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N Dunlop

Publications and source records attributed to N Dunlop.

At least 19 recordsLinked to original sources
PubMed2000

The flavonoid baicalin exhibits anti-inflammatory activity by binding to chemokines.

Baicalin (BA) is a flavonoid compound purified from the medicinal plant Scutellaria baicalensis Georgi and has been reported to possess anti-inflammatory and anti-viral activities. In order to elucidate the mechanism(s) of action of BA, we tested whether BA could interfere with chemokines or chemokine receptors, which are critical mediators of inflammation and infection. We observed that BA inhibited the binding of a number of chemokines to human leukocytes or cells transfected to express specific chemokine receptors. This was associated with a reduced capacity of the chemokines to induce cell migration. Co-injection of BA with CXC chemokine interleukin-8 (IL-8) into rat skin significantly inhibited IL-8 elicited neutrophil infiltration. BA did not directly compete with chemokines for binding to receptors, but rather acted through its selective binding to chemokine ligands. This conclusion was supported by the fact that BA cross-linked to oxime resin bound chemokines of the CXC (stromal cell-derived factor (SDF)-1alpha, IL-8), CC (macrophage inflammatory protein (MIP)-1beta, monocyte chemotactic protein (MCP)-2), and C (lymphotactin (Ltn)) subfamilies. BA did not interact with CX3C chemokine fractalkine/neurotactin or other cytokines, such as TNF-alpha and IFN-gamma, indicating that its action is selective. These results suggest that one possible anti-inflammatory mechanism of BA is to bind a variety of chemokines and limit their biological function.

Animals↗
PubMed1999

HIV-specific immunity following immunization with HIV synthetic envelope peptides in asymptomatic HIV-infected patients.

OBJECTIVE: A phase I trial was conducted to evaluate the safety and immunogenicity of an HIV synthetic peptide vaccine in HIV-seropositive individuals. The immunogens used in this study were PCLUS 3-18MN and PCLUS 6.1-18MN envelope peptides. METHODS: Eight HIV-infected patients received six subcutaneous injections of 160 microg PCLUS 3-18MN in Montanide ISA 51 and were followed longitudinally for a year after the first immunization. Peripheral blood mononuclear cells (PBMC) were tested for peptide-specific T helper and cytotoxic T cell (CTL) responses, HIV-1MN neutralizing antibodies and antibodies against HIV PCLUS 3 and P18 MN peptides. RESULTS: PCLUS 3-1 8MN-specific T helper responses were significantly increased at 36 weeks (P < 0.05, after adjustment for multiple comparisons) following initial immunization with PCLUS 3-18MN. A P18MN-specific CTL response, not present prior to vaccination, was observed after immunization in one patient. Serum HIV-1 MN-neutralizing antibody titers increased in each of the three patients who had low titers prior to immunization. Plasma HIV RNA levels and CD4 cell counts did not change appreciably during the study period. CONCLUSIONS: This trial demonstrates that both peptides can be safely administered to HIV-infected individuals and that PCLUS 3-18MN induces increases in HIV peptide-specific immune responses.

AIDS Vaccines↗
PubMed1998

Organization of the human glucokinase regulator gene GCKR.

Glucokinase plays an important role in regulating insulin secretion in response to changes in blood glucose levels. As a result, one form of maturity onset diabetes of the young (MODY) results from haploinsufficiency of glucokinase. In both liver and pancreatic islet, glucokinase is allosterically regulated by an inhibitory protein (glucokinase regulatory protein, GCKR). GCKR has therefore become an important gene for functional analysis in type 2 diabetes. To allow genetic assessment of any such role, we have determined the structure of the human GCKR gene. Characterization of P1 and YAC clones containing GCKR shows it to consist of 19 exons spanning 27 kb. RT-PCR, RACE, and RNase protection experiments defined a transcriptional start site for GCKR 66 bp upstream of the initiation codon, but provided no evidence for islet cell specific alternative splicing in the rat. By SSCP screening, a common polymorphic sequence variant has been defined within exon 15 of human GCKR, at nt 1400 of the cDNA. This alters amino acid residue 446 from proline, conserved in rat and Xenopus, to leucine.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Dose-limiting neurotoxicity in a phase I study of penclomedine (NSC 388720, CRC 88-04), a synthetic alpha-picoline derivative, administered intravenously.

