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Background The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P) Epidemiology, Surveillance and Preparedness of the Novel SARS-CoV-2 Epidemic (RESPONSE) seroprevalence study conducted monthly cross-sectional testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood donors in 6 US metropolitan regions to estimate the extent of SARS-CoV-2 infections over time. Methods During March–August 2020, approximately ≥1000 serum specimens were collected monthly from each region and tested for SARS-CoV-2 antibodies using a well-validated algorithm. Regional seroprevalence estimates were weighted based on demographic differences compared with the general population. Seroprevalence was compared with reported coronavirus disease 2019 (COVID-19) case rates over time. Results For all regions, seroprevalence was, SARS-CoV-2 serosurveillance of blood donors in 6 US regions enabled population weighted seroprevalence estimates. Seroprevelance rates were higher in younger, non-Hispanic Black, and Hispanic donors and correlated with regional case rates. The study has expanded to a national serosurveillance program.

IntroductionThe REDS-IV-P Epidemiology, Surveillance and Preparedness of the Novel SARS-CoV-2 Epidemic (RESPONSE) seroprevalence study conducted monthly cross-sectional testing for SARS-CoV-2 antibodies on blood donors in six U.S. metropolitan regions to estimate the extent of SARS-COV-2 infections over time.Study Design/MethodsDuring March-August 2020, approximately ≥1,000 serum specimens were collected monthly from each region and tested for SARS-CoV-2 antibodies using a well-validated algorithm. Regional seroprevalence estimates were weighted based on demographic differences with the general population. Seroprevalence was compared with reported COVID-19 case rates over time.Results/FindingsFor all regions, seroprevalence was ConclusionIncreases in seroprevalence were found in all regions, with the largest increase in New York. Seroprevalence was higher in non-Hispanic Black and Hispanic blood donors than in non-Hispanic White blood donors. SARS-CoV-2 antibody testing of blood donor samples can be used to estimate the seroprevalence in the general population by region and demographic group. The methods derived from the RESPONSE seroprevalence study served as the basis for expanding SARS-CoV-2 seroprevalence surveillance to all 50 states and Puerto Rico.SummarySARS-CoV-2 serosurveillance data from blood donors in 6 US regions were used to estimate population weighted seroprevalence. Seroprevelance rates were higher in case rates. The study was expanded to a national donor serosurveillance program.DisclaimerThe content is solely the responsibility of the authors and does not represent the policy of the National Institutes of Health or the Department of Health and Human Services. Any specific brandnames included in this manuscript are for identification purposes only and are not intended to represent an endorsement by CDC. The findings and conclusions in this report are those of the authorsand do not necessarily represent the official position of the Centers of Disease Control and Prevention.

Molecular methods have greatly increased our understanding of the previously cryptic spatial ecology of bumble bees (Bombus spp.), with knowledge of the spatial ecology of these bees being central to conserving their essential pollination services. Bombus hypnorum, the Tree Bumble Bee, is unusual in that it has recently rapidly expanded its range, having colonized much of the UK mainland since 2001. However, the spatial ecology of B. hypnorum has not previously been investigated. To address this issue, and to investigate whether specific features of the spatial ecology of B. hypnorum are associated with its rapid range expansion, we used 14 microsatellite markers to estimate worker foraging distance, nest density, between‐year lineage survival rate and isolation by distance in a representative UK B. hypnorum population. After assigning workers to colonies based on full or half sibship, we estimated the mean colony‐specific worker foraging distance as 103.6 m, considerably less than values reported from most other bumble bee populations. Estimated nest density was notably high (2.56 and 0.72 colonies ha−1 in 2014 and 2015, respectively), estimated between‐year lineage survival rate was 0.07, and there was no evidence of fine‐scale isolation by distance. In addition, genotyping stored sperm dissected from sampled queens confirmed polyandry in this population (mean minimum mating frequency of 1.7 males per queen). Overall, our findings establish critical spatial ecological parameters and the mating system of this unusual bumble bee population and suggest that short worker foraging distances and high nest densities are associated with its rapid range expansion.

Introduction Bicondylar tibial plateau fractures with meta-diaphyseal comminution commonly have a compromised soft tissue envelope. Combined plate-nail fixation is an emerging technique that utilizes a limited anterolateral approach for plate application and percutaneous incisions for placement of an intramedullary nail. This technique alleviates the need for a separate medial approach to the proximal tibia. We report a series of patients treated with this approach and outline the steps for implementation. Methods We performed a retrospective review of 18 consecutive patients treated with combination lateral locked plating and intramedullary nailing at a single academic institution from 2016 to 2019. Of these, 16 patients met inclusion criteria and were included in this study. All patients had AO/OTA type 41C2/C3 fractures. Primary outcomes included coronal plane and sagittal plane alignment at latest follow up, rate of articular subsidence at latest follow up, and rate of postoperative infection. Results Of the 16 patients included, average followup was 8.2 months (range 0.1-29.7 months). At latest follow-up, average coronal alignment ranged from 0.8±1.2 degrees of varus (maximum 4.0 degrees) to 1.4±1.7 degrees of valgus (maximum 4.0 degrees). Average sagittal alignment ranged from 0.8±1.1 degrees of procurvatum (maximum 3.0 degrees) to 0.6±1.2 degrees of recurvatum (maximum 4.0 degrees). There was no radiographic evidence of articular subsidence at latest follow up for any patient. One patient (5.9%) presented at 141 days postoperatively with a draining wound and infection. Conclusions Combination plate-nail fixation is a viable option for treating patients with select bicondylar tibial plateau fractures with meta-diaphyseal comminution. This case series with short-term followup demonstrates acceptable radiographic and clinical outcomes, as well as rates of postoperative infection and implant removal similar to those currently reported in the literature for other techniques used to treat these fractures.

Genetic bottlenecks can limit the success of populations colonizing new ranges. However, successful colonizations can occur despite bottlenecks, a phenomenon known as the genetic paradox of invasion. Eusocial Hymenoptera such as bumblebees (Bombusspp.) should be particularly vulnerable to genetic bottlenecks, since homozygosity at the sex-determining locus leads to costly diploid male production (DMP). The Tree Bumblebee (Bombus hypnorum) has rapidly colonized the UK since 2001 and has been highlighted as exemplifying the genetic paradox of invasion. Using microsatellite genotyping, combined with the first genetic estimates of DMP in UKB. hypnorum, we tested two alternative genetic hypotheses (‘bottleneck’ and ‘gene flow’ hypotheses) forB. hypnorum's colonization of the UK. We found that the UK population has not undergone a recent severe genetic bottleneck and exhibits levels of genetic diversity falling between those of widespread and range-restrictedBombusspecies. Diploid males occurred in 15.4% of reared colonies, leading to an estimate of 21.5 alleles at the sex-determining locus. Overall, the findings show that this population is not bottlenecked, instead suggesting that it is experiencing continued gene flow from the continental European source population with only moderate loss of genetic diversity, and does not exemplify the genetic paradox of invasion.

Summary A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation., Graphical Abstract, Highlights • Particles partition to the air-water-interface even during fast (ms) grid making • Higher grid-making speeds can result in greater angular spread of the specimen • Higher grid-making speeds may reduce particle damage and subunit dissociation • Different grid-making apparatus can affect sample concentration in the ice, Here, we take advantage of new grid-making procedures to investigate the effect of speed in cryoEM grid production on protein particle behavior. These results allow us to better understand how different grid-making approaches can affect the resultant grid with respect to particle damage, preferred orientation, and resultant concentration.

