis. These features resemble changes occurring in regenerating retinae of optic nerves which were subjected only to crush injury. Retinae of tectal ablated nerves maintained their ability to sprout in culture longer than retinae of crushed-injured optic nerves. This may indicate a possible absence of the machinery which regulates growth termination as reflected in this case, by the enduring growth activity in culture. Among the changes in protein synthesis which occurred in retinae following either tectal ablation or crush injury, the most pronounced were in tubulin and in some other polypeptides of the following molecular weights: 46-49, 65 and 74 kdalton. Results are discussed with respect to the possible role of the target in initiating regeneration upon disconnection of the nerve, and in terminating growth upon reconnection.

Antigenic determinants detected on adenoid lymphocytes.

This study was undertaken to detect the antigenic determinants expressed by adenoid lymphocytes. For this purpose, adenoid and peripheral blood lymphocytes obtained from the same patient were subsequently tested with human and hetero antisera. The research was based upon microlymphocytotoxicity where the various lymphocyte subsets were incubated with the antiserum and complement. The reactions were scored by the dye exclusion technique. The results indicated that: 1) compared with peripheral blood, the expression of the major histocompatibility gene products is enhanced by adenoid lymphocytes; 2) adenoid T lymphocytes express DRw antigens. These antigens were thought to be restricted to B lymphocytes and macrophages but have later been detected in T lymphocytes activated in vitro. Thus, lymphocytes derived from hypertrophied adenoids may simulate a culture activated in vivo; 3) because of their higher sensitivity to antibodies and complement-mediated cell lysis, adenoid lymphocytes may be useful in detecting minor histocompatibility gene products and differentiation antigens. It is concluded that adenoids represent a highly active lymphoid organ probably actively participating in the host's immunological defence mechanism.

Regenerating goldfish retinal explants: induction and maintenance of neurites by conditioned medium from cells originated in the nervous system.

Fiber outgrowth from goldfish regenerating retinas can be induced by conditioned medium of cloned cells which originated in the nervous system, i.e. glioma and neuroblastoma. Dilution of the released factor(s) was required to achieve optimal effect; high concentrations are detrimental. The fibers can be maintained for at least 2 weeks in vitro, and reach a length of several millimeters. This system may provide a means to purify and characterize neurotrophic factors involved in nerve regeneration.

Comparative studies of the aromatization of testosterone and epitestosterone by human placental aromatase.

The aromatization of epitestosterone (17 alpha-hydroxy-4-androsten-3-one) and testosterone by lyophilized human placental microsomes was studied. Upon incubation of epitestosterone, 12% was converted to 17 alpha-estradiol, 15% to 19-keto-epitestosterone (17 alpha-hydroxy-4-oxo-4-androsten-19-al), 10% to 19-hydroxyepitestosterone (17 alpha, 19-dihydroxy-4-androsten-3-one), and about 10% to several unidentified products. A similar incubation with testosterone resulted in 60% conversion to 17 beta-estradiol; 30% was unchanged. At increasing substrate concentrations (0.1-50 microM), the aromatization rate of epitestosterone increased gradually and did not reach a plateau, whereas aromatization rate of testosterone plateaued at about 3 microM. The presence of either testosterone or 17 beta-estradiol in concentrations 0.1-10 times the concentration of epitestosterone inhibited the aromatization of epitestosterone by about 70%, while the aromatization of testosterone was not inhibited by either epitestosterone or 17 alpha-estradiol. Lyophilization of fresh microsomes or storage of the lyophilized microsomes at -20 C greatly reduced the aromatizing activity upon epitestosterone but not upon testosterone. These results suggest that the aromatizing system for epitestosterone is different from that for testosterone.

Histocompatibility (HLA) antigens in heterotopic ossification associated with neurological injury.

Twenty patients with heterotopic ossification were HLA typed. The group consisted of 12 patients with severe cranio-cerebral injury and 8 with spinal cord injury. No significant differences in the frequency of any HLA antigens were found in these patients when compared to 631 healthy matched controls. None of the patients was B27 positive.