Stephen J. Jordan

T Cell Glycolipid-Enriched Membrane Domains Are Constitutively Assembled as Membrane Patches That Translocate to Immune Synapses

In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.

Transient Association of Ku with Nuclear Substrates Characterized Using Fluorescence Photobleaching

The autoantigen Ku, composed of subunits Ku70 and Ku86, is necessary for repair of DNA double-strand breaks by nonhomologous end joining. Similarly, Ku participates in repair of DNA double-strand breaks that occur during V(D)J recombination, and it is therefore required for the development of B and T lymphocytes. Although previous studies have identified the DNA-binding activities of Ku, little is known concerning its dynamics, such as the mobility of Ku in the nucleus and its rate of association with substrates. To address this question, fluorescence photobleaching experiments were performed using HeLa cells and B cells expressing a green fluorescent protein (GFP) fusion construct of either Ku70 or Ku86. The results show that Ku moves rapidly throughout the nucleus even following irradiation of the cells. However, the rate of diffusion of Ku was ∼100-fold slower than that predicted from its size. Association of Ku-GFP with a filamentous nuclear structure was also evident, and nuclear extraction experiments suggest that this represents nuclear matrix. A central domain of Ku70 containing its DNA-binding and heterodimerization regions and its nuclear localization signal shows that this alone is sufficient for the observed mobility of Ku70-GFP and its association with nuclear matrix. These data suggest the mobility of Ku is characterized by a transient, high flux association with nuclear substrates that includes both DNA and the nuclear matrix and may represent a mechanism for repair of double-strand breaks using the nuclear matrix as a scaffold.

A device for precision positioning and alignment of room lasers to diminish their contribution to patient setup errors

This report presents an analysis of patient setup errors resulting from inaccurately positioned wall lasers. It suggests that laser beams should agree within 0.2 degree or better with the machine axes that they are delineating. For typical simulator and treatment rooms having wall‐to‐isocenter distances of 3 m, this requirement is satisfied when the beam‐emitting aperture is mounted within about 1.0 cm from the intersection of the respective machine axis with the wall. To achieve the required precision, we developed and clinically tested a simple, inexpensive tool, the Laser Placer (LP). The essential component of the LP is a cube with mirror surfaces that is aligned with the machine axes using built‐in spirit levels and the light field and crosshairs of the collimator. Wall, ceiling, and sagittal lasers are installed and aligned according to reflections of their beams by the cube, and reference lines provided by the LP. Measurements showed that, even in new accelerator installations performed by highly experienced technicians, wall lasers are often mounted off target by more than 1.5 cm. Such inaccuracies can contribute systematic errors of 2 mm or more to the random setup errors attributable to interfraction movement in patient anatomy. To keep setup errors to a minimum, medical physicists should check beam orthogonality in addition to beam congruence at isocenter as recommended by the TG‐40 quality assurance protocol from the American Association of Physicists in Medicine. PACS number: 87.53.‐j

Malaria Immunoepidemiology in Low Transmission: Correlation of Infecting Genotype and Immune Response to Domains of Plasmodium falciparum Merozoite Surface Protein 3

ABSTRACT Malaria caused by Plasmodium falciparum is a major cause of global infant mortality, and no effective vaccine currently exists. Multiple potential vaccine targets have been identified, and immunoepidemiology studies have played a major part in assessing those candidates. When such studies are carried out in high-transmission settings, individuals are often superinfected with complex mixtures of genetically distinct P. falciparum types, making it impossible to directly correlate the genotype of the infecting antigen with the antibody response. In contrast, in regions of low transmission P. falciparum infections are often genetically simple, and direct comparison of infecting genotype and antigen-specific immune responses is possible. As a test of the utility of this approach, responses against several domains and allelic variants of the vaccine candidate P. falciparum merozoite surface protein 3 (PfMSP3) were tested in serum samples collected near Iquitos, Peru. Antibodies recognizing both the conserved C-terminal and the more variable N-terminal domain were identified, but anti-N-terminal responses were more prevalent, of higher titers, and primarily of cytophilic subclasses. Comparing antibody responses to different PfMSP3 variants with the PfMSP3 genotype present at the time of infection showed that anti-N-terminal responses were largely allele class specific, but there was some evidence for responses that cross-reacted across allele classes. Evidence for cross-reactive responses was much stronger when variants within one allele class were tested, which has implications for the rational development of genotype-transcending PfMSP3-based vaccines.

Azithromycin for rectal chlamydia: is it time to leave azithromycin on the shelf?...Not yet.

