Biomedical subjects

R P Sekaly

Publications and source records attributed to R P Sekaly.

At least 19 recordsLinked to original sources

PubMed2003

Changes in immunological and virological parameters in HIV-1 infected subjects following leukapheresis.

In order to assess immune responses during HIV-1 therapeutic immunization, a large number of blood mononuclear cells (PBMC) are needed. Clinical tolerance and safety, as well as changes in immunological and virological parameters, were assessed, following leukapheresis in HIV-1 infected subjects with CD4(+) cell count >200 x 10(6)/l. PBMC were collected using a Fenwal CS3000 cell separator in 29 subjects with mean CD4(+) cell counts of 503 x 10(6)/l (range 172-1,119) and viral load of 2.5 log(10) copies/ml (range <1.7-5.4). Twenty-four (83%) subjects were on antiretroviral therapy while 5 (17%) were untreated. The blood volume processed was 7 L over a period of 3 hours. A mean value (+/- standard error) of 82 +/- 26 x 10(9)/l lymphocytes was collected by a single apheresis in a mean volume of 200 +/- 1.8 ml, containing 9.0 +/- 1.3 x 10(9)/l CD4(+) and 10.2 +/- 1.3 x 10(9)/l CD8(+) cells. The leukapheresis procedures were well tolerated and no immediate or delayed side effects were observed within 90 days of follow-up. No changes from blood pre-leukapheresis values were detected for white blood cells, lymphocytes, monocytes, CD8(+), CD34(+), naive and memory CD4(+) cell counts immediately after, 1 h, 7 days, or within 90 days after leukapheresis. However, absolute CD4(+) cell counts and percentage significantly increased from pre-leukapheresis values after 1 h (530 +/- 43 vs. 700 +/- 75 cell x 10(6)/l; 32.6 +/- 1.6 vs. 36.9 +/- 1.9%; P < 0.001 for both paired t-tests) before returning to pre-leukapheresis levels on day 7. No significant changes in viral load from pre-leukapheresis levels in treated or untreated subjects were detected at any time points. We conclude that leukapheresis in HIV-1 infected subjects with CD4(+) cell counts >200 x 10(6)/l is safe and induces a transient increase in the absolute and percentage of CD4(+) cell count without enhancing viral replication.

Adult↗

PubMed2002

HIV-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation.

Infection of T cells with HIV-1 induces apoptosis and modulates apoptosis regulatory molecules. Similar effects occur following treatment of cells with individual HIV-1 encoded proteins. While HIV-1 protease is known to be cytotoxic, little is known of its effect on apoptosis and apoptosis regulatory molecules. The ability of HIV-1 protease to kill cells, coupled with the degenerate substrate specificity of HIV-1 protease, suggests that HIV-1 protease may activate cellular factor(s) which, in turn, induce apoptosis. We demonstrate that HIV-1 protease directly cleaves and activates procaspase 8 in T cells which is associated with cleavage of BID, mitochondrial release of cytochrome c, activation of the downstream caspases 9 and 3, cleavage of DFF and PARP and, eventually, to nuclear condensation and DNA fragmentation that are characteristic of apoptosis. The effect of HIV-1 protease is not seen in T cell extracts which have undetectable levels of procaspase 8, indicating a specificity and requirement for procaspase 8.

Apoptosis↗

PubMed2001

Substantial differences in specificity of HIV-specific cytotoxic T cells in acute and chronic HIV infection.

Cytotoxic T lymphocytes (CTLs) play a vital part in controlling viral replication during human viral infections. Most studies in human infections have focused on CTL specificities in chronic infection and few data exist regarding the specificity of the initial CTL response induced in acute infection. In this study, HIV-1 infection in persons expressing human histocompatibility leukocyte antigen (HLA)-A*0201 was used as a means of addressing this issue. In chronic infection, the dominant HLA-A*0201-restricted CTL response is directed towards the epitope SLYNTVATL ("SL9") in p17 Gag (residues 77-85). This epitope is targeted by 75% of HLA-A*0201-positive adults, and the magnitude of this A*0201-SL9 response shows a strong negative association with viral load in progressive infection. Despite using the highly sensitive peptide-major histocompatibility complex tetramer and intracellular cytokine assays, responses to the SL9 epitope were not detectable in any of 11 HLA-A*0201-positive subjects with acute HIV-1 infection (P = 2 x 10(-6)), even when assays were repeated using the SL9 peptide variant that was encoded by their autologous virus. In contrast, multiple responses (median 3) to other epitopes were evident in 7 of the 11 A*0201-positive subjects. Longitudinal study of two subjects confirmed that the A*0201-SL9 response emerged later than other CTL responses, and after viral set point had been reached. Together, these data show that the CTL responses that are present and that even may dominate in chronic infection may differ substantially from those that constitute the initial antiviral CTL response. This finding is an important consideration in vaccine design and in the evaluation of vaccine candidates.