Historically chloroquine was used to treat the most deadly form of malaria, caused by the parasite Plasmodium falciparum. The selective pressure of chloroquine therapy led to the rapid emergence of chloroquine resistant parasites. Resistance has been attributed to the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT), an integral membrane protein of unknown structure. A PfCRT structure would provide new insights into how the protein confers chloroquine resistance and thereby also yield novel opportunities for developing anti-malarial therapies. Although PfCRT is an attractive target for characterisation and structure determination, very little work has been published on its expression and purification. Here we present a medium throughput protocol, employing Sf9 insect cells, for testing the expression, stability and purification yield of rationally designed PfCRT mutant constructs and constructs of a PfCRT orthologue from Neospora caninum (NcCRT). We have identified a conserved cysteine residue in PfCRT that results in elevated protein stability when mutated. Combining this mutation with the insertion of T4-lysozyme into a specific surface loop further augments PfCRT protein yield and thermostability. Screening also identified an NcCRT construct with an elevated purification yield. Furthermore it was possible to purify both PfCRT and NcCRT constructs at milligram-scales, with high purities and with size exclusion chromatography profiles that were consistent with monodispersed, homogeneous protein.

Iron deficiency anemia is a common clinical condition often treated with tablets containing 65 mg of elemental iron. Such doses can elicit gastrointestinal side effects lowering patient compliance. Oral iron supplements also increase hepcidin production causing decreased fractional absorption of subsequent doses. Frequent blood donors often become iron deficient. Therefore, they were enrolled in a two-year study involving continued blood donations and randomization to receive no pill, placebo, 19, or 38 mg ferrous gluconate for 60 days. Total body iron (TBI) did not change for the subset of donors in the no pill and placebo groups who completed both enrollment and final visits (p=0.21 and p=0.28, respectively). However, repeated measures regression analysis on the complete dataset estimated a significant decrease in TBI of 52 mg/year for the placebo and no pill groups (p=0.001). The effects of 19 and 38 mg iron supplementation on TBI were indistinguishable (p=0.54). TBI increased by 229 mg after the initial 60 days of iron supplementation (p

BACKGROUND Repeated blood donation alters the iron balance of blood donors. We quantified these effects by analyzing changes in body iron as well as calculating iron absorbed per day for donors enrolled in a prospective study. STUDY DESIGN AND METHODS For 1308 donors who completed a final study visit, we calculated total body iron at the enrollment and final visits and the change in total body iron over the course of the study. Taking into account iron lost from blood donations during the study and obligate losses, we also calculated the average amount of iron absorbed per day. RESULTS First-time/reactivated donors at enrollment had iron stores comparable to previous general population estimates. Repeat donors had greater donation intensity and greater mean iron losses than first-time/reactivated donors, yet they had little change in total body iron over the study period, whereas first-time/reactivated donors had an average 35% drop. There was higher estimated iron absorption in the repeat donors (men: 4.49 mg/day [95% confidence interval [CI], 4.41-4.58 mg/day]; women: 3.75 mg/day [95% CI, 3.67-3.84 mg/day]) compared with estimated iron absorption in first-time/reactivated donors (men: 2.89 mg/day [95% CI, 2.75-3.04 mg/day]; women: 2.76 mg/day [95% CI, 2.64-2.87 mg/day]). The threshold for negative estimated iron stores (below “0” mg/kg stores) was correlated with the development of anemia at a plasma ferritin value of 10 ng/mL. CONCLUSIONS These analyses provide quantitative data on changes in estimated total body iron for a broad spectrum of blood donors. In contrast to using ferritin alone, this model allows assessment of the iron content of red blood cells and the degree of both iron surplus and depletion over time.

Selective degradation of proteins by proteolysis targeting chimeras (PROTACs) offers a promising potential alternative to protein inhibition for therapeutic intervention. Current PROTAC molecules incorporate a ligand for the target protein, a linker, and an E3 ubiquitin ligase recruiting group, which bring together target protein and ubiquitinating machinery. Such hetero-bifunctional molecules require significant linker optimization and possess high molecular weight, which can limit cellular permeation, solubility, and other drug-like properties. We show here that the hetero-bifunctional molecule can be formed intracellularly by bio-orthogonal click combination of two smaller precursors. We designed a tetrazine tagged thalidomide derivative which reacts rapidly with a trans-cyclo-octene tagged ligand of the target protein in cells to form a cereblon E3 ligase recruiting PROTAC molecule. The in-cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully used to degrade two key oncology targets, BRD4 and ERK1/2. ERK1/2 degradation was achieved using a CLIPTAC based on a covalent inhibitor. We expect this approach to be readily extendable to other inhibitor-protein systems because the tagged E3 ligase recruiter is capable of undergoing the click reaction with a suitably tagged ligand of any protein of interest to elicit its degradation., Tetrazine-tagged thalidomide derivatives undergo click reaction with trans-cyclo-octene-tagged protein ligands in cells: the resulting click-formed proteolysis-targeting chimeric molecule elicits ubiquitination and degradation of the target protein.

BACKGROUND The historical approach of offering dietary advice to donors with low hemoglobin (Hb) is ineffective for preventing iron deficiency in frequent donors. Alternative approaches to maintaining donor iron status were explored. STUDY DESIGN AND METHODS Frequent blood donors were randomly assigned into five arms for 2 years of follow-up. Three double-blinded arms provided 60 once-daily pills after each donation (38, 19, or 0 mg of iron). Two single-blinded arms provided iron status (ferritin) or no information letters after each donation. Ferritin, soluble transferrin receptor, and complete blood count were measured at each donation. RESULTS There were 692 subjects enrolled and 393 completed the study. Subjects in pill groups deenrolled more than those in letter groups (39% vs. 7%). Adverse events occurred equally in subjects receiving iron or placebo pills. Of those completing the study, the prevalence of ferritin of less than 12 or less than 26 ng/mL declined by more than 50% and was statistically indistinguishable in the three intervention groups (19 or 38 mg of iron; iron status letter). Longitudinal analyses of all subjects showed improved iron status in iron pill groups and worsening iron status in control groups (placebo; no information letter). The iron pill groups experienced a net increase of approximately 0.6 g/dL Hb compared to control groups. The iron status letter group had little change in Hb. CONCLUSION Providing 19 or 38 mg of daily iron or iron status information were effective and mostly equivalent interventions for mitigating iron deficiency in regular donors when compared at the end of the 2-year longitudinal phase of the study. Donors without intervention had worsened iron deficiency with continued donation.

Lay Summary We manipulated factors that normally constrain the social mate choice of female Seychelles warblers to test if, when less constrained, these females chose partners based on variation at specific immune genes that have been linked to mate choice in other species. We found that older and more generally genetically diverse males were more likely to end up paired with females, but contrary to our predictions, variation at the immune genes did not appear to influence social pairing., The prevalence and significance of precopulatory mate choice remains keenly debated. The major histocompatibility complex (MHC) plays a key role in vertebrate adaptive immunity, and variation at the MHC influences individual survival. Although MHC-dependent mate choice has been documented in certain species, many other studies find no such pattern. This may be, at least in part, because in natural systems constraints may reduce the choices available to individuals and prevent full expression of underlying preferences. We used translocations to previously unoccupied islands to experimentally reduce constraints on female social mate choice in the Seychelles warbler (Acrocephalus sechellensis), a species in which patterns of MHC-dependent extrapair paternity (EPP), but not social mate choice, have been observed. We find no evidence of MHC-dependent social mate choice in the new populations. Instead, we find that older males and males with more microsatellite heterozygosity are more likely to have successfully paired. Our data cannot resolve whether these patterns in pairing were due to male–male competition or female choice. However, our research does suggest that female Seychelles warblers do not choose social mates using MHC class I to increase fitness. It may also indicate that the MHC-dependent EPP observed in the source population is probably due to mechanisms other than female precopulatory mate choice based on MHC cues.

