Do genetic variations in proximal tubule transporters influence tenofovir-induced renal dysfunction? An exploratory study in a Ghanaian population.

OBJECTIVES: To assess associations between polymorphisms within genes encoding proximal tubule transporters implicated in tenofovir renal clearance and kidney tubular dysfunction (KTD), chronic kidney disease (CKD) and individual biochemical parameters. PATIENTS AND METHODS: The study included a cohort of HIV-positive Ghanaians receiving regimens containing tenofovir disoproxil fumarate (n = 66) for at least 6 months prior to study enrolment. SNPs in ABCC10, ABCC2 and ABCC4 were selected for analysis based on previous published associations. All SNPs were genotyped by real-time PCR allelic discrimination. Creatinine clearance (CLCR), serum and urine creatinine concentrations and biochemical measures of KTD were assessed. Statistical significance was determined through univariate linear or binary logistical regression (P ≤ 0.05). RESULTS: None of the SNPs evaluated was associated with CKD or KTD. A trend between body weight and higher incidence of CKD (P = 0.012, OR = 0.9) was observed. ABCC10 2843T>C (rs2125739) was significantly associated with lower log10 baseline creatinine (P = 0.001, ß= -0.4), higher baseline CLCR (P = 0.008, ß = 65.2) and lower CLCR after 1 year (P = 0.024, ß= -26.6). CONCLUSIONS: This study demonstrates an association of ABCC10 rs2125739 with indicators of declining renal function and builds on current knowledge of this interaction within a Ghanaian cohort.

EBV-Specific Immune Response: Early Research and Personal Reminiscences.

Early research on Epstein-Barr virus (EBV) developed from serological observations that were made soon after the discovery of the virus. Indeed, the definition of the humoral response to a variety of EBV proteins dominated the early literature and was instrumental in providing the key evidence for the association of the virus with infectious mononucleosis (IM), Burkitt's lymphoma (BL), and nasopharyngeal carcinoma (NPC). Each of these disease associations involved a distinct pattern of serological reactivity to the EBV membrane antigens (MA), early antigens (EA), and the EBV nuclear antigen (EBNA). When it became generally accepted that the marked lymphocytosis , which is a hallmark of acute IM, was dominated by T cells, considerable effort was directed toward untangling the specificities that might be associated with restricting the proliferation of newly infected B cells. Early evidence was divided between support for both EBV non-specific and/or HLA non-restricted components. However, all results needed to be reassessed in light of the observation that T cells died by apoptosis within hours of separation from fresh blood from acute IM patients. The observation that EBV-infected cultures from immune (but not non-immune) individuals began to die (termed regression) about 10 days post-seeding, provided the first evidence of a specific memory response which was apparently capable of controlling the small pool of latently infected B cells which all immune individuals possess. In this early era, CD8(+) T cells were thought to be the effector population responsible for this phenomenon, but later studies suggested a role for CD4(+) cells. This historical review includes reference to key early observations in regard to both the specific humoral and cellular responses to EBV infection from the time of the discovery of the virus until 1990. As well, we have included personal recollections in regard to the events surrounding the discovery of the memory T cell response since we believe they add a human dimension to a chapter focussed on early history.

Mid-infrared nonlinear optical response of Si-Ge waveguides with ultra-short optical pulses.

We characterize the nonlinear optical response of low loss Si(0.6)Ge(0.4) / Si waveguides in the mid-infrared between 3.3 µm and 4 µm using femtosecond optical pulses. We estimate the three and four-photon absorption coefficients as well as the Kerr nonlinear refractive index from the experimental measurements. The effect of multiphoton absorption on the optical nonlinear Kerr response is evaluated and the nonlinear figure of merit estimated providing some guidelines for designing nonlinear optical devices in the mid-IR. Finally, we compare the impact of free-carrier absorption at mid-infrared wavelengths versus near-infrared wavelengths for these ultra-short pulses.

Nonlinear optical response of low loss silicon germanium waveguides in the mid-infrared.

We have investigated the nonlinear optical response of low loss Si(0.6)Ge(0.4) / Si waveguides in the mid-infrared wavelength range from 3.25- 4.75µm using picosecond optical pulses. We observed and measured the three and four-photon absorption coefficients as well as the Kerr nonlinear refractive index. The dynamics of the spectral broadening suggests that, in addition to multiphoton absorption, the corresponding higher order nonlinear refractive phenomena also needs to be included when high optical pulse intensities are used at mid-infrared wavelengths in this material.

