Guidelines on good clinical laboratory practice: bridging operations between research and clinical research laboratories.

A set of Good Clinical Laboratory Practice (GCLP) standards that embraces both the research and clinical aspects of GLP were developed utilizing a variety of collected regulatory and guidance material. We describe eleven core elements that constitute the GCLP standards with the objective of filling a gap for laboratory guidance, based on IND sponsor requirements, for conducting laboratory testing using specimens from human clinical trials. These GCLP standards provide guidance on implementing GLP requirements that are critical for laboratory operations, such as performance of protocol-mandated safety assays, peripheral blood mononuclear cell processing and immunological or endpoint assays from biological interventions on IND-registered clinical trials. The expectation is that compliance with the GCLP standards, monitored annually by external audits, will allow research and development laboratories to maintain data integrity and to provide immunogenicity, safety, and product efficacy data that is repeatable, reliable, auditable and that can be easily reconstructed in a research setting.

Isolation of a sealed homogeneous population of inner membrane fragments with inverted orientation from rat liver mitochondria using specific lectin immunoprecipitation.

A novel method for the isolation of well-defined populations of inside-out vesicles from rat liver mitochondria is described. The technique utilizes specific immunoprecipitation of vesicles with accessible carbohydrate residues from a mixed population of inner membrane fragments using wheat germ agglutinin and anti-wheat germ agglutinin IgG. The unprecipitated fraction comprises 30--50% of the original population and exhibits little or no cytochrome c oxidase activity as estimated with exogenous cytochrome c as substrate. Addition of deoxycholate to promote membrane disruption results in an 8--10-fold increase in enzymic activity compared to only 1.5--2.0-fold stimulation in standard preparations of submitochondrial particles. It is concluded that the lectin affinity-purified membranes represent a sealed homogeneous (90--95% pure) population of inside-out inner membrane vesicles.

Adenine nucleotide efflux in mitochondria induced by inorganic pyrophosphate.

The efflux of mitochondrial adenine nucleotide which is induced by addition of PPi to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 microM, Vmax being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The Km for PPi of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PPi.

Lectins as biochemical agents for the isolation of sealed membrane vesicles of defined polarity.

The asymmetric distribution of carbohydrate on biological membranes has provided the basis for the development of lectin-affinity methodology which permits the isolation of sealed, inside-out membrane fractions from heterogeneous populations of vesicles. Optimal conditions for these separations have been assessed employing purified right-side-out and inside-out vesicles derived from the plasma membrane of human erythrocytes as a model system. In this special case, homogeneous populations of defined polarity can be produced by varying the ionic conditions during formation of the vesicles. Surface-specific enzymic markers exist also for monitoring the integrity and orientation of a given population. Multivalent lectins such as wheat germ agglutinin and soya bean agglutinin which induce direct agglutination of erythrocyte membrane fragments containing accessible carbohydrate residues, selectively remove more than 90% of right-side-out and non-sealed membrane from a mixed population, a reaction which is inhibited by GluNAc or GalNAc, respectively. Non-agglutinating lectins, e.g. concanavalin A, immobilized on an inert matrix such as Sepharose 4B, may be employed to adsorb out specifically vesicles with exposed glycopeptides on their surface. In this technique, it is necessary normally to remove the non-sealed membranes on Dextran density gradients prior to the final preparation of inside-out vesicles on Con A-Sepharose. Finally, selective immunoprecipitation of fragments with accessible sugars may also be achieved after treatment with a non-agglutinating lectin (concanavalin A) followed by incubation with anti-concanavalin A IgG which promotes rapid aggregation of membrane containing exposed receptors for the lectin. These procedures should prove generally suitable for the isolation of tightly-sealed, inside-out membrane populations in a variety of biological systems. Pure populations of vesicles, exhibiting reversed polarity, are valuable in surface-labelling studies for investigating the structure, function and transmembrane distribution of integral membrane proteins/glycoproteins.

Evaluation of monoclonal antibodies to HIV-1 by neutralization and serological assays: an international collaboration. Collaborating Investigators.