Author(s): Sabino, EC; Ribeiro, AL; Lee, TH; Oliveira, CL; Carneiro-Proietti, AB; Antunes, AP; Menezes, MM; Ianni, BM; Salemi, VM; Nastari, L; Fernandes, F; Sachdev, V; Carrick, DM; Deng, X; Wright, D; Goncalez, TT; Murphy, EL; Custer, B; Busch, MP; Chagas Study Group of the NHLBI Retrovirus Epidemiology Donor Study-II, International Component | Abstract: BackgroundThe significance of detection of Trypanosoma cruzi DNA in blood of antibody-positive patients for risk of development of Chagas heart disease is not well established. The objective of this study was to compare detection of T. cruzi DNA with known clinical and laboratory markers of Chagas cardiomyopathy (CC) severity.MethodsThis is a case-control study nested within a retrospective cohort developed in Brazil to understand the natural history of Chagas disease. The study enrolled 499 T. cruzi seropositive blood donors (SP-BD) and 488 frequency matched seronegative control donors (SN-BD) who had donated between 1996 and 2002, and 101 patients with clinically diagnosed CC. In 2008-2010 all enrolled subjects underwent a health questionnaire, medical examination, electrocardiograms and echocardiograms and polymerase chain reaction (PCR) analyses. A blinded panel of three cardiologists adjudicated the outcome of CC. Trypanosoma cruzi kinetoplast minicircle sequences were amplified by real-time PCR using an assay with a sensitivity of one parasite per 20 mL of blood. All testing was performed on coded samples.ResultsRates of PCR detection of T. cruzi DNA were significantly (P = 0.003) higher in CC patients and SP-BD diagnosed with CC (79/105 [75.2 %]) compared with SP-BD without CC (143/279 [51.3%]). The presence of parasitaemia was significantly associated with known markers of disease progression such as QRS and QT interval duration, lower left ventricular ejection fraction, higher left ventricular index mass, and elevated troponin and NTpro-BNP levels.ConclusionTrypanosoma cruzi PCR positivity is associated with presence and severity of cardiomyopathy, suggesting a direct role of parasite persistence in disease pathogenesis.

Background and Objectives Repeated blood donation produces iron deficiency. Changes in dietary iron intake do not prevent donation-induced iron deficiency. Prolonging the interdonation interval or using oral iron supplements can mitigate donation-induced iron deficiency. The most effective operational methods for reducing iron deficiency in donors are unknown. Materials and Methods ‘Strategies To Reduce Iron Deficiency’ (STRIDE) was a two-year, randomized, placebo-controlled study in blood donors. 692 donors were randomized into one of two educational groups or one of three interventional groups. Donors randomized to educational groups either received letters thanking them for donating, or, suggesting iron supplements or delayed donation if they had low ferritin. Donors randomized to interventional groups either received placebo, 19-mg or 38-mg iron pills. Results Iron deficient erythropoiesis was present in 52·7% of males and 74·6% of females at enrolment. Adverse events within 60 days of enrolment were primarily mild gastrointestinal symptoms (64%). The incidence of de-enrolment within 60 days was more common in the interventional groups than in the educational groups (P = 0·002), but not more common in those receiving iron than placebo (P = 0·68). Conclusion The prevalence of iron deficient erythropoiesis in donors enrolled in the STRIDE study is comparable to previously described cohorts of regular blood donors. De-enrolment within 60 days was higher for donors receiving tablets, although no more common in donors receiving iron than placebo.

Background Few studies were conducted on hepatitis B and C virus (HBV and HCV, respectively) risk factors among Chinese blood donors in recent years since voluntary donors replaced commercial donors. Study Design and Methods A case-control survey was conducted in HBV- or HCV-positive and -negative donors from five blood centers in China between September 2009 and April 2011. Case status was defined by having a reactive result on Monolisa HBsAg Ultra (Bio-Rad) for HBV and Ortho anti-HCV EIA 3.0 (Johnson & Johnson) for HCV. Controls were randomly selected qualified blood donors matched to cases by donation month and blood center. Specific test-seeking, medical-related, and behavioral risk factors were compared by HBV and HCV status using chi-square tests or Fisher's exact tests with Bonferroni correction. Results A total of 364 HBV cases, 174 HCV cases, and 689 controls completed the survey; response rates were 66.2, 47.3, and 82%, respectively. HCV-positive donors were significantly more likely to report having a blood transfusion history (23.4% vs. 3.0%, p

BACKGROUND Donor behaviors in STRIDE (Strategies to Reduce Iron Deficiency), a trial to reduce iron deficiency, were examined. STUDY DESIGN AND METHODS Six hundred ninety-two frequent donors were randomized to receive either 19 or 38 mg iron for 60 days or an educational letter based on their predonation ferritin. Compliance with assigned pills, response to written recommendations, change in donation frequency, and future willingness to take iron supplements were examined. RESULTS Donors who were randomized to receive iron pills had increased red blood cell donations and decreased hemoglobin deferrals compared with controls or with pre-STRIDE donations. Donors who were randomized to receive educational letters had fewer hemoglobin deferrals compared with controls. Of those who received a letter advising of low ferritin levels with recommendations to take iron supplements or delay future donations, 57% reported that they initiated iron supplementation, which was five times as many as those who received letters lacking a specific recommendation. The proportion reporting delayed donation was not statistically different (32% vs. 20%). Of donors who were assigned pills, 58% reported taking them “frequently,” and forgetting was the primary reason for non-compliance. Approximately 80% of participants indicated that they would take iron supplements if provided by the center. CONCLUSIONS Donors who were assigned iron pills had acceptable compliance, producing increased red blood cell donations and decreased low hemoglobin deferrals compared with controls or with pre-STRIDE rates. The majority of donors assigned to an educational letter took action after receiving a low ferritin result, with more donors choosing to take iron than delay donation. Providing donors with information on iron status with personalized recommendations was an effective alternative to directly providing iron supplements.

Genetic factors play an important role in the long-term persistence of populations and species, and conservation strategies should take such factors into account. We use neutral molecular markers to assess diversity and divergence between the three remaining island populations of a little-studied endemic passerine, the Cape Verde warbler Acrocephalus brevipennis. Variation at both microsatellite loci and the cytochrome b gene reveal low diversity within the species overall, but considerable divergence among the populations on Santiago, Fogo and Sao Nicolau islands. The genetic markers show a gradient of genetic diversity with population size, with the smallest population of S. Nicolau being the least diverse, and the largest, Santiago, the most diverse. The more geographically isolated population on S. Nicolau is also more divergent from Santiago and Fogo than these two are from each other. The Cape Verde warbler diverged from its sister species, the greater swamp warbler Acrocephalus rufescens, within the last million years, and the three populations became isolated from one another 165,000–199,000 years ago. There is also evidence of population bottlenecks, especially on S. Nicolau. This population seems to have decreased during the last century, with potential for further decreases and even extinction. As the three populations are genetically distinct, with no evidence of gene flow between them, we argue they should be treated as separate management units for the successful conservation of this species.

