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Biomedical subjects

B D Walker

Publications and source records attributed to B D Walker.

At least 55 records · Page 3Linked to original sources
PubMed2000

Antiretroviral resistance during successful therapy of HIV type 1 infection.

HIV type 1 (HIV-1) drug resistance mutations were selected during antiretroviral therapy successfully suppressing plasma HIV-1 RNA to <50 copies/ml. New resistant mutant subpopulations were identified by clonal sequencing analyses of viruses cultured from blood cells. Drug susceptibility tests showed that biological clones of virus with the mutations acquired during successful therapy had increased resistance. Each of the five subjects with new resistant mutants had evidence of some residual virus replication during highly active antiretroviral therapy (HAART), based on transient episodes of plasma HIV-1 RNA > 50 copies/ml and virus env gene sequence changes. Each had received a suboptimal regimen before starting HAART. Antiretroviral-resistant HIV-1 can be selected from residual virus replication during HAART in the absence of sustained rebound of plasma HIV-1 RNA.

Anti-HIV Agents↗
PubMed2000

Impaired CTL recognition of cells latently infected with Kaposi's sarcoma-associated herpes virus.

Kaposi's sarcoma-associated herpes virus (KSHV) is a recently identified human gamma2-herpesvirus associated with Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease. We reasoned that CTL responses may provide host defense against this virus, and consequently, KSHV may have evolved strategies to evade the CTL-mediated immune surveillance. In this study six B cell lines latently infected with KSHV were found to express reduced levels of HLA class I surface molecules compared with B cell lines transformed by the related gamma-herpesvirus EBV. KSHV-infected cells also required higher concentrations of soluble peptides to induce efficient CTL-mediated lysis than control cell lines and were unable to process and/or present intracellularly expressed Ag. Incubation of the KSHV-infected cell lines with high concentrations of soluble HLA class I binding peptides did not restore the deficient HLA class I surface expression. To assess the underlying mechanisms of these phenomena, TAP-1 and TAP-2 gene expression was analyzed. While no attenuation in TAP-2 expression was observed, TAP-1 expression was significantly reduced in all KSHV cell lines compared with that in controls. These results indicate that KSHV can modulate HLA class I-restricted Ag presentation to CTL, which may allow latently infected cells to escape CTL recognition and persist in the infected host.

Antigen Presentation↗
PubMed2000

Analysis of successful immune responses in persons infected with hepatitis C virus.

Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-gamma, a "stunned" phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.

Acute Disease↗
PubMed2000

Comparative effects of azimilide and ambasilide on the human ether-a-go-go-related gene (HERG) potassium channel.

OBJECTIVE: To evaluate the effects of azimilide and ambasilide on the biophysical properties of the human-ether-a-go-go-related (HERG) channel. METHODS: HERG was stably transfected into Chinese hamster ovary (CHO-K1) cells and currents were measured using a whole cell, voltage-clamp technique. RESULTS: Azimilide had a 'dual effect', inhibiting current at voltage steps above -40 mV and augmenting current at -40 and -50 mV. Tail current inhibition following a step to +30 mV did not vary with temperature (IC(50) 610 nM at 22 degrees C and 560 nM at 37 degrees C). The agonist effect at -50 mV was concentration-dependent and correlated with a hyperpolarizing shift in the V(1/2) of activation (r=0.98, P<0.05). Time constants of inactivation were faster and there was a -10 mV shift in the V(1/2) of steady state inactivation suggestive of open and inactivated state binding. By comparison, ambasilide inhibited HERG channels with lower potency (IC(50) 3.6 microM), in a voltage- and time-dependent but frequency-independent manner (0.03-1 Hz). Ambasilide had no effect on activation or inactivation gating but prolonged both fast and slow components of deactivation consistent with unbinding from the open state. The net effect of both drugs was similar during a voltage ramp which simulated a cardiac action potential. CONCLUSIONS: Inhibition of HERG channels by azimilide and ambasilide exhibits a similar time and voltage-dependence. While both exhibit affinity for the open state, azimilide also binds to inactivated channels.

Aminobenzoates↗
PubMed2000

Modulation of host immune responses by clinically relevant human DNA and RNA viruses.

Numerous mechanisms allow viruses to evade host immune surveillance, and new evasion strategies continue to be identified. In addition to interference with antigen processing and presentation, direct viral modulation of host immune responses can also be achieved by altering the host cytokine milieu and the development of immunoregulatory cells. A better understanding of these viral evasion strategies will help to define critical host defense mechanisms and will lead to novel immune-based therapeutic strategies in the future.

DNA Virus Infections↗
PubMed2000

Inhibition of HERG potassium channels by the antimalarial agent halofantrine.

Halofantrine is a widely used antimalarial agent which has been associated with prolongation of the 'QT interval' of the electrocardiogram (ECG), torsades de pointes and sudden death. Whilst QT prolongation is consistent with halofantrine-induced increases in cardiac ventricular action potential duration, the cellular mechanism for these observations has not been previously reported. The delayed rectifier potassium channel, I(Kr), is a primary site of action of drugs causing QT prolongation and is encoded by the human-ether-a-go-go-related gene (HERG). We examined the effects of halofantrine on HERG potassium channels stably expressed in Chinese hamster ovary (CHO-K1) cells. Halofantrine blocked HERG tail currents elicited on repolarization to -60 mV from +30 mV with an IC(50) of 196.9 nM. The therapeutic plasma concentration range for halofantrine is 1.67-2.98 microM. Channel inhibition by halofantrine exhibited time-, voltage- and use-dependence. Halofantrine did not alter the time course of channel activation or deactivation, but inactivation was accelerated and there was a 20 mV hyperpolarizing shift in the mid-activation potential of steady-state inactivation. Block was enhanced by pulses that render channels inactivated, and channel blockade increased with increasing duration of depolarizing pulses. We conclude that HERG channel inhibition by halofantrine is the likely underlying cellular mechanism for QT prolongation. Our data suggest preferential binding of halofantrine to the open and inactivated channel states.