In a National Institutes of Health (NIH)/World Health Organization (WHO)-sponsored collaboration, 26 laboratories characterized a coded panel of monoclonal antibodies (MAb) to HIV-1 envelope protein. The MAb were evaluated by serological [radioimmunoprecipitation, immunoblot, enzyme-linked immunosorbent assay (ELISA) and peptide mapping] and neutralization assays. Although laboratories used diverse neutralization assays that vary considerably in sensitivity, qualitatively similar data were obtained. The MAb were classified into three neutralization specificities: type-specific for MN and SF2, type-specific for IIIB, and group-specific for MN, SF2, and IIIB. The group-specific MAb displayed much lower neutralizing titers than the type-specific MAb. The specificity of MAb for neutralization was greater than for serological recognition of gp120 protein or peptide epitopes. Some MAb that bound to the same or closely overlapping linear epitopes had very different neutralization properties. The distinction between serological recognition and neutralization may result from differences in affinity of the MAb or may indicate that MAb can neutralize by interactions at a site distinct from the antibody binding site.

Evaluation of monoclonal antibodies to HIV-1 envelope by neutralization and binding assays: an international collaboration.

OBJECTIVE: To characterize a purified panel of monoclonal antibodies (MAb) to epitopes in HIV-1 envelope V3, CD4-binding region, C4 and gp41. DESIGN: Neutralization and/or binding activity data were obtained from 21 laboratories on a coded panel consisting of seven human MAb, seven mouse MAb, recombinant human CD4 immunoadhesin [CD4-immunoglobulin G (IgG)], normal human and normal murine Ig. METHODS: Laboratories performed a variety of neutralization assays and antigen binding assays with HIVIIIB, HIVMN and other laboratory strains of HIV-1. RESULTS: For a single MAb, there was up to a 10(3) range of neutralizing antibody titers between laboratories. The range in titers appeared to depend on the sensitivity of the neutralization assay. Two methods were used to consolidate the data from all laboratories, the geometric mean titer (GMT) and the median neutralizing titer (MNT). The panel of MAb were also analyzed by a variety of assays that measure binding activity to native or denatured epitopes. The relative binding activity of the MAb did not appear to correlate with neutralizing activity. CONCLUSION: Neutralization results from any single laboratory did not correlate with the collective data. The relative potency (rank order) of the MAb in the panel were equivalent when determined by GMT or MNT. These values may be useful to individual laboratories for estimating the sensitivity of their neutralization assays. The study also identified potential reference reagents with which neutralizing activity could be compared.

Neutralization of primary HIV-1 isolates by anti-envelope monoclonal antibodies.

OBJECTIVE: To evaluate human monoclonal antibodies (MAb) for neutralizing activity against primary HIV-1 isolates in peripheral blood mononuclear cells. DESIGN: Neutralization activity data were obtained from 11 laboratories on a coded panel consisting of six human MAb to HIV envelope V3, CD4-binding region or gp41. Hyperimmune globulin against HIV-1 and normal human immunoglobulin G were supplied as controls. Each laboratory received pre-titered virus for use in the studies. METHODS: Each laboratory measured neutralization of the MAb against laboratory strain HIVMN, genomic clone HIVJR-CSF, two subtype B and one subtype D primary isolates. RESULTS: The titers of the centrally supplied virus stocks as determined by re-titration or back-titration varied among laboratories and were generally 10-100-fold less than provided. The neutralizing activity of each MAb varied by as much as a 1000-fold among laboratories. These differences may result from varying sensitivity in neutralization assay protocols and the differing susceptibility of primary cells to infection with HIV-1. CONCLUSIONS: To consolidate the data from multiple laboratories, the neutralization titers were compared by classifying antibodies as neutralizing if the antibody concentration for 50% virus inhibition was < or = 10 micrograms/ml. By this criterion, the CD4-binding region and gp41 MAb neutralized all four subtype B viruses and the subtype D isolate in a few of the laboratories. The V3 MAb neutralized only HIVMN and the closely related HIVJR-CSF viruses.

Lysosomal membrane glycoproteins: properties of LAMP-1 and LAMP-2.

Several properties of the lysosomal membrane glycoproteins LAMP-1 and LAMP-2 have been analysed. Each molecule was strongly associated with lysosome membranes and was extracted only in the presence of detergent. Studies of the biosynthesis and processing of the glycoproteins showed that each contained a polypeptide core of approx. 43,000 Da as identified by use of tunicamycin and endoglycosidase H. Nascent glycoproteins pulse-labelled for 5 min with [35S]methionine were approx. 92,000 Da. These precursor molecules were processed in 30 min to highly heterogeneous mature glycoproteins of approx. 110,000 Da(LAMP-1) and 105,000 Da(LAMP-2). Concomitant with the increase in apparent Mr the molecules became endoglycosidase H resistant and acquired sialic acid residues, indicating that they were converted to complex-type oligosaccharides. The final maturation of the glycoproteins was blocked by monensin. Immunohistochemical analysis of tissues from Balb/c and Beige/J mice showed that the molecules were present on many types of cells, consistent with their presence in lysosomes. The patterns of tissue expression of LAMP-1 and LAMP-2 in the two mouse strains were the same except that the intensity of staining of LAMP-2 was less than that of LAMP-1. LAMP-2, but not LAMP-1, gave a decreased immunofluorescent staining intensity in transformed HaNIH as compared with NIH/3T3 cells. The marked similarities between the LAMP proteins raise the consideration of common functions, possibly associated with the high oligosaccharide content of the molecules.