3,5-Dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine (penclomedine, NSC 338720, CRC 88-04) is an alpha-picoline derivative with anti-tumour activity in preclinical models. Penclomedine administration by 1-h intravenous infusion on 5 consecutive days was repeated 3 weekly in the absence of dose-limiting toxicity (DLT) or disease progression. Five dose levels were investigated (22.5-340 mg m(-2) day[-1]). Eight men and eight women were entered, median age 59 years (range 39-73 years), with good performance status (ECOG 0/1) in 11 patients. A total of 13 out of 16 patients had received previous chemotherapy. Common toxicity criteria grade (CTCg) II vomiting was recorded at all dose levels. Neurotoxicity (cerebellar ataxia and dizziness) was the DLT, CTCg III toxicity occurring in three out of three patients treated at 340 mg m(-2) day(-1). CTCg III dizziness was noted in one out of three patients at 250 mg m(-2) day(-1). Neurotoxicity developed during the 1-h infusion and persisted for a variable period (maximum 5 h) after infusion. Prophylactic antiemetic drugs appeared to reduce associated vomiting but did not prevent ataxia. No antiproliferative toxicities were noted and no anti-tumour responses were documented. Penclomedine pharmacokinetic studies confirmed preclinical evidence of extensive apparent distribution (93 l m[-2]) and rapid clearance (41 l h[-1] m[-2]). Purkinje cell loss has been identified in preclinical models after intraperitoneal administration (O'Reilly et al, 1996a) and further clinical development of penclomedine will focus on oral administration.

Adult↗
PubMed1998

Organisation of the human PAX4 gene and its exclusion as a candidate for the Wolcott-Rallison syndrome.

Neonatal diabetes mellitus is a rare condition, the causes of which are mostly unknown. One well defined though very rare entity is the autosomal recessive Wolcott-Rallison syndrome, in which permanent neonatal diabetes, osteopenia, and epiphyseal dysplasia occur. Only five previous families have been reported, and here we describe the second in which parental consanguinity was present. The proband was born to first cousin parents and died at 2 years from the sequelae of poorly controlled diabetes. To test the hypothesis that mutation of PAX4, required in the mouse for pancreatic islet beta cell development, might cause WRS, the structure of the human PAX4 gene was deduced and DNA from two unrelated WRS patients sequenced. No PAX4 mutation was present, though the entire coding region was sequenced in both patients. It therefore appears unlikely that PAX4 is involved in the aetiology of Wolcott-Rallison syndrome, though it remains a good candidate for other forms of neonatal diabetes mellitus.

Abnormalities, Multiple↗
PubMed1997

Cytokine-in-adjuvant steering of the immune response phenotype to HIV-1 vaccine constructs: granulocyte-macrophage colony-stimulating factor and TNF-alpha synergize with IL-12 to enhance induction of cytotoxic T lymphocytes.

To enhance and steer immune responses to synthetic peptide vaccines toward selected functional types and to understand the cooperative action of cytokines in fine-tuning the immune response, we attempted to influence the in vivo cytokine environment by delivering cytokines directly to the microenvironment in which the immune response is initiated. Here we study the effects of IL-2, IL-4, IL-7, IL-1beta, IL-12, IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) incorporated with peptide in adjuvant on a variety of responses elicited: CTL, T cell proliferation, cytokine production and message, and Ab isotype. We show GM-CSF to be the single most effective cytokine for enhancing both cellular and humoral immunity to two previously characterized HIV-1 MN vaccine constructs. Novel synergies were also detected. GM-CSF synergized with IL-12 for CTL induction in BALB/c mice concomitant with suppression of Th2 cytokines IL-4 and IL-10. TNF-alpha also synergized with IL-12, but by a different mechanism, inducing IFN-gamma production in BALB/c mice and thus shifting the response to a Th1 phenotype. The results presented here suggest that in addition to IL-2, optimum induction of CD8+ CTL in vivo requires a combination of cytokines, including GM-CSF (probably acting to enhance Ag presentation and CD4+ cell help) and IL-12 (steering the Th response toward Th1 cytokines).