Animals↗
PubMed2000

Marked differences in quantity of infectious human immunodeficiency virus type 1 detected in persons with controlled plasma viremia by a simple enhanced culture method.

Culture of autologous CD4 lymphocytes from peripheral blood mononuclear cells compared favorably with two other methods for the measurement of cell-associated human immunodeficiency virus type 1 (HIV-1). For subjects with undetectable HIV-1 RNA levels in plasma, there was a 10,000-fold range of cell-associated virus detected. This method provides a simple and reproducible means for monitoring cell-associated HIV-1.

CD4-Positive T-Lymphocytes↗
PubMed2000

HLA-B57-restricted cytotoxic T-lymphocyte activity in a single infected subject toward two optimal epitopes, one of which is entirely contained within the other.

Viral peptides are recognized by cytotoxic T lymphocytes (CTL) as a complex with major histocompatibility complex (MHC) class I molecules, but the extent to which a single HLA allele can accommodate epitope peptides of different length and sequence is not well characterized. Here we report the identification of clonal CTL responses from the same donor that independently recognize one of two HLA-B57-restricted epitopes, KAFSPEVIPMF (KF11; p24(Gag) residues 30 to 40) and KAFSPEVI (KF8; p24(Gag) residues 30 to 37). Although lysis studies indicated that the KF11 peptide stabilized the HLA-B57-peptide complex more efficiently than the KI8 peptide, strong clonal responses were directed at each epitope. In samples from a second donor, the same phenomenon was observed, in which distinct CTL clones recognized peptide epitopes presented by the same HLA class I allele (in this case, HLA-A3) which were entirely overlapping. These data are relevant to the accurate characterization of CTL responses, which is fundamental to a detailed understanding of MHC class I-restricted immunity. In addition, these studies demonstrate marked differences in the length of peptides presented by HLA-B57, an allele which is associated with nonprogressive human immunodeficiency virus infection.

Adolescent↗
PubMed2000

Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected African and caucasoid adults and children.

Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17(Gag) and p24(Gag) sequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17(Gag) peptide residues 16 to 30 (38 versus 12%; P < 0.01), while p24(Gag) peptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%; P < 0.005). Within this 20-mer p24(Gag), an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.

Adult↗
PubMed2000

Inhibition of human immunodeficiency virus type 1 replication in primary CD4(+) T lymphocytes, monocytes, and dendritic cells by cytotoxic T lymphocytes.

We demonstrate that human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) cytotoxic T lymphocytes (CTL) suppress HIV-1 replication in primary lymphocytes, monocytes, and dendritic cells individually. Viral inhibition is significantly diminished in lymphocyte-dendritic cell clusters, suggesting that these clusters in vivo could be sites where viral replication is more difficult to control by CTL.

CD4-Positive T-Lymphocytes↗
PubMed2000

Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay.

Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1.

Adult↗
PubMed2000

Distinct trafficking pathways mediate Nef-induced and clathrin-dependent major histocompatibility complex class I down-regulation.

The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.

Clathrin↗
PubMed2000

Unusual polymorphisms in human immunodeficiency virus type 1 associated with nonprogressive infection.

Factors accounting for long-term nonprogression may include infection with an attenuated strain of human immunodeficiency virus type 1 (HIV-1), genetic polymorphisms in the host, and virus-specific immune responses. In this study, we examined eight individuals with nonprogressing or slowly progressing HIV-1 infection, none of whom were homozygous for host-specific polymorphisms (CCR5-Delta32, CCR2-64I, and SDF-1-3'A) which have been associated with slower disease progression. HIV-1 was recovered from seven of the eight, and recovered virus was used for sequencing the full-length HIV-1 genome; full-length HIV-1 genome sequences from the eighth were determined following amplification of viral sequences directly from peripheral blood mononuclear cells (PBMC). Longitudinal studies of one individual with HIV-1 that consistently exhibited a slow/low growth phenotype revealed a single amino acid deletion in a conserved region of the gp41 transmembrane protein that was not seen in any of 131 envelope sequences in the Los Alamos HIV-1 sequence database. Genetic analysis also revealed that five of the eight individuals harbored HIV-1 with unusual 1- or 2-amino-acid deletions in the Gag sequence compared to subgroup B Gag consensus sequences. These deletions in Gag have either never been observed previously or are extremely rare in the database. Three individuals had deletions in Nef, and one had a 4-amino-acid insertion in Vpu. The unusual polymorphisms in Gag, Env, and Nef described here were also found in stored PBMC samples taken 3 to 11 years prior to, or in one case 4 years subsequent to, the time of sampling for the original sequencing. In all, seven of the eight individuals exhibited one or more unusual polymorphisms; a total of 13 unusual polymorphisms were documented in these seven individuals. These polymorphisms may have been present from the time of initial infection or may have appeared in response to immune surveillance or other selective pressures. Our results indicate that unusual, difficult-to-revert polymorphisms in HIV-1 can be found associated with slow progression or nonprogression in a majority of such cases.

Amino Acid Sequence↗