HIV-mediated defects in immune regulation.

The Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases (NIAID), sponsored a Workshop on HIV-Mediated Defects in Immune Regulation on September 29-30, 1993. Workshop participants included investigators in basic research of immune regulation, animal models of HIV disease, HIV epidemiology, and HIV clinical research and treatment. The purpose of the workshop was to describe and evaluate biological mechanisms of HIV-mediated immune deficiency other than direct killing of infected CD4+ cells. The workshop focused on HIV-mediated dysfunction in signal transduction and in T cell development and maturation. Mechanisms by which HIV has been proposed to influence signal transduction include gp120 ligation to CD4, HIV superantigen(s), and HIV-mediated perturbations in signal pathway components (e.g., receptors, kinases, phosphatases, cytokines, and cyclins). As a result of signal dysfunction, cells may fail to respond to foreign antigens (anergy) or become predisposed to enter suicide pathways, otherwise known as programmed cell death or apoptosis. Programmed cell death is a normal immune regulatory mechanism that is activated to prevent anti-self responses and also to delete expanded but no longer needed cell populations. In the immune system, new cells are constantly produced from stem cells to replace those that die from age, pathological response, or programmed cell death. Dysfunction in these new cells may occur if HIV causes changes in the structural environment of the thymus and lymph nodes, or in cytokine signals.

Early phases of HIV type 1 infection.

A workshop entitled "Early Phases of HIV-1 Infection" was held to review current research on the immunological and virological aspects of early phases of HIV infection in humans and in animal models, to identify studies for future research, and to foster collaborations among investigators in the biomedical community. In infections of adults, the appearance of cytotoxic T lymphocyte activity, when present, coincides with a decrease in viral load as measured by plasma viremia. In neonatal infections, however, an initial decrease in viral load has been observed months before cytotoxic T lymphocytes are detected. Immunological data, from a limited number of patients, indicated that CD8+ cytotoxic T lymphocytes detected early after HIV-1 infection may recognize epitopes in any of several HIV-1 proteins: Env, Gag, Pol, Tat, and Nef. With regard to the humoral antibody response, anti-Env binding antibodies appear before neutralizing antibodies and do not predict the appearance of neutralizing activity. The time at which neutralizing antibody appears is variable and unpredictable. Preliminary data indicate that early viral peak load does not predict disease progression in many cases, and the phenotype or virulence of the virus appears to be a critical variable. However, the quantity of HIV-1 RNA in plasma is a strong CD4+ T cell-independent predictor of outcome following HIV-1 seroconversion in homosexual men. Early, high virus load with sustained viremia is often accompanied, in both adults and infants, by the inability to mount an effective immune response, resulting in rapid disease progression.

International collaboration comparing neutralization and binding assays for monoclonal antibodies to simian immunodeficiency virus.

Thirteen laboratories characterized a coded panel of 10 MAbs to SIVmac251 envelope protein in a collaboration organized by the National Institute of Allergy and Infectious Diseases (NIAID). The MAbs were examined against SIV isolates in neutralization and radioimmune precipitation, immunoblot, enzyme-linked immunosorbent, and radioimmune assays. Although laboratories employed diverse neutralization assays that varied in sensitivity there was agreement on the relative ability of the MAbs to neutralize SIVmac251. Additionally, even though the quantity of any single MAb required to neutralize SIVmac251 varied between laboratories, there was agreement on the rank-order strength fo the five neutralizing MAbs. Based on the data from this study, the MAbs were classified according to their neutralization potential as high efficiency (MAb concentration, < 5 micrograms/ml), low efficiency (MAb concentration, 5-100 micrograms/ml), or nonneutralizing (MAb concentration, > 100 micrograms/ml). The MAbs could be assigned to four serological groups based on ability to cross-neutralize and bind different SIV isolates. The distinction between groups I, II, and III were based on the limited neutralization data obtained with the sooty mangabey isolate.