AIDS Vaccines↗
PubMed1996

Candidate HIV type 1 multideterminant cluster peptide-P18MN vaccine constructs elicit type 1 helper T cells, cytotoxic T cells, and neutralizing antibody, all using the same adjuvant immunization.

Cytotoxic T lymphocytes and Th1 cells have been suggested to play a critical role in the control of HIV infection. It is therefore considered that a vaccine that induces a strong Th1 response and CTL response would be more efficacious than one that does not in providing protection against infection and progression toward AIDS. In this study we show that immunization with vaccine constructs consisting of multideterminant cluster peptides containing Th epitopes from the HIV-1IIIB envelope colinearly synthesized to peptide 18MN, is capable of inducing a Th1 response in mice and, dependent on this help, both cytotoxic T cell responses and neutralizing antibody toward the homologous strain of HIV. Moreover, the cytotoxic T cell response elicited by immunization with a mixture of cluster peptide-P18MN vaccine constructs was at least as cross-reactive against known viral variant P18 target sequences as a CTL line produced by immunization with a vaccinia construct expressing recombinant gp160 MN. Four adjuvants were compared to optimize both CTL and antibody responses. A single adjuvant formulation of peptide in ISA 51 could elicit all three: Th1 cells, CTLs, and neutralizing antibody. Thus, immunization directed toward the development of a cytotoxic T cell response does not preclude the development of neutralizing antibody and vice versa, i.e., the responses are not mutually exclusive. The immunization protocol described here should be directly applicable for study in clinical trials aimed at HIV-1 immunotherapy or prophylaxis.

AIDS Vaccines↗
PubMed1994

Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody.

The ability of antibodies to neutralize diverse primary isolates of human immunodeficiency virus-type 1 in vitro has been questioned, with implications for the likely efficacy of vaccines. A recombinant human antibody to envelope glycoprotein gp120 was generated and used to show that primary isolates are not refractory to antibody neutralization. The recombinant antibody neutralized more than 75 percent of the primary isolates tested at concentrations that could be achieved by passive immunization, for example, to interrupt maternal-fetal transmission of virus. The broad specificity and efficacy of the antibody implies the conservation of a structural feature on gp120, which could be important in vaccine design.

AIDS Vaccines↗
PubMed1994

In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity.

A method is described that allows for the improvement of antibody affinity. This method, termed complementary-determining region (CDR) walking, does not require structural information on either antibody or antigen. Complementary-determining regions are targeted for random mutagenesis followed by selection for fitness, in this case increased binding affinity, by the phage-display approach. The current study targets a human CD4-binding-site anti-gp120 antibody that is potently and broadly neutralizing. Evolution of affinity of this antibody demonstrates in this case that affinity can be increased while reactivity to variants of human immunodeficiency virus type 1 is broadened. The neutralizing ability of this antibody is improved, as assayed with laboratory and primary clinical isolates of human immunodeficiency virus type 1. The ability to produce human antibodies of exceptional affinity and broad neutralizing ability has implications for the therapeutic and prophylactic application of antibodies for human immunodeficiency virus type 1 infection.

Amino Acid Sequence↗
PubMed1993

Construction of an HIV-1 peptide vaccine containing a multideterminant helper peptide linked to a V3 loop peptide 18 inducing strong neutralizing antibody responses in mice of multiple MHC haplotypes after two immunizations.