Acute Disease↗

PubMed2000

Comparison of clinical features of acute HIV-1 infection in patients infected sexually or through injection drug use. The Investigators of the Québec Primary HIV Infection Study.

Acute HIV-1 infection (AHI) may present with a clinical picture that represents a diagnostic challenge. We tested the hypothesis that two different routes of infection, that is, sexual versus parenteral, might be associated with a difference in the clinical features of AHI. A prospective cohort of seroconvertors was established in Montréal in private medical clinics and hospitals from February 1996 to May 1999. The prevalence of the symptomatic presentation was almost overlapping within the two groups of newly infected individuals 69% (42 of 61) for men having sex with men (MSM) and 69% (18 of 26) for injection drug users (IDUs; p =.98). Comparison of all types of symptoms and signs as well as their duration was also similar in both groups. Of particular interest, the site of lymph node enlargement was not different despite the estimated sites of intravenous inoculation. Oral and anal ulcers were more frequently observed in MSM than in IDUs (6 versus 0 and 4 versus 1, respectively). Neither the mean CD4+ count (514.8 and 414.7 cells/mm3; p =.14) nor the mean viral load (4.45 and 4.70 log copies/ml; p =.40) were different between the two groups at the time of the first study visit. Our study results clearly indicate that health care workers can expect similar clinical presentation of AHI in MSM and in IDUs despite the different routes of infection.

Adolescent↗

PubMed2000

Prevalence of HIV-1 resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or injecting drug use. Investigators of the Quebec Primary Infection Study.

OBJECTIVE: Prolonged treatment with antiretroviral drugs results in the selection of HIV-1 variants with mutations conferring resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI) or to protease inhibitors (PI). There is serious concern about transmission of resistant viruses to newly infected persons. This study monitored the prevalence of resistant viruses in individuals undergoing primary HIV infection. DESIGN: Resistance testing was performed on 81 individuals infected between 1997 and 1999 by injecting drug use (n =21), sexual (n = 56), or unknown (n = 4) transmission. METHODS: Automated sequencing was used to genotype the reverse transcriptase (RT) and protease regions of virus isolated from patients' plasma. The phenotypic susceptibility of stimulated peripheral blood mononuclear cells to antiretroviral drugs was assayed. Line probe assays detected quasispecies variations in wild-type and mutated RT codons. RESULTS: A high prevalence of PI and RT genotypic variants, associated with high-level resistance to antiretroviral drugs, was observed in individuals newly infected by injecting drug use (PI = 24%, RT = 24%) or sexual transmission (PI = 12%, RT = 22%). The PI mutations, L101, V82A, and L90M, were found in 10.5, 3 and 4% of cases, respectively; whereas for RT, primary mutations at positions T215Y (zidovudine), M184V (lamivudine), T69D/A (zalcitabine), and K103N (multi-NNRTI) were present in 8, 5, 4, and 4% of subjects, respectively. Resistance to NRTI was demonstrated by phenotypic, genotypic, and line probe analyses. Transmission of multidrug (NRTI/NNRTI/PI) resistance in eight subjects (9.9%) was confirmed by showing that source partners possessed viruses of similar genotype. CONCLUSIONS: The transmission of drug-resistant HIV is a serious problem that merits further attention by public health officials as well as virologists and clinicians.

Anti-HIV Agents↗

PubMed2000

Superantigen presenting capacity of human astrocytes.

We found that human fetal astrocytes (HFA) are able to support superantigen (SAG) staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1)-induced activation of immediately ex vivo allogenic human CD4 T cells. Using radiolabelled toxins, we demonstrate that both SEB and TSST-1 bind with high affinity to MHC class II antigen expressing astrocytes; binding is displaceable with excess cold toxin. Competition experiments further indicate that TSST-1 and SEB at least partially compete with each other for binding to astrocytes suggesting they bind to the same HLA-DR region on these cells. Our study supports the hypothesis that SAG would be capable of stimulating immune responses within the human CNS and contribute to persistence or recurrence of inflammatory responses within this compartment.

Antigen-Presenting Cells↗

PubMed2000

The extracellular domain of CD4 regulates the initiation of T cell activation.