Neutralization of HIV-1.

A Workshop on Neutralization of HIV-1: Technology and reagents for analysis of prophylactic vaccines clinical trials, sponsored by the Food and Drug Administration (FDA) and the Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), was held on April 19-20, 1993, in Bethesda, Maryland. This workshop brought together researchers who are involved in the development, testing, and evaluation of HIV-1 prophylactic vaccines. The major objectives were (1) to discuss critically the different neutralization and binding assays that are currently used in the evaluation of immune sera; (2) to identify assays that will measure the "most relevant" antibodies, which are likely to predict neutralization of primary isolates; and (3) to identify well-characterized reference reagents, which could be used to standardize neutralization assays used in laboratories around the world.

Cellular and anatomical reservoirs of HIV-1 in patients receiving potent antiretroviral combination therapy.

The eradication of human immunodeficiency virus 1 (HIV-1) from infected persons is the ultimate goal of HIV therapeutic interventions. Great strides have been made in developing potent antiretroviral regimens that greatly suppress HIV-1 replication. Despite these therapeutic advances, major obstacles remain to eradicating HIV-1. Reservoirs of HIV-1 have been identified that represent major impediments to eradication. Conceptually, there are 2 types of sanctuaries for HIV-1, cellular and anatomical. Cellular sanctuaries may include latent CD4+ T cells containing integrated HIV-1 provirus; macrophages, which may express HIV-1 for prolonged periods; and follicular dendritic cells, which may hold infectious HIV-1 on their surfaces for indeterminate lengths of time. The key anatomical reservoir for HIV-1 appears to be the central nervous system. An understanding of the nature of HIV within these reservoirs is critical to devising strategies to hasten viral eradication.

A kinetic analysis of biosynthesis and localization of a lysosome-associated membrane glycoprotein.

The biosynthesis and subcellular distribution of a major lysosomal membrane glycoprotein of mouse embryo 3T3 cells, LAMP-1, have been examined by [35S]methionine pulse-labeling, sucrose density gradient fractionation, and oligosaccharide analysis. Mature LAMP-1, immunoprecipitated after labeling for 4 h, had a molecular mass of about 110,000 Da. It comigrated during sucrose density fractionation with lysosomal markers, consistent with previous electron microscopic evidence for its localization in lysosomal membranes. Precursor molecules, pulse-labeled for 5 min and extracted during the first 15 min of post-translational processing, were concentrated in the rough endoplasmic reticulum fraction as a species of 92,000 Da. Within 30 min after synthesis, LAMP-1 was found in fractions enriched in Golgi and lysosomal marker enzyme activities as the mature 110,000-Da glycoprotein. Oligosaccharide processing was complete by 1 h after synthesis, and the mature glycoprotein remained in a fraction bearing lysosomal markers. Treatment of the 92,000-Da precursor with endo-beta-N-acetyl-glucosaminidase H produced a core polypeptide of 43,000 Da. Pulse-labeling in the presence of tunicamycin yielded a 42,000-Da form of LAMP-1, which was converted within 30 min to a 43,000-Da molecule. Bio-Gel column chromatography and hexosamine/hexosaminitol analyses indicated that the mature 110,000-Da molecule contained both complex-type and high-mannose N-linked oligosaccharides.

Neutralizing antibodies against HIV determined by amplification of viral long terminal repeat sequences from cells infected in vitro by nonneutralized virions.

Based on the earliest intracellular synthesis of nascent HIV-1 long terminal repeat (LTR) fragments, we have established a heminested polymerase chain reaction (HNPCR) amplification of the 5' LTR sequences (LTR-HNPCR) for molecular assay of virus-neutralizing antibodies (VNAb). We incubated HIV antibodies with virus isolates for an hour, followed by addition of lymphoid cells (H9 or peripheral blood mononuclear cells [PBMC]) and further incubation for an hour. After washing the cells three times for thorough removal of free virions and antibodies, LTR-HNPCR consistently revealed HIV DNA in H9 cells after 15 minutes, in PBMC after 4 hours, and corresponding virion expression after 7 days in culture. Replication-competent HIV detected by LTR-HNPCR following overnight culture of infected PBMC for 16 to 18 hours was comparable with tissue culture infectivity measured by p24 antigen expression at 7 days. After establishing a molecular assay for in vitro HIV neutralization by HIV Ig, a panel of five HIV isolates tested with 6 monoclonal antibodies and HIV Ig revealed that LTR-HNPCR was comparable with other VNAb assays. These preliminary data indicate that the molecular assay for HIV neutralization has a clear-cut end point, is specific, reliable, and more rapid than other VNAb assays. Therefore, it offers potential utility in evaluating immune response to candidate vaccines.