Chlamydia trachomatis infection (‘‘chlamydia’’) remains the most prevalent bacterial sexually transmitted infection worldwide. Although chlamydia most often occurs in the genital tract, it can also occur in the rectum. Rectal chlamydia is a relatively common infection in men who have sex with men (MSM), with reported prevalence rates of approximately 8%. Rectal chlamydia can lead to symptomatic proctitis and can increase the risk of HIV acquisition or transmission. A fundamental component of chlamydia control efforts is the provision of highly effective therapy. The Centers for Disease Control and Prevention (CDC) currently recommends either azithromycin 1-g single dose or doxycycline 100 mg twice daily for 7 days for treatment of rectal chlamydia, but what is the evidence for this recommendation? The CDC recommendation stems not from robust studies showing equivalent efficacy of these treatment regimens for rectal chlamydia but, instead, from extrapolation of solid evidence supporting the efficacy of these regimens for urogenital chlamydia treatment and from clinical experience and expert consultation. A previous meta-analysis by Lau and Qureshi of 12 randomized clinical trials (RCTs) of azithromycin versus doxycycline for urogenital chlamydia treatment demonstrated these regimens to be highly efficacious, with microbial cure rates of 97% and 98%, respectively. However, most of these studies used culture for test of cure rather than the more sensitive nucleic acid amplification tests (NAATs) that are now CDC recommended for chlamydia testing. Unfortunately, to date, there have been no RCTs comparing the effectiveness of azithromycin versus doxycycline for rectal chlamydia, limiting our knowledge of the true efficacy of these treatment regimens for rectal chlamydia. However, there have been a limited number of clinical studies of these regimens for rectal chlamydia treatment whose findings collectively suggest that the doxycycline regimen may have a higher cure rate than the azithromycin regimen. Three were retrospective studies that evaluated just one of the treatment regimens and had significant limitations. For example, Steedman and McMillan reported an estimated azithromycin efficacy of 87% among 68 MSM, although the study was limited in that 8 of the 9 MSM with a repeat positive chlamydia test (polymerase chain reaction) after azithromycin treatment reported sexual activity between treatment and repeat chlamydia testing and also 3 of the 9 MSM with a repeat positive chlamydia test had their repeat test performed less than 21 days after therapy, introducing the possibility of a false-positive chlamydia test due to residual chlamydial DNA. Only one previous study has compared both azithromycin and doxycycline regimens for rectal chlamydia. Although not an RCT, Hathorn et al. compared both treatment regimens in a small prospective observational rectal chlamydia treatment study in men and women. They reported a difference in efficacy of these regimens for rectal chlamydia: 79% for azithromycin and 100% for doxycycline after adjusting for possible reinfection risk; however, the study also had significant limitations, including a high lost-to-follow-up rate (only approximately 50% of patients with rectal chlamydia returned to the clinic for a repeat chlamydia test). The limited prior evidence suggesting that the CDC-recommended doxycycline regimen may have a higher efficacy rate for rectal chlamydia than the recommended azithromycin regimen has now been further strengthened by findings from the study by Khosropour and colleagues reported in this issue. In a large retrospective study, Khosropour et al. evaluated rectal chlamydia treatment outcomes in a retrospective cohort of MSM diagnosed as having rectal chlamydia at a Seattle STD clinic between EDITORIAL

The Predominant CD4+ Th1 Cytokine Elicited to Chlamydia trachomatis Infection in Women is Tumor Necrosis Factor-Alpha, not Interferon-Gamma.

ABSTRACT Chlamydia trachomatis infection is the most prevalent bacterial sexually transmitted infection and can cause significant reproductive morbidity in women. There is insufficient knowledge of C. trachomatis-specific immune responses in humans, which could be important in guiding vaccine development efforts. In contrast, murine models have clearly demonstrated the essential role of T helper type 1 (Th1) cells, especially interferon gamma (IFN-γ)-producing CD4+ T cells, in protective immunity to chlamydia. To determine the frequency and magnitude of Th1 cytokine responses elicited to C. trachomatis infection in humans, we stimulated peripheral blood mononuclear cells from 90 chlamydia-infected women with C. trachomatis elementary bodies, Pgp3, and major outer membrane protein and measured IFN-γ-, tumor necrosis factor alpha (TNF-α)-, and interleukin-2 (IL-2)-producing CD4+ and CD8+ T-cell responses using intracellular cytokine staining. The majority of chlamydia-infected women elicited CD4+ TNF-α responses, with frequency and magnitude varying significantly depending on the C. trachomatis antigen used. CD4+ IFN-γ and IL-2 responses occurred infrequently, as did production of any of the three cytokines by CD8+ T cells. About one-third of TNF-α-producing CD4+ T cells coproduced IFN-γ or IL-2. In summary, the predominant Th1 cytokine response elicited to C. trachomatis infection in women was a CD4+ TNF-α response, not CD4+ IFN-γ, and a subset of the CD4+ TNF-α-positive cells produced a second Th1 cytokine.