Peptide constructs comprised of multideterminant Th peptides from the envelope glycoprotein of HIV previously identified to induce proliferative responses in four different haplotypes of mice and IL-2 responses in 52 to 73% of HIV positive, Ag-responsive patients, were colinearly synthesized with the peptide 18 of the V3 loop of HIV-1 gp 160, corresponding to the principal neutralizing determinant of HIV-IIIB. The segments containing clusters of overlapping Th epitopes were called cluster peptides. Cognate help for peptide 18 antibody was elicited after a single immunization in all strains of mice that had previously responded to a T cell epitope encompassed by the cluster peptides. Animals boosted with cluster peptide-peptide 18 constructs 36 to 52 wk later displayed secondary antibody responses. Cluster peptide 3-peptide 18 induced antibody that neutralized homologous virus in one strain of mice although strong peptide 18 antibody responses were detected in all four strains of mice. The most promising construct, cluster peptide 6-peptide 18, induced neutralizing antibody in all strains of mice tested, and in two strains the level of neutralizing antibody achieved was comparable to levels adequate for protection from homologous viral challenge in chimpanzees. After a single boost, antibody titers for 90% neutralization in the range of 1/1000 to 1/16,000 were achieved. These neutralizing titers against the homologous viral strain, after just two immunizations, are at least four- to eightfold higher than the highest titered other polyclonal V3-specific immune sera we have ever observed in our laboratories. We also asked why some sera neutralized and others with similar ELISA titers did not. No correlation was found between neutralization and isotype or affinity for peptide or gp 120. We could not account for neutralization by antibodies to the helper sites. Substitutions made in the central loop region of peptide 18, amino acid residues PGRAF, dramatically reduced binding of both neutralizing and non-neutralizing sera although some fine specificity differences between neutralizing and nonneutralizing sera were noted. These results have implications for the design of synthetic peptide vaccines for HIV.

AIDS Vaccines↗
PubMed1993

HIV experimental vaccines based on the iscom technology using envelope and GAG gene products.

In previous experiments gp160 incorporated into iscom was shown to induce neutralizing antibodies to the homologous as well as the heterologous isolates of HIV-1 (Akerblom et al., AIDS Res., 1991). In the present work we have incorporated into iscoms three defined recombinant DNA products of HIV-1. The carboxy-terminal part of gp120 expressed in E. Coli-PB-1; a chimera containing parts of both p24 and p15 expressed in E. coli-GAG; and baculovirus gp160 cloned in baculovirus and produced in insect cells. Immune responses were induced by the iscom preparations to the homologous antigen as well as to defined recombinant products and to the synthetic peptide RP135 (aa 304-328) harboring a neutralizing epitope. Sera from mice immunized with PB1-iscoms and gp160 (baculo) iscoms were tested in a syncytie inhibition assay. The serum from a mouse immunized with PB1 iscoms reacted strongly with the synthetic peptide RP135 and also neutralized the homologous isolate HIV-1/IIIB with a neutralization titer of 1/64. Three gp160 (baculo) iscom antisera were tested, of which two reacted strongly with the synthetic peptide RP135 but did not neutralize the homologous isolate HIV-1/IIIB. High serum titers were induced in mice by the gp160 iscoms (2 micrograms) to homologous antigen and the recombinant DNA E. coli construct p121 covering part of gp41. The ceilings of the antibody responses were reached after two immunizations. The PB1- and GAG-iscoms required three immunizations to reach the ceiling of the antibody response.

AIDS Vaccines↗
PubMed1992

Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies.

Amino Acid Sequence↗
PubMed1992

Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons.

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.

AIDS Vaccines↗
PubMed1991

Cross-neutralizing antibodies to HIV-1 in mice after immunization with gp160 iscoms. Dissection of the immune response.

Gp160 expressed in vaccinia and produced in vero cells was integrated into iscoms. Gp160 iscoms elicited a high serum antibody response in mice, and after two immunizations a ceiling was reached. The serum antibody response was dissected by the use of defined recombinant DNA products, representing different regions of the gp160 molecule. High antibody titers to the peptid RP135 (a.a. 296-332) correlated with induction of neutralizing serum antibodies. In some animals, gp160 (IIIB) iscoms elicited cross-neutralizing antibodies that also neutralized the distantly related RF isolate.

AIDS Vaccines↗
PubMed1990

Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees.