Labeling of succinate-cytochrome c reductase with 125I. Accessibility of the peptides to the aqueous phases on the cytosolic and matrix sides of the mitochondrial membrane.

Lactoperoxidase-catalyzed radioiodination was used to study the arrangement of the component peptides of succinate-cytochrome c reductase with respect to the aqueous phases on each side of the mitochondrial inner membrane. Mitochondria depleted of their outer membrane and inside-out vesicles purified from submitochondrial particles by the lectin-affinity procedure (D'Souza, M. P., and Lindsay, J. G. (1981) Biochim. Biophys. Acta 640, 463-472) were iodinated using immobilized preparations of lactoperoxidase. The labeled membranes were solubilized in detergent and the succinate-cytochrome c reductase was purified by immunoprecipitation with specific IgG. Analysis of the radioiodine distribution after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparison with peptide stain patterns show that bands 2 (64 kilodaltons), 6 (30 kilodaltons), 9 (15 kilodaltons), and 11 (less than 10 kilodaltons) are labeled from the cytoplasmic surface of the membrane. Bands 1 (72 kilodaltons), 4 (48 kilodaltons), and 8 (20 kilodaltons) appear to be labeled on the matrix side of the membrane, while bands 3 (52 kilodaltons), 5 (35 kilodaltons), 7 (25 kilodaltons), and 10 (11 kilodaltons) are labeled from both sides of the membrane. Tentative identification of the labeled bands suggests that band 1 is the large subunit of succinate dehydrogenase. Bands 3 and 4 represent proteins which have been referred to as core proteins I and II. Bands 5 and 6 are the proteins associated with cytochromes b and c1, respectively; band 7 is the Rieske iron-sulfur protein.

Evaluation of monoclonal antibodies to human immunodeficiency virus type 1 primary isolates by neutralization assays: performance criteria for selecting candidate antibodies for clinical trials. AIDS Clinical Trials Group Antibody Selection Working Group.

A collaborative study was organized to identify monoclonal antibodies (MAbs) that may be broadly and potently neutralizing for a panel of human immunodeficiency virus type 1 (HIV-1) low-passaged adult and pediatric primary isolates in peripheral blood mononuclear cells. Five laboratories evaluated a coded panel of seven human MAbs to HIV-1 subtype B envelope V3, CD4 binding region, gp41, and other conformationally sensitive determinants. Each laboratory measured neutralizing activity of the MAbs against the laboratory isolate HIV(MN) and a panel of 9 subtype B primary isolates. Antibodies were classified as suitable candidates for future clinical studies if they could neutralize at least half of the 9 primary isolates at a concentration of < or = 25 microg/mL for 90% viral inhibition. The study identified three MAbs that met stated performance criteria: IgG1b12, 2G12, and 2F5. These results may provide a rationale for examining the clinical efficacy, either singly or in combination, of the three MAbs.

Structural analysis of human adult and fetal alkaline phosphatases by cyanogen bromide peptide mapping.

The adult and fetal forms of human intestinal alkaline phosphatase (ALPase; orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) are indistinguishable by a variety of analytical procedures. However, they differ electrophoretically and can be differentiated by binding studies with monoclonal antibodies. In this report, these two enzymes along with placental and liver ALPases are compared by the technique of CNBr peptide mapping, and the role of carbohydrate in generating these patterns is investigated. NaDodSO4/PAGE of CNBr digests of radiolabeled ALPases from fetal and adult intestine shows that these two isozymes share five of seven common-sized CNBr fragments. Placental ALPase shares only one common-sized fragment with either intestinal enzyme. Liver ALPase has no CNBr fragments in common with any of the others. These data indicate that fetal intestinal ALPase is not a heterodimer of one subunit each of intestinal ALPase and placental ALPase as has been postulated. CNBr digests of neuraminidase-treated enzymes reveal a change of mobility of only one CNBr band in each of fetal intestinal, placental, and liver ALPases, indicating the presence of sialic acid residues in these fragments. Periodic acid/Schiff reagent staining (specific for carbohydrate) of CNBr digests of fetal and adult intestinal ALPases reacts with only one band in each enzyme, which is the same band from the fetal enzyme shown to contain sialic acid. However, fetal and adult intestinal ALPases each contain at least one CNBr fragment of unique size that is apparently nonglycosylated.