Bazedoxifene acetate (BZA) is a selective estrogen receptor modulator that is approved in a number of countries for the prevention and/or treatment of osteoporosis in postmenopausal women. To assess carcinogenic potential, BZA was administered ad libitum in the diet to male and female rats for 2 years. The achieved mean dosages of BZA were approximately 1.31 to 56.9 mg/kg/day at dietary concentrations of 0.003% to 0.1%. BZA treatment resulted in a reduction and a delayed onset in total tumor burden in both male and female rats. Survival rates were enhanced due to decreased pituitary and mammary tumors and decreased body weight gain in BZA-treated animals compared with controls. In male rats only, an increase in renal tubular tumors was observed. The greater increase in tumor incidence in male rats given BZA was associated with the increased survival and increased time for development of late onset tumors. These findings are consistent with a non-genotoxic mechanism, unique to male rats, that involves test article–induced corticomedullary mineralization, renal tubular injury, and exacerbation of naturally occurring chronic progressive nephropathy in aged male rats that led to a sequela of proliferative changes and tumor formation.

Bazedoxifene Acetate (BZA) is a selective estrogen receptor modulator (SERM) that is approved for the prevention and/or treatment of osteoporosis in postmenopausal women. To assess for carcinogenic potential, BZA was administered ad libitum in the diet to rats for 2 years. BZA caused an increase in benign ovarian tumors in female rats and decreased incidences of mammary tumors (females) and pituitary tumors (males and females). In addition, BZA provided a significant survival benefit at all dosages tested, which correlated with a significant reduction in pituitary and mammary gland tumors and decreased body weight gain (both genders). Additional studies were subsequently conducted in rats and monkeys to further explore the mechanisms likely responsible for the observed effects. Results from studies in hypophysectomized and chemically castrated female rats indicated that BZA did not directly stimulate formation of ovarian cysts, but an intact pituitary was required for cyst formation. Further, BZA increased estradiol concentrations in rats and monkeys. In monkeys, BZA increased concentrations of luteinizing hormone (LH) after onset of treatment and prohibited the preovulatory surge of LH until after cessation of treatment. These hormonal changes suggest that BZA inhibited both the positive and negative feedback effects of estrogen on gonadotropins and the resulting increase in LH caused formation and persistence of ovarian cysts, which eventually transformed into benign ovarian granulosa cell tumors in the rat carcinogenicity study. These results also suggest that the reductions in pituitary and mammary gland tumors were attributed to BZA-related antagonism of endogenous estrogens at the estrogen receptors.

OBJECTIVE The purpose of this study was to assess the effect of interleukin (IL)-13 deficiency on fertility and reproductive performance of adult mice and on morphological and behavioral development of the offspring. METHODS Wild-type and homozygous IL-13-deficient (KO) mice were grouped by genotype, and male and female mice were mated within each group. Adult (F0) mice were evaluated for reproductive performance, and development was assessed in F1 fetuses on gestation day 18, and in F1 pups to postnatal day 35. RESULTS In F0 males, there were no differences in the number of males that mated or impregnated females, or in total sperm count or sperm motility, between the wild-type and KO groups. In F0 females, there were no observed genotype-related differences in fertility, length of gestation, number of viable fetuses per litter, or viability of offspring. There were no differences in embryo-fetal development (external/palate, skeletal, visceral) of the F1 fetuses between genotypes. Similarly, IL-13 deficiency had no impact on any postnatal parameters assessed including reflex, sexual maturation, learning, and memory. CONCLUSIONS IL-13 deficiency had no observed effect on reproductive performance or morphological and behavioral development in mice.

Background. Genetic variations of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) can affect diagnostic assays and therapeutic interventions. Recent changes in prevalence of subtypes/genotypes and drug/immune-escape variants were characterized by comparing recently infected vs more remotely infected blood donors. Methods. Infected donors were identified among approximately 34 million US blood donations, 2006‐2009; incident infections were defined as having no or low antiviral antibody titers. Viral genomes were partially sequenced. Results. Of 321 HIV strains (50% incident), 2.5% were non-B HIV subtypes. Protease and reverse transcriptase (RT) inhibitor resistance mutations were found in 2% and 11% of infected donors, respectively. Subtypes in 278 HCV strains (31% incident) yielded 1a.1b.3a.2b.2a.4a.6d, 6e: higher frequencies of 3a in incident cases vs higher frequencies of 1b in prevalent cases were found (P 5 .04). Twenty subgenotypes among 193 HBV strains (26% incident) yielded higher frequencies of A2 in incident cases and higher frequencies of A1, B2, and B4 in prevalent cases (P 5 .007). No HBV drug resistance mutations were detected. Six percent of incident vs 26% of prevalent HBV contained antibody neutralization escape mutations (P 5 .01). Conclusions. Viral genetic variant distribution in blood donors was similar to that seen in high-risk US populations. Blood-borne viruses detected through large-scale routine screening of blood donors can complement molecular surveillance studies of highly exposed populations.

Frequent blood donors become iron deficient. HFE mutations are present in over 30% of donors. A 24-month study of 888 first time/reactivated donors and 1537 frequent donors measured haemoglobin and iron status to assess how HFE mutations impact the development of iron deficiency erythropoiesis. Donors with two HFE mutations had increased baseline haemoglobin and iron stores as did those with one mutation, albeit to a lesser extent. Over multiple donations haemoglobin and iron status of donors with HFE mutations paralleled those lacking mutations. The prevalence of HFE mutations was not increased in higher intensity donors. Thus, in general, HFE mutations do not temper donation-induced changes in haemoglobin and iron status. However, in Black donors there was an increase of H63D carriers at baseline, from 3·7% in first time/reactivated donors to 15·8% in frequent donors, suggesting that the relative effects of HFE mutations on iron absorption may vary between racial/ethnic groups. In secondary analyses, venous haemoglobin decreased more slowly in donors with ferritin ≥12μg/l; and haemoglobin recovery time was shorter in donors with reticulocyte haemoglobin (CHr) ≥32·6pg, indicating that these biochemical measures are better indicators of a donor's response to phlebotomy than their HFE mutation status.

Author(s): Cable, Ritchard G; Glynn, Simone A; Kiss, Joseph E; Mast, Alan E; Steele, Whitney R; Murphy, Edward L; Wright, David J; Sacher, Ronald A; Gottschall, Jerry L; Tobler, Leslie H; Simon, Toby L; NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II) | Abstract: BackgroundBlood donors are at risk of iron deficiency. We evaluated the effects of blood donation intensity on iron and hemoglobin (Hb) in a prospective study.Study design and methodsFour cohorts of frequent and first-time or reactivated (FT/RA) blood donors (no donation in 2 years), female and male, totaling 2425, were characterized and followed as they donated blood frequently. At enrollment and the final visit, ferritin, soluble transferrin receptor (sTfR), and Hb were determined. Models to predict iron deficiency and Hb deferral were developed. Iron depletion was defined at two levels: iron deficiency erythropoiesis (IDE) [log(sTfR/ferritin) ≥ 2.07] and absent iron stores (AIS; ferritin l 12 ng/mL).ResultsAmong returning female FT and RA donors, 20 and 51% had AIS and IDE at their final visit, respectively; corresponding proportions for males were 8 and 20%. Among female frequent donors who returned, 27 and 62% had AIS and IDE, respectively, while corresponding proportions for males were 18 and 47%. Predictors of IDE and/or AIS included a higher frequency of blood donation in the past 2 years, a shorter interdonation interval, and being female and young; conversely, taking iron supplements reduced the risk of iron depletion. Predictors of Hb deferral included female sex, black race, and a shorter interdonation interval.ConclusionsThere is a high prevalence of iron depletion in frequent blood donors. Increasing the interdonation interval would reduce the prevalence of iron depletion and Hb deferral. Alternatively, replacement with iron supplements may allow frequent donation without the adverse outcome of iron depletion.