Emergence in two chimpanzees of human immunodeficiency virus type 1 (HIV-1) IIIB variants resistant to neutralization by the preexisting antibody is described. Viruses isolated from the HIV-1 IIIB gp120-vaccinated and -challenged animal were more resistant to neutralization by the chimpanzee's own serum than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. However, all reisolated viruses were 16- to 256-fold more neutralization resistant than the inoculum virus to antibodies binding to the third variable domain (V3) of the HIV-1 external envelope. Early chimpanzee serum samples that neutralized the inoculum strain but not the reisolated viruses were found to bind an HIV-1 IIIB common nonapeptide (IQRGPGRAF) derived from the gp120 isolate-specific V3 domain shown to induce isolate-specific neutralization in other animals. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo-infected animals indicated that early resistance to neutralization by an HIV-1 IIIB monoclonal antibody (0.5 beta) was conferred by changes outside the direct binding site for the selective neutralizing antibody. The reisolated neutralization-resistant isolates consisted of the lower-replication-competent virus subpopulations of the HIV-1 IIIB stock, as confirmed by biological and sequence analyses. In vitro passage of the HIV-1 IIIB stock through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody resulted in homogenic amplification of the more-replication-competent subpopulation preexisting in the original viral stock, suggesting a role for the immune system in suppressing the more-replication-competent viruses.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Evidence for rapid selection and deletion of HIV-1 subpopulations in vivo by V3-specific neutralizing antibody: a model of humoral-associated selection.

Emergence in two chimpanzees of HIV-1 HTLV-IIIB variants resistant to neutralization by the pre-existing antibody is described. Viruses isolated from the HTLV-IIIB gp120 vaccinated and challenged animal were more resistant to neutralization, had more heterogenic genomes and showed more pronounced antigenic drift as determined by serotypic neutralization analysis than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. The earliest selecting antibody population detected in the chimpanzees appeared to be conformationally dependent. This was demonstrated by its ability to neutralize only the most replication-competent sub-populations of the HTLV-IIIB inoculum strain, yet was found to bind a HTLV-IIIB common nonapeptide (IQRGPGRAF) derived from the gp120 isolate-specific 3rd variable domain (V3) also known to induce isolate-specific neutralization in multiple species. Each of the recovered isolates had become resistant to neutralization by both a monoclonal (0.5 beta) and polyclonal HTLV-IIIB gp120 V3-specific antibody by as much as 16 to 256-fold. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo infected animals indicated that resistance to neutralization was conferred by changes outside the direct binding site for the selective neutralizing antibody. Further studies indicated that apparent neutralization-resistant variants, yielded after in vitro passage through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody, result from the homogenic amplification of the more replication competent sub-population pre-existing in the original viral stock. These faster replicating sub-populations appear to dominate initially and therefore are the initial prime targets recognized by the chimpanzee humoral immune system upon infection in vivo and thus eliminated. The described finding of virus escape due to a conformationally-induced flexibility of the major neutralization epitope termed "conformational-V3 hypervariability", coupled to the known primary amino acid hypervariability of this epitope (V3) termed "linear-V3 hypervariability", may be all that is required by the virus to survive in an otherwise effective humoral immune system.

Amino Acid Sequence↗
PubMed1990

Heat-labile, complement-like factor(s) of animal sera prevent(s) HIV-1 infectivity in vitro.

We studied inactivation of HIV-1 by fresh sera of animals. We found that while fresh sera of humans and chimpanzees (among others) did not have antiviral activity, fresh sera of several other mammals, especially those of rodents and felines, showed a dose-dependent viral-inactivating property against cell-free HIV-1; these sera were also capable of inactivating virus preadsorbed to cells, similar to neutralizing antibody. The activity was destroyed by heating to 56 degrees C, required Ca2+ but no antiviral antibody, and therefore apparently involves the classical complement pathway. The activity could not be ascribed to any single fraction of sera separated on a size exclusion HPLC column. Mouse serum (the only one tested) also inactivated HIV-2. The data are consistent with classical C-mediated pathway inactivation of HIV-1 and HIV-2 by animal sera and is the first report of any "complement-like" antilentiviral serum factor. Elucidation of this mechanism may aid in understanding the lack of activity of human serum against HIV-1 and may prove useful in combined interventive strategies against HIV-1.

Animals↗