A meta-analysis of the prevalence of attention deficit hyperactivity disorder in incarcerated populations.

BACKGROUND: Studies report the variable prevalence of attention deficit hyperactivity disorder (ADHD) in incarcerated populations. The aim of this meta-analysis was to determine the prevalence of ADHD in these populations. METHOD: Primary research studies reporting the prevalence (lifetime/current) of ADHD in incarcerated populations were identified. The meta-analysis used a mixed log-binomial model, including fixed effects for each covariate and a random study effect, to estimate the significance of various risk factors. RESULTS: Forty-two studies were included in the analysis. ADHD prevalence was higher with screening diagnoses versus diagnostic interview (and with retrospective youth diagnoses versus current diagnoses). Using diagnostic interview data, the estimated prevalence was 25.5% and there were no significant differences for gender and age. Significant country differences were noted. CONCLUSIONS: Compared with published general population prevalence, there is a fivefold increase in prevalence of ADHD in youth prison populations (30.1%) and a 10-fold increase in adult prison populations (26.2%).

Seroepidemiology of Toxoplasma in a coastal region of Haiti: multiplex bead assay detection of immunoglobulin G antibodies that recognize the SAG2A antigen.

Toxoplasma gondii is a globally distributed parasitic protozoan that infects most warm-blooded animals. We incorporated a bead coupled with recombinant SAG2A protein into our Neglected Tropical Disease (NTD) multiplex bead assay (MBA) panel and used it to determine Toxoplasma infection rates in two studies in Haiti. In a longitudinal cohort study of children aged 0-11 years, the infection rate varied with age reaching a maximum of 0·131 infections/year in children aged 3 years [95% confidence interval (CI) 0·065-0·204]. The median time to seroconversion was estimated to be 9·7 years (95% CI 7·6-∞). In a cross-sectional, community-wide survey of residents of all ages, we determined an overall seroprevalence of 28·2%. The seroprevalence age curve from the cross-sectional study also suggested that the force of infection varied with age and peaked at 0·057 infections/year (95% CI 0·033-0·080) at age 2·6 years. Integration of the Toxoplasma MBA into NTD surveys may allow for better estimates of the potential burden of congenital toxoplasmosis in underserved regions.

Interactions between tenofovir and nevirapine in CD4+ T cells and monocyte-derived macrophages restrict their intracellular accumulation.

OBJECTIVES: There is no pharmacokinetic interaction between tenofovir and nevirapine, but a higher emergence rate of resistance mutations has been reported when these drugs are coadministered. We sought to examine if there is a potential intracellular interaction that may account for the emergence of resistant virus. METHODS: Primary CD4+ and CD14+ cells were isolated from healthy volunteer blood. Monocyte-derived macrophages were differentiated from CD14+ cells. Accumulation of radiolabelled tenofovir and nevirapine was then assessed in these cells. RESULTS: We show here that tenofovir and nevirapine immune cell intracellular concentrations are lower when coincubated in CD4+ cells and monocyte-derived macrophages, but not in CD14+ cells. CONCLUSIONS: These data indicate a potential intracellular drug-drug interaction between these drugs that warrants further investigation.

Blackbody emission from light interacting with an effective moving dispersive medium.

Intense laser pulses excite a nonlinear polarization response that may create an effective flowing medium and, under appropriate conditions, a blocking horizon for light. Here, we analyze in detail the interaction of light with such laser-induced flowing media, fully accounting for the medium dispersion properties. An analytical model based on a first Born approximation is found to be in excellent agreement with numerical simulations based on Maxwell's equations and shows that when a blocking horizon is formed, the stimulated medium scatters light with a blackbody emission spectrum. Based on these results, diamond is proposed as a promising candidate medium for future studies of Hawking emission from artificial, dispersive horizons.

Amorphous silicon nanowires combining high nonlinearity, FOM and optical stability.

We demonstrate optically stable amorphous silicon nanowires with both high nonlinear figure of merit (FOM) of ~5 and high nonlinearity Re(γ) = 1200W(-1)m(-1). We observe no degradation in these parameters over the entire course of our experiments including systematic study under operation at 2 W coupled peak power (i.e. ~2GW/cm(2)) over timescales of at least an hour.

All-optical wavelength conversion for 10 Gb/s DPSK signals in a silicon ring resonator.