Donor-based risk reduction interventions for transfusion-related acute lung injury (TRALI) have been widely adopted in the United States and elsewhere in the past several years.1–5 These strategies are based on data indicating that TRALI has continued to be the leading cause of blood transfusion-related deaths in the United States,6 the consensus that approximately 80% of TRALI cases are mediated by donor human leukocyte antigen (HLA) antibodies,7 and surveillance system findings that TRALI cases are reduced by use of such risk reduction strategies.2,8,9 Thus, as of 2009, almost all US blood centers are transfusing plasma supplied primarily by male or never-pregnant female donors, and many centers are screening previously pregnant platelet (PLT) and/or plasma apheresis donors for HLA antibody.1,9 Most transfused components containing HLA antibody do not result in recipients developing TRALI. Reasons for this include transfusion to a recipient who does not have a cognate HLA antigen profile, as well as less established factors such as the titer and/or other undefined characteristics of the specific transfused HLA antibody or the existence of underlying predisposing recipient risk factors (first hit). Most current theories of TRALI pathogenesis agree that two hits are usually necessary for TRALI to occur: the first is a recipient condition that results in neutrophil priming and the second is the infusion of a biologic response modifier that activates primed neutrophils. The biologic response modifier can be an HLA antibody, an HNA antibody, or several candidate nonantibody substances, such as bioactive lipids.10–12 In terms of this model, HLA antibody–containing components may fail to cause TRALI in the absence of a first hit. At least four studies have assessed outcomes in recipients of previously donated components from HLA antibody–positive donors who were implicated as causing TRALI in an index recipient.13–16 In aggregate, these studies detected five new cases of TRALI that were not reported to the blood bank when records of 171 patients were reviewed (2.9%). In addition, two lookback studies were reported on donors who were HLA antibody positive but had not been implicated in TRALI. In one of these studies, 62 HLA antibody–positive female donors gave a total of 211 blood components, and only one case of TRALI (which had previously been reported to the blood bank) was identified.17 In the second study, no TRALI cases were detected in 167 recipients of transfusions from four HLA antibody–positive donors or in 295 recipients from 12 HLA antibody–negative donors. However, this conclusion appeared to be based solely on review of transfusion service records.18 One additional study did not detect TRALI in any of 265 recipients of either HLA antibody–positive or HLA antibody–negative plateletpheresis units but did not clearly delineate the number of recipients in each group.19 Taken as a group, these lookback studies have significant limitations in that they used differing methods for HLA antibody screening, reviewing recipient outcome data and for diagnosing TRALI; they had small sample sizes considering the low incidence of TRALI; and most did not evaluate TRALI occurrence in recipients of control components using a blinded study design. Since it is well known that TRALI is both underrecognized and underre-ported,20 it is unclear if the findings in these studies can be generalized because methods for ascertainment were not robust. As part of the Retrovirus Epidemiology Donor Study-II (REDS-II), we previously conducted the Leukocyte Antibody Prevalence Study-I (LAPS-I), which identified 1032 whole blood and apheresis donors with HLA antibody.21 Since LAPS-I was conducted before the implementation of TRALI risk reduction policies, donations from these HLA antibody–positive donors were transfused to recipients. Given the limitations of previous studies noted above and the recognition that the recent implementation of TRALI risk reduction strategies will reduce the possibility of conducting future large-scale TRALI lookback studies, we designed a large multicenter retrospective cohort study termed the Leukocyte Antibody Prevalence Study-II (LAPS-II). Based on surveillance data showing a higher TRALI risk from transfused plasma components, LAPS-II was confined to high-plasma-volume components (fresh-frozen plasma and other transfusable plasma manufactured from whole blood or apheresis as well as plateletpheresis components).2,8,9 LAPS-II was designed to evaluate a primary endpoint of combined incidence of TRALI and possible TRALI in study recipients of at least one HLA antibody–positive high-plasma-volume component versus control recipients of at least one HLA antibody–negative high-plasma-volume component. Secondary endpoints were the association of HLA antibody class, HLA antibody specificity, HLA antibody screening test signal strength, and concomitant HNA antibody on TRALI occurrence.

The interaction between murine double minute (MDM2) and p53 is a major target in anticancer drug design. Several potent compound series, including the nutlins and spirooxindoles, have previously been established as high-affinity antagonists of MDM2. In this paper, we describe the interaction of isoindolinone inhibitors with MDM2, as characterized by nuclear magnetic resonance spectroscopy. Isoindolinone inhibitors bind specifically to the MDM2 p53 binding site and exploit all sub-pockets used by p53, nutlins and spirooxindoles. Furthermore, isoindolinones bind with low micromolar to high nanomolar affinities, with the best compound approaching the potency of nutlin-3.

Author(s): Cable, Ritchard G; Glynn, Simone A; Kiss, Joseph E; Mast, Alan E; Steele, Whitney R; Murphy, Edward L; Wright, David J; Sacher, Ronald A; Gottschall, Jerry L; Vij, Vibha; Simon, Toby L; NHLBI Retrovirus Epidemiology Donor Study-II | Abstract: BackgroundRegular blood donors are at risk of iron deficiency, but characteristics that predispose to this condition are poorly defined.Study design and methodsA total of 2425 red blood cell donors, either first-time (FT) or reactivated donors (no donations for 2 years) or frequent donors, were recruited for follow-up. At enrollment, ferritin, soluble transferrin receptor (sTfR), and hemoglobin were determined. Donor variables included demographics, smoking, dietary intake, use of iron supplements, and menstrual and/or pregnancy history. Models to predict two measures of iron deficiency were developed: Absent iron stores (AIS) were indicated by a ferritin level of less than 12 ng/mL and iron-deficient erythropoiesis (IDE) by a log(sTfR/ferritin) value of 2.07 or greater.ResultsA total of 15.0% of donors had AIS and 41.7% IDE. In frequent donors, 16.4 and 48.7% of males had AIS and IDE, respectively, with corresponding proportions of 27.1 and 66.1% for females. Donation intensity was most closely associated with AIS and/or IDE (odds ratios from 5.3 to 52.2 for different donation intensity compared to FT donors). Being female, younger, and/or menstruating also increased the likelihood of having AIS and/or IDE, as did having a lower weight. Marginally significant variables for AIS and/or IDE were being a nonsmoker, previous pregnancy, and not taking iron supplements. Dietary variables were in general unrelated to AIS and/or IDE, as was race and/or ethnicity.ConclusionA large proportion of both female and male frequent blood donors have iron depletion. Donation intensity, sex and/or menstrual status, weight, and age are important independent predictors of AIS and/or IDE. Reducing the frequency of blood donation is likely to reduce the prevalence of iron deficiency among blood donors, as might implementing routine iron supplementation.

This report discusses the principles of developmental and reproductive toxicity (DART) testing for biopharmaceuticals. Biopharmaceuticals are large-molecular-weight proteins or peptides produced by modern biotechnology techniques incorporating genetic engineering and hybridoma technologies. The principles of DART testing for biopharmaceuticals are similar to those for small-molecule pharmaceuticals and in general follow the regulatory guidance outlined in International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) document S5(R2). However, because many biopharmaceuticals are species-specific, alternate approaches may be needed to evaluate DART potential as outlined in ICH S6. For molecules that show species-specific cross-reactivity restricted to non-human primates (NHP), some aspects of DART may require NHP testing. For biopharmaceuticals that are uniquely specific and only active on intended human targets or human and chimpanzee targets, surrogate molecules that cross-react with the more traditional rodent species may need to be developed and used for DART testing. Alternatively, genetically modified transgenic animals may also need to be considered. Surrogate molecules and transgenic animals may also be considered for DART testing even if the biopharmaceutical is active in NHPs in order to reduce the use of NHPs. Because of the unique properties of biopharmaceuticals, a case-by-case approach is needed for DART and general toxicity evaluation, which requires consideration of specific product attributes including biochemical and biophysical characteristics, pharmacological activity, and intended clinical indication.

Background The dynamics of the early stages of West Nile virus (WNV) infection can be assessed by follow-up studies of viremic blood donors. Methods A total of 245 donors with WNV viremia were followed up weekly for 4 weeks and then monthly for up to 6 additional months or until seroconversion. Plasma samples were tested for WNV RNA by transcription-mediated amplification (TMA) and for WNV-specific IgM and IgG antibodies. RNA persistence was investigated by 6 replicate TMA tests; samples that were viremic for >40 days were tested for WNV-neutralizing activity. Follow up of 35 additional viremic donors for up to 404 days was conducted to evaluate persistence of WNV-specific antibody. Results The median time from RNA detection to IgM seroconversion was 3.9 days; to IgG seroconversion, 7.7 days; to RNA negativity by single-replicate TMA, 13.2 days; and to RNA negativity by 6-replicate TMA, 6.1 additional days after results of single-replicate TMA are negative. For 4 donors in whom RNA persisted for >40 days after the index donation, all samples obtained after this threshold were also positive for WNV IgG and neutralizing activity. The mean times to IgM and IgA negativity were 156 and 220 days, respectively. Conclusions IgM and IgG develop rapidly after viremia and before RNA levels become undetectable, which occurred a mean of 13.2 days after the index donation among donors in this study. WNV RNA detection by replicate TMA rarely persists for >40 days after the index donation and is accompanied by WNV-specific neutralizing antibody, consistent with an absence of WNV transmission via transfusion of seropositive blood components.

Between 30% and 100% of HIV-infected patients are coinfected with hepatitis C virus (HCV) [1–4]. A number of studies have negatively associated HIV with the progression of HCV disease [5–7]. HIV accelerates HCV-associated complications, such as liver cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The impact of HCV on the progression of HIV disease is less clear—there have been a number of discordant findings. Some studies have reported that HCV accelerates the progression of HIV disease [8–11], whereas others found no impact [12, 13]. Variation in the definition of the progression of HIV disease—based on incidences of opportunistic infections or decreases in CD4+ T cell count, sample size, duration of follow-up, and other cofounding variables—may have contributed to these conflicting findings. Few studies have evaluated the effect of HCV co-infection on immune recovery after the initiation of highly active antiretroviral therapy (HAART). In those studies, immune recovery was defined by either the magnitude or rate of increases in CD4+ T cell counts over time, with controversial findings. Some studies have reported that the level and rate of increases in CD4+ T cell counts are lessened and slower in coinfected than in monoinfected patients [10, 14, 15], whereas others did not find a negative association between HCV coinfection and CD4+ T cell responses after the initiation of HAART [16–18]. None of these studies, however, evaluated a larger breadth of immunophenotypic markers relevant to immune function. Through the Women’s Interagency HIV Study (WIHS), a multicenter cohort established in 1993 in the United States, where ~39% of HIV-infected women are coinfected with HCV, we retrospectively evaluated the impact of HIV/HCV coinfection on immune recovery after the initiation of HAART between HIV-monoinfected women and women coinfected with HCV who have either cleared or did not clear HCV. Immune recovery was evaluated by examining alterations in CD4+ and CD8+ T cell counts and alterations in the dynamics of activation and naive/memory T cell status within the CD4+ and CD8+ T cell compartments.

Imagery can improve sport performance, although its efficacy is mediated by the ease with which athletes can generate images. Establishing techniques that improve this imagery ability factor is desirable to enhance the effectiveness of imagery interventions. Twenty-seven club-level female golfers were assigned to imagery, action observation, or physical practice training groups (n = 9). Changes in image generation ease were assessed using the Revised Movement Imagery Questionnaire over an 8-week period. Imagery and action observation training were both effective in improving aspects of imagery ability. Action observation can provide sport psychologists with an effective tool for improving visual imagery ability in athletes.

The importance of evolutionary conservation – how understanding evolutionary forces can help guide conservation decisions – is widely recognized. However, the historical demography of many endangered species is unknown, despite the fact that this can have important implications for contemporary ecological processes and for extinction risk. Here, we reconstruct the population history of the Seychelles warbler (Acrocephalus sechellensis) – an ecological model species. By the 1960s, this species was on the brink of extinction, but its previous history is unknown. We used DNA samples from contemporary and museum specimens spanning 140 years to reconstruct bottleneck history. We found a 25% reduction in genetic diversity between museum and contemporary populations, and strong genetic structure. Simulations indicate that the Seychelles warbler was bottlenecked from a large population, with an ancestral Ne of several thousands falling to

SUMMARY Length-biased sampling occurs in renewal processes when the probability that an interval is selected is proportional to the length of the interval. This can occur when intervals are selected because they contain an event that is independent of the renewal process and occurs with constant hazard. For example, if the times between donations for repeat blood donors are independent and identically distributed, and if the donor seroconverts to HIV (develops antibodies that indicate infection with human immunodeficiency virus), then the interval between the last HIV seronegative and first HIV seropositive test is expected to be longer than that donor’s previous time intervals between donations. We develop hypothesis tests to determine if the relationship between the typical and length-biased intervals is as expected, or if there is departure from length-biased sampling. We further develop a regression method to determine if there are covariates that explain the departure from length-biased sampling. Our approach is motivated by the question of whether there is evidence that repeat blood donors who develop antibodies to HIV or other viral infections change their donation pattern in some way because of seroconversion.

Objectives The characterization of primary HIV infection by the analysis of serial plasma samples from newly infected persons using multiple standard viral assays. Design A retrospective study involving two sets of archived samples from HIV-infected plasma donors. (A) 435 samples from 51 donors detected by anti-HIV enzyme immunoassays donated during 1984-1994; (B) 145 specimens from 44 donors detected by p24 antigen screening donated during 1996-1998. Setting Two US plasma products companies. Main outcome measures The timepoints of appearance of HIV-1 markers and viral load concentrations during primary HIV infection. Results The pattern of sequential emergence of viral markers in the 'A' panels was highly consistent, allowing the definition and estimation of the duration of six sequential stages. From the 'B' panels, the viral load at p24 antigen seroconversion was estimated by regression analysis at 10 000 copies/ml (95% CI 2000-93 000) and the HIV replication rate at 0.35 log copies/ml/day, corresponding to a doubling time in the preseroconversion phase of 20.5 h (95% CI 18.2-23.4 h). Consequently, an RNA test with 50 copies/ml sensitivity would detect HIV infection approximately 7 days before a p24 antigen test, and 12 days before a sensitive anti-HIV test. Conclusion The sequential emergence of assay reactivity allows the classification of primary HIV-1 infection into distinct laboratory stages, which may facilitate the diagnosis of recent infection and stratification of patients enrolled in clinical trials. Quantitative analysis of preseroconversion replication rates of HIV is useful for projecting the yield and predictive value of assays targeting primary HIV infection.

Imagery is commonly used by both athletes and musicians as an additional training aid to enhance performance. Although considerable research attention has been paid to this psychological intervention technique in sporting settings, studies investigating the beneficial effects of imagery on musical performance are less common. Imagery research that has been conducted in musical settings has tended to show that imagery is beneficial in improving performance, although few guidelines exist in the music literature regarding how musicians could use imagery to improve their performance. In this article, we review the research which has investigated the use of imagery as a performance enhancing technique in musical settings to date. We then outline a model for designing imagery interventions (known as the PETTLEP model) which has produced beneficial performance effects when implemented in sporting settings, and discuss how it could be used by musicians to enhance performance.

Translocations are an increasingly common tool in conservation. The maintenance of genetic diversity through translocation is critical for both the short- and long-term persistence of populations and species. However, the relative spatio-temporal impacts of translocations on neutral and functional genetic diversity, and how this affects genetic structure among the conserved populations overall, have received little investigation. We compared the impact of translocating different numbers of founders on both microsatellite and major histocompatibility complex (MHC) class I diversity over a 23-year period in the Seychelles warbler (Acrocephalus sechellensis). We found low and stable microsatellite and MHC diversity in the source population and evidence for only a limited loss of either type of diversity in the four new populations. However, we found evidence of significant, but low to moderate, genetic differentiation between populations, with those populations established with fewer founders clustering separately. Stochastic genetic capture (as opposed to subsequent drift) was the main determinant of translocated population diversity. Furthermore, a strong correlation between microsatellite and MHC differentiation suggested that neutral processes outweighed selection in shaping MHC diversity in the new populations. These data provide important insights into how to optimize the use of translocation as a conservation tool.

To evaluate the effect of the menstrual cycle in HIV-positive women on plasma and genital cytokine levels, interrelationships between vaginal and plasma cytokines, CD4 and CD8 T cell fluctuations, and genital and plasma viral loads.Plasma and cervicovaginal lavage specimens were collected from 55 HIV-positive women with CD4 cell counts350 cells/microl during phases of the menstrual cycle. Samples were assayed for IL-1beta, IL-6, IL-4, IL-8, IL-10, TGFbeta, TNFalpha, INFgamma, MIP1alpha, MIP1beta, RANTES, and TNFR-II using enzyme-linked immunosorbent assays. CD4 and CD8 T cell expression was evaluated by flow cytometry. Repeated measures regression models were used to assess the effect of the menstrual cycle on cytokines and viral load. Multivariate repeated regression models were used to assess the correlation among selected cytokines and between selected cytokines and HIV viral load.Vaginal IL-1beta, IL-4, IL-6, IL-8, IL-10, MIP1beta, RANTES, TGFbeta, and TNFR-II were significantly elevated during menses but were not altered during other phases. Plasma cytokine levels were not altered during the menstrual cycle. A positive Candida culture increased vaginal IL-8 during menses, whereas vaginal discharge was associated with a reduction in vaginal IL-4, IL-10, and RANTES. CD4 and CD8 cell numbers did not vary with the menstrual cycle. Vaginal cytokine levels correlated only with vaginal viral load, in a sampling method-dependent manner.We provide evidence of elevated vaginal cytokine levels during menses, which appear to regulate vaginal and not plasma HIV shedding, suggesting that a menstrual cycle pattern exists for cytokine production in HIV-positive women impacting vaginal shedding of HIV.

OBJECTIVE To assess the variation in HIV-1 over the menstrual cycle, including RNA levels in the female genital tract, plasma HIV-1-RNA levels, CD4 cell counts, and culturable virus. DESIGN A prospective analysis of 55 HIV-1-infected women. METHODS Blood and genital tract specimens were collected weekly over 8 weeks, spanning two complete menstrual cycles. Applying repeated-measures models that used menses as the reference level, the variation in viral RNA levels was compared in endocervical canal fluid and cells (collected by Sno-strips and cytobrush, respectively) and ectocervicovaginal lavage (CVL) fluid. Repeated-measures models were also used to assess the variation in plasma CD4 cell counts and viral load. RESULTS Shedding patterns differed among the three sampling methods, independent of genital tract co-infections. Genital tract HIV-1-RNA levels from CVL fluid and endocervical canal cytobrush specimens were highest during menses and lowest immediately thereafter (P = 0.001 and P = 0.04). The HIV-1-RNA level in endocervical canal fluid was highest in the week preceding menses (P = 0.003). The menstrual cycle had no effect on blood levels of RNA (P = 0.62), culturable virus (P = 0.34), or CD4 cell counts (P = 0.55). HIV-1-RNA levels were higher in endocervical canal fluid than in peripheral blood plasma during the late luteal phase (P = 0.03). CONCLUSION HIV-1-RNA levels vary with the menstrual cycle in the female genital tract but not the blood compartment. HIV-1-RNA levels are higher in endocervical canal fluid than in blood plasma. These findings may have important implications for sex-specific pathogenesis, heterosexual transmission, and contraceptive hormone interventions in HIV-1-infected women.

To understand the impact of the menstrual cycle on immunologic parameters, we measured the level of cytokines and chemokines from plasma, cervicovaginal lavage (CVL), and saliva samples of 6 premenopausal women during the follicular and luteal phases of the ovulatory cycle. We demonstrate that the level of plasma interleukin-8 (IL-8) was 4-fold higher during the follicular phase than the luteal phase (p = 0.004), whereas plasma IL-1beta, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and TNF receptor II (TNFR II) were not altered during the ovulatory cycle. In the vaginal compartment, as measured from CVL samples, the levels of IL-6 and IL-1beta were both 5-fold higher in the follicular than the luteal phase (p = 0.0002 and 0.03, respectively). Salivary cytokine and chemokine samples were similar when measured during the luteal and the follicular phases. Additional analysis of lymphocyte subsets for phenotypic and functional markers indicated that they were not influenced by the ovulatory cycle. Collectively, these data suggest that IL-6, IL-8, and IL-1beta are differentially regulated during the ovulatory cycle.

Context.—The human T-lymphotropic viruses types 1 and 2 (HTLV-1 and HTLV-2) are highly prevalent among injection drug users in the United States. However, the clinical course of infection has not been well characterized. Objective.—To understand HTLV-1–and HTLV-2–associated laboratory abnormalities, which may provide insights into their underlying pathophysiology. Design.—Cohort study. Setting.—Five US blood centers. Participants.—A total of 133 HTLV-1–and 332 HTLV-2–seropositive former blood donors and 717 HTLV-seronegative donors followed up prospectively since 1991. Main Outcome Measures.—Selected serum chemistry tests and complete blood cell counts were analyzed at enrollment and approximately 2 years later in participants. Repeated-measures analyses were conducted to evaluate the effect of HTLV infection on laboratory measures. Results.—Compared with seronegative subjects, HTLV-1–seropositive subjects had 13% higher creatine kinase (P = .02) and slightly elevated lactate dehydrogenase (P = .03) levels at follow-up. The HTLV-2–seropositive participants had 11% higher absolute lymphocyte counts than seronegative subjects (P = .0001). Infection with HTLV-2 also appeared to be associated with slightly higher hemoglobin levels (P = .03) and hematocrit (P = .03) and with lower albumin levels (P = .01). Conclusions.—These results further our understanding of the biological mechanisms underlying HTLV and suggest that HTLV-associated laboratory changes are unlikely to alter clinical evaluation or counseling of otherwise healthy HTLV-infected subjects.

The objective of this study was to assess the effect of menstrual phase on the ability to quantitate HIV-1 in vaginocervical secretions (VCS) through reconstruction experiments with HIV seronegative VCS collected throughout the menstrual cycle. Measurement of HIV-1 inoculated into both fresh and frozen VCS was undertaken by quantitative micro co-culture, p24 antigen assay and polymerase chain reaction (PCR) for both HIV-1 RNA and pro-viral DNA. Two laboratories carried out these assays over a range of viral concentrations. The study involved a randomized factorial design and the factors were: (1) diluents (phases of the menstrual cycle and controls); (2) laboratories; (3) stock concentrations; and (4) frozen versus fresh VCS samples. Each assay was assessed independently using a random effects analysis of variance (ANOVA) model. No statistical differences due to menstrual cycle were seen in the assay results of p24 antigen (P=0.08), PBMC culture (P=0.74), plasma culture (P=0.13), cell-free RNA (P=0.44), cell-associated RNA (P=0.58) and cell-associated DNA (P=0.43). Inter-laboratory differences were statistically significant for cell-free RNA (P

INTRODUCTION To investigate the coordinate developmental expression of low-density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, sterol carrier protein 2 (SCP2), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450,sc) enzyme messages throughout the pig estrous cycle, RNase protection analysis was performed using homologous (partially cloned) porcine sequences. Total RNA was isolated from ovarian tissues from unstimulated prepubertal gilts and gilts stimulated with eCG (Day -3) and hCG (Day 0) to induce follicular growth and ovulation. Specific transcripts (relative to 18S rRNA) were quantified in immature ovaries, preovulatory follicles (- 5 mm), corpora lutea (CL), and corpora albicantia. As an index of steroidogenesis, tissue progesterone content (per microgram protein) was low in the unstimulated ovary and preovulatory follicles, and it began to increase 4 days post-hCG, peaked at 12 days, and returned to preovulatory concentrations by 20 days post-hCG. HMG-CoA reductase mRNA was expressed at low levels and did not change significantly throughout the estrous cycle. The amount of LDL receptor mRNA increased approximately 6-fold after eCG stimulation and was expressed at similar concentrations in both preovulatory follicles and functional CL. Expression of SCP2 mRNA did not differ among the four tissue types but tended to be highest in midcycle (Day 12) CL compared other stages of CL (p = 0.007). StAR mRNA expression was minimal in unstimulated ovaries, was higher in preovulatory follicles (p = 0.014), and then rose again in CL (p = 0.009 compared with unstimulated ovary). P450sc mRNA concentrations were low in unstimulated ovaries, increased in preovulatory follicles (p = 0.044), and increased further in CL (p = 0.001 compared with preovulatory follicles). P450sc, and StAR mRNA levels correlated with progesterone levels (r = +0.37, p = 0.025, and r = +0.71, p < 0.001, respectively). The expression of LDL receptor, StAR, and P450sc, messages showed a dramatic decline by Day 20 post-hCG (p = 0.002, p = 0.003, p = 0.006, respectively, compared with CL) corresponding with functional regression of the CL. In summary, P450,. and StAR message expression are coordinately amplified during the pig follicular and luteal phase, whereas LDL receptor message after an initial increase is expressed at constitutively high levels, thus indicating a differential regulation of ovarian sterol-metabolizing genes during the steroidogenic life of the follicle and CL.

We are grateful that our article has stimulated interest in and discussion of the incidence of clinical cardiomyopathy among asymptomatic persons infected with Trypanosoma cruzi .1 Rassi and Rassi comment that without more frequent interval examinations, we are unable to determine whether the incidence density of cardiomyopathy was constant over the 10 years of follow-up. This is true. However, their comment that “T cruzi–infected individuals who develop cardiomyopathy usually do so within 20 years after being infected” is not referenced in their letter; in a recent review article, the same authors postulated a 10- to 30-year incubation period.2 We are therefore unsure that the question of a constant versus declining incidence density has been settled. We also caution that the 18-year estimate for duration of exposure to T cruzi in our article was derived from an analysis of subject-reported risk factors and residence in endemic areas and should not be overinterpreted. However, we agree that the question of whether incidence density is constant or declines with time is important, and …

Dietary iron absorption is regulated by hepcidin, an iron regulatory protein produced by the liver. Hepcidin production is regulated by iron stores, erythropoiesis and inflammation, but its physiology when repeated blood loss occurs has not been characterized. Hepcidin was assayed in plasma samples obtained from 114 first-time/reactivated (no blood donations in preceding 2 years) female donors and 34 frequent (≥3 red blood cell donations in preceding 12 months) male donors as they were phlebotomized ≥4 times over 18-24 months. Hepcidin levels were compared to ferritin and hemoglobin levels using multivariable repeated measures regression models. Hepcidin, ferritin and hemoglobin levels declined with increasing frequency of donation in the first-time/reactivated females. Hepcidin and ferritin levels correlated well with each other (Spearman's correlation of 0.74), but on average hepcidin varied more between donations for a given donor relative to ferritin. In a multivariable repeated measures regression model the predicted inter-donation decline in hemoglobin varied as a function of hepcidin and ferritin; hemoglobin was 0.51 g/dL lower for subjects with low (>45.7 ng/mL) or decreasing hepcidin and low ferritin (>26 ng/mL), and was essentially zero for other subjects including those with high (>45.7 ng/mL) or increasing hepcidin and low ferritin (>26 ng/mL) levels (P

Transcranial magnetic stimulation (TMS) studies indicate that the observation of other people's actions influences the excitability of the observer's motor system. Motor evoked potential (MEP) amplitudes typically increase in muscles which would be active during the execution of the observed action. This 'motor resonance' effect is thought to result from activity in mirror neuron regions, which enhance the excitability of the primary motor cortex (M1) via cortico-cortical pathways. The importance of TMS intensity has not yet been recognised in this area of research. Low-intensity TMS predominately activates corticospinal neurons indirectly, whereas high-intensity TMS can directly activate corticospinal axons. This indicates that motor resonance effects should be more prominent when using low-intensity TMS. A related issue is that TMS is typically applied over a single optimal scalp position (OSP) to simultaneously elicit MEPs from several muscles. Whether this confounds results, due to differences in the manner that TMS activates spatially separate cortical representations, has not yet been explored. In the current study, MEP amplitudes, resulting from single-pulse TMS applied over M1, were recorded from the first dorsal interosseous (FDI) and abductor digiti minimi (ADM) muscles during the observation of simple finger abductions. We tested if the TMS intensity (110% vs. 130% resting motor threshold) or stimulating position (FDI-OSP vs. ADM-OSP) influenced the magnitude of the motor resonance effects. Results showed that the MEP facilitation recorded in the FDI muscle during the observation of index-finger abductions was only detected using low-intensity TMS. In contrast, changes in the OSP had a negligible effect on the presence of motor resonance effects in either the FDI or ADM muscles. These findings support the hypothesis that MN activity enhances M1 excitability via cortico-cortical pathways and highlight a methodological framework by which the neural underpinnings of action observation can be further