Limited variation in vaccine candidate Plasmodium falciparum Merozoite Surface Protein-6 over multiple transmission seasons

BackgroundPlasmodium falciparum Merozoite Surface Protein-6 (PfMSP6) is a component of the complex proteinacious coat that surrounds P. falciparum merozoites. This location, and the presence of anti-PfMSP6 antibodies in P. falciparum-exposed individuals, makes PfMSP6 a potential blood stage vaccine target. However, genetic diversity has proven to be a major hurdle for vaccines targeting other blood stage P. falciparum antigens, and few endemic field studies assessing PfMSP6 gene diversity have been conducted. This study follows PfMSP6 diversity in the Peruvian Amazon from 2003 to 2006 and is the first longitudinal assessment of PfMSP6 sequence dynamics.MethodsParasite DNA was extracted from 506 distinct P. falciparum infections spanning the transmission seasons from 2003 to 2006 as part of the Malaria Immunology and Genetics in the Amazon (MIGIA) cohort study near Iquitos, Peru. PfMSP6 was amplified from each sample using a nested PCR protocol, genotyped for allele class by agarose gel electrophoresis, and sequenced to detect diversity. Allele frequencies were analysed using JMP v.8.0.1.0 and correlated with clinical and epidemiological data collected as part of the MIGIA project.ResultsBoth PfMSP6 allele classes, K1-like and 3D7-like, were detected at the study site, confirming that both are globally distributed. Allele frequencies varied significantly between transmission seasons, with 3D7-class alleles dominating and K1-class alleles nearly disappearing in 2005 and 2006. There was a significant association between allele class and village location (p-value = 0.0008), but no statistically significant association between allele class and age, sex, or symptom status. No intra-allele class sequence diversity was detected.ConclusionsBoth PfMSP6 allele classes are globally distributed, and this study shows that allele frequencies can fluctuate significantly between communities separated by only a few kilometres, and over time in the same community. By contrast, PfMSP6 was highly stable at the sequence level, with no SNPs detected in the 506 samples analysed. This limited diversity supports further investigation of PfMSP6 as a blood stage vaccine candidate, with the clear caveat that any such vaccine must either contain both alleles or generate cross-protective responses that react against both allele classes. Detailed immunoepidemiology studies are needed to establish the viability of these approaches before PfMSP6 advances further down the vaccine development pipeline.

Meatal Swabs Contain Less Cellular Material and Are Associated with a Decrease in Gram Stain Smear Quality Compared to Urethral Swabs in Men

ABSTRACT Urethral swabs are the samples of choice for point-of-care Gram stain testing to diagnose Neisseria gonorrhoeae infection and nongonococcal urethritis (NGU) in men. As an alternative to urethral swabs, meatal swabs have been recommended for the collection of urethral discharge to diagnose N. gonorrhoeae and Chlamydia trachomatis infection in certain populations by nucleic acid amplification testing (NAAT), as they involve a less invasive collection method. However, as meatal swabs could be sampling a reduced surface area and result in fewer collected epithelial cells compared to urethral swabs, the adequacy of meatal swab specimens to collect sufficient cellular material for Gram stain testing remains unknown. We enrolled 66 men who underwent either urethral or meatal swabbing and compared the cellular content and Gram stain failure rate. We measured the difference in swab cellular content using the Cepheid Xpert CT/NG sample adequacy control crossing threshold (SACCT) and determined the failure rate of Gram stain smears (GSS) due to insufficient cellular material. In the absence of discharge, meatal smears were associated with a significant reduction in cellular content (P = 0.0118), which corresponded with a GSS failure rate significantly higher than that for urethral swabs (45% versus 3%, respectively; P < 0.0001). When discharge was present, there was no difference among results from urethral and meatal swabs. Therefore, if GSS testing is being considered for point-of-care diagnosis of N. gonorrhoeae infection or NGU in men, meatal swabs should be avoided in the absence of a visible discharge.

Antibodies directed against merozoite surface protein-6 are induced by natural exposure to Plasmodium falciparum in a low transmission environment

Malaria caused by Plasmodium falciparum is a major cause of global infant mortality, and there is currently no licensed vaccine that provides protection against infection or disease. Several P. falciparum vaccine targets have undergone early testing, but many more candidates remain with little data to support their development. Plasmodium falciparum Merozoite Surface Protein 6 (PfMSP6) is a candidate of particular interest because it is a member of the PfMSP3 multi‐gene family, raising the possibility that vaccine‐induced immune responses could cross‐react across multiple family members. However, few immunoepidemiological studies of PfMSP6 have been carried out to measure domain‐specific anti‐PfMSP6 responses. This study investigated anti‐PfMSP6 responses in P. falciparum‐infected individuals from the Peruvian Amazon, using two different PfMSP6 N‐terminal allele antigens and a single C‐terminal domain antigen, and compared the responses with both PfMSP6 genotyping data and anti‐PfMSP3 response data that had been previously generated for the same samples. Anti‐PfMSP6 responses were detected despite the low transmission setting, but were less frequent and of considerably lower intensity than anti‐PfMSP3 responses. There was a positive correlation between anti‐PfMSP3 and PfMSP6 responses, suggesting that the possibility that PfMSP3 family antigens could induce cross‐reactive responses requires further detailed investigation.

Utilization of the Cepheid Xpert® CT/NG Sample Adequacy Control to Determine the Influence of the Urethral Swab on Cellular Content in Post-Swab versus Pre-Swab Urine.

Chlamydia trachomatis/Neisseria gonorrhoeae assay performance in males is typically determined using post-swab urine, though pre-swab urine is used in practice. We collected swabs and urine from men and used the Cepheid Xpert® CT/NG sample adequacy control to determine the effect of swab collection on urine cellular content. No difference was observed.