To assess the respective contribution of the extracellular and intracellular domains of CD4 in regulating early T cell activation events, we have used a CD4-independent murine T cell clone transfected with human CD4. Stimulation of CD4 positive clones could only be observed if CD4 molecules associated to lck were co-aggregated with the TCR complex, confirming that the simultaneous interaction of MHC class II molecules with the CD4/lck complex and the TCR is required to initiate T cell activation. To assess the involvement of the extracellular portion of CD4 in this process, we transfected a chimeric molecule (EGFRCD4) consisting of the extracellular portion of the epidermal growth factor receptor (EGFR), and of the transmembrane and cytoplasmic domains of human CD4. Although this chimeric molecule associates with lck, transfected clones were induced to proliferate by mAb specific for TCR in the absence of co-aggregation. A new regulatory role for the extracellular domain of CD4 which is independent of its interaction with MHC class II molecules is thus revealed in these experiments. Taken together, our results demonstrate that, in a CD4-independent cell line, two domains of CD4 regulate early T cell activation events: (1) its association with lck and (2) its extracellular domain, independently of its interaction with MHC class II molecules.

Animals↗

PubMed2000

Resistance to antiretroviral drugs in patients with primary HIV-1 infection. Investigators of the Quebec Primary Infection Study.

The widespread use of antiretroviral agents (ARVs) and the growing occurrence of HIV strains resistant to these drugs have given rise to serious concerns regarding the transmission of resistant viruses to newly infected persons. Plasma viral RNA from 80 individuals newly infected between 1997 and 1999 was genotyped by automated sequencing to analyze the profile of viruses resistant to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) and to protease inhibitors (PIs). The prevalence of mutations that conferred primary resistance to PIs (L10I, D30Y, V82A, L90M) was 15% of the cohort. RT genotypic variants, associated with high-level resistance to ARVs, were observed in 21% of individuals, including NRTI, NNRTI and multidrug (MDR) resistance in 6, 5, and 10% of cases, respectively. The phenotypic susceptibility of viral isolates to ARVs was also assayed and showed transmission of high-level resistance to ZDV, 3TC, and PIs in those individuals with MDR. The transmission of drug-resistant HIV genotypic variants is a serious problem that merits further attention by public health officials, virologists, and clinicians.

Anti-HIV Agents↗

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↗

PubMed1998

Induction of endogenous mammary tumor virus in lymphocytes infected with murine acquired immunodeficiency syndrome virus.

Mice infected with murine acquired immunodeficiency syndrome (MAIDS) virus developed lymphoadenopathy and profound immunodeficiency. Concomitantly the expression of endogenous mammary tumor virus (MTV) mRNA increased significantly, especially for the 1.7-kb 3' open reading frame (ORF) mRNA encoding MTV superantigen. B cell lines that are established from MAIDS mice and exhibit superantigen activity also express a high level of 1.7-kb endogenous MTV and mRNA. Infection of a B cell tumor line in vitro with retrovirus containing the cloned MAIDS virus gene induced superantigen activity and this cell line also expressed the 1.7-kb superantigen coding MTV 3' ORF mRNA. These results strongly suggest a link between MAIDS virus infection and the induction of endogenous superantigen activity. This may play an important role in the pathogenesis of the MAIDS virus.

Amino Acid Sequence↗

PubMed1997

Evidence for rapid disappearance of initially expanded HIV-specific CD8+ T cell clones during primary HIV infection.

Down-regulation of the initial burst of viremia during primary HIV infection is thought to be mediated predominantly by HIV-specific cytotoxic T lymphocytes, and the appearance of this response is associated with major perturbations of the T cell receptor repertoire. Changes in the T cell receptor repertoire of virus-specific cytotoxic T lymphocytes were analyzed in patients with primary infection to understand the failure of the cellular immune response to control viral spread and replication. This analysis demonstrated that a significant number of HIV-specific T cell clones involved in the primary immune response rapidly disappeared. The disappearance was not the result of mutations in the virus epitopes recognized by these clones. Evidence is provided that phenomena such as high-dose tolerance or clonal exhaustion might be involved in the disappearance of these monoclonally expanded HIV-specific cytotoxic T cell clones. These findings should provide insights into how HIV, and possibly other viruses, elude the host immune response during primary infection.

Amino Acid Sequence↗

PubMed1997

Paracrine transfer of mouse mammary tumor virus superantigen.

Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive V beta s. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific V beta s led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II-specific mAbs. Moreover, injection of vSAG7+ class II+ cells in mice led to expansion of V beta 6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II+ and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the alpha 1 domain of HLA-DR.

Animals↗

PubMed1997

The qualitative nature of the primary immune response to HIV infection is a prognosticator of disease progression independent of the initial level of plasma viremia.

Following infection of the host with a virus, the delicate balance between virus replication/spread and the immune response to the virus determines the outcome of infection, i.e., persistence versus elimination of the virus. It is unclear, however, what relative roles immunologic and virologic factors play during primary viral infection in determining the subsequent clinical outcome. By studying a cohort of subjects with primary HIV infection, it has been demonstrated that qualitative differences in the primary immune response to HIV, but not quantitative differences in the initial levels of viremia are associated with different clinical outcomes.

CD4 Lymphocyte Count↗

PubMed1997

Analysis of the contact sites on the CD4 molecule with class II MHC molecule: co-ligand versus co-receptor function.

The CD4 molecule interacts with the alpha2 and beta2 domains of the MHC class II molecules. However, the class II contact sites on CD4 are less clear. Involvement of different regions throughout D1, D2, and D3 domains have been suggested. To further delineate the class II MHC contact sites on CD4, a crystal structure-informed mutagenesis was performed. Alanine scan mutants were generated for exposed residues located throughout D1 and the FG loop of D2, and in the "hinge-like" region, a short and flexible region between D2 and D3. Mutants were tested in a co-ligand (Dd stimulation) and a co-receptor (staphylococcal enterotoxin B stimulation) assay. In the co-ligand assay, TCR and CD4 interact with two distinct ligands (Dd or HLA-DR), while in the co-receptor assay both molecules interact with the same ligand, namely HLA-DR. Results show that residues from both lateral faces of D1 and the FG loop of D2 are implicated in interaction with class II, although a bigger surface of CD4 was involved in co-receptor compared with co-ligand function. The potential involvement of residues on both the top and two side faces of CD4 is consistent with a bivalent model, which involves the interaction between a single CD4 and two class II molecules. Alternatively, our results can be interpreted with a model of a specifically organized CD4 and/or class II oligomerization event. Finally, results from the hinge region mutants revealed a very important role in maintaining the overall structural integrity of CD4, its topology, and function.

CD4 Antigens↗

PubMed1997

V alpha domain modulates the multiple topologies of mouse T cell receptor V beta20/staphylococcal enterotoxins A and E complexes.

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major histocompatibility complex (MHC) class II molecules on two sites located on the alpha and beta chains. We have investigated the role of the T cell receptor (TCR) alpha chain in the modulation of the various topologies of TCR/SEA (or SEE)/class II complexes. For this purpose, we have used three mouse V beta20 T cell lines expressing different V alpha domains and two T cell hybridomas expressing mouse V beta1 or V beta11 segments. The response of these T cells to SEA and SEE was studied in the context of presentation by wild-type human MHC class II molecules; or by mutants on MHC, in each of the two superantigen binding sites (position alpha39K and beta81H) to which the superantigens can still bind but with an altered conformation. Although V beta20 T cell lines are efficiently stimulated using SEA and SEE presented by wild-type HLA-DR1 molecules, our results show that the nature of the TCR V alpha domain can affect differently the recognition of the toxins bound to mutant class II molecules. This suggests that various functional topologies exist for both SEA and SEE/class II complexes and that the T cell response to each of these complexes can be modulated by the V alpha domain of the TCR. Interestingly, the recognition of SEA and SEE is achieved in different fashions by a given V beta20 T cell line.

Animals↗

PubMed1997

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection.

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.

Antigen Presentation↗

PubMed1997

MHC class II-dependent peptide antigen versus superantigen presentation to T cells.

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.

Antigen Presentation↗

PubMed1996

TCR binding differs for a bacterial superantigen (SEE) and a viral superantigen (Mtv-9).

Both superantigens (SAG) and many anti-TCR monoclonal antibodies (mAb) have specificity for the V beta region of the TCR encoded by TCRBV genes. For instance the bacterial SAG staphylococcal enterotoxin E (SEE), the retroviral SAG MTV-9 and the mAb OT145 each react with human T cells expressing BV6S7. This BV gene encodes two common alleles. We found that SEE and the mAb preferentially activate T cells expressing BV6S7*1 as opposed to BV6S7*2, but Mtv-9 activates T cells expressing either allele. Thus binding to the TCR differs between the two SAGs. A mutation in the TCR HVR-4 region of BV6S7*1 (G72E), where the two BV6S7 alleles differ, indicated that HVR-4 is a component of the binding site for SEE and for the mAb OT145. BV6S7*2 has a charged E72 which may result in electrostatic repulsion of SEE, as SEE contains a similarly acidic aspartic acid residue at a TCR interaction site (204D).

Alleles↗