We demonstrate all-optical wavelength conversion at 10 Gb/s for differential phase-shift keyed (DPSK) data signals in the C-band, based on four-wave mixing (FWM) in a silicon ring resonator. Error-free operation with a system penalty of ~4.1 dB at 10⁻9 BER is achieved.

All-optical XOR logic gate for 40Gb/s DPSK signals via FWM in a silicon nanowire.

We demonstrate an all-optical XOR logic function for 40Gb/s differential phase-shift keyed (DPSK) data signals in the C-band, based on four-wave mixing (FWM) in a silicon nanowire. Error-free operation with a system penalty of ~3.0dB and ~4.3dB at 10⁻9 BER is achieved.

Ultrafast strain-induced current in a GaAs Schottky diode.

Picosecond acoustic pulses generated by femtosecond laser excitation of a metal film induce a transient current with subnanosecond rise time in a GaAs/Au Schottky diode. The signal consists of components due to the strain pulse crossing the edge of the depletion layer in the GaAs and also the GaAs/Au interface. A theoretical model is presented for the former and is shown to be in very good agreement with the experiment.

New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1.

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

An alloresponse in humans is dominated by cytotoxic T lymphocytes (CTL) cross-reactive with a single Epstein-Barr virus CTL epitope: implications for graft-versus-host disease.

The phenomenon of T cell allorecognition is difficult to accommodate within the framework of a T cell repertoire positively selected in the thymus, unless allorecognition results from the cross-reactions of self-major histocompatibility complex restricted T cells. Herein, we demonstrate the dual specificity of cytotoxic T lymphocyte (CTL) clones for the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, presented on HLA-B8, and the alloantigen HLA-B*4402. CTL which recognized peptide FLRGRAYGL in association with HLA-B8 could be reactivated in vitro from healthy individuals who had been exposed previously to EBV, using stimulator cells expressing the cross-reacting alloantigen HLA-B*4402. Limiting dilution analysis of the alloresponse to HLA-B*4402 in eight healthy individuals revealed that HLA-B8+, EBV-sero+ donors had higher CTL precursor frequencies for alloantigen HLA-B*4402 than EBV-sero- control donors. It is surprising that the majority (65-100%) of anti-HLA-B*4402 CTL, generated in limiting dilution mixed lymphocyte reactions between responder cells from HLA-B8+, EBV-sero+ individuals and HLA-B*4402+ stimulators, also recognized the EBV CTL epitope FLRGRAYGL/HLA-B8. In contrast to previous studies showing extensive diversity in the T cell repertoire against individual alloantigens, these data demonstrate that the response to an alloantigen can be dominated by CTL cross-reactive with a single viral epitope, thus illustrating a possible mechanism for the frequent clinical association between herpesvirus exposure and graft-versus-host disease after bone marrow transplants.

T cell-T cell killing is induced by specific epitopes: evidence for an apoptotic mechanism.

Epstein-Barr virus-specific cytotoxic T lymphocyte clones were shown to be an effective target for their own lysis when incubated in the presence of their specific epitopes but not in the presence of irrelevant epitopes. The mode of cell killing appeared to be by apoptosis and was prevented by previously described inhibitors of the process. Degranulation, as measured by serine esterase activity, was involved in this form of T cell-T cell killing. This is the first report of T cell-T cell killing by apoptosis and is only observed in the presence of a specific epitope. This result may be of significance in the use of peptide-based vaccines.

An Epstein-Barr virus-specific cytotoxic T cell epitope in EBV nuclear antigen 3 (EBNA 3).

Epstein-Barr virus (EBV)-specific CTL clones were isolated that recognized A-type EBV transformants but not B-type transformants. These A-type-specific CTL clones (HLA B8 restricted) were used to screen peptides derived from the EBV nuclear antigens (EBNAs) 2, 3, 4, and 6 as potential CTL epitopes. Of the 76 peptides screened, one sequence from EBNA 3 (residues 329-353) was recognized by A-type-specific CTL clones after absorption onto target cells (either autologous B-type transformants or PHA blasts). This report is the first description of an EBV target epitope recognized by specific CTL clones.

T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen.

Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self-reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area that mimics a structure presented on HLA B*4402. Thus, tolerance to a background major histocompatibility antigen can effectively diversify the TCR repertoire for a foreign epitope by deflecting the response away from an immunodominant combination of TCR-binding residues.

Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.

There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type 1 and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA 1 as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the down-regulation of latent antigens can be reversed.

Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing.

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus-specific CTL response was shown to include specificities for two HLA-B8-restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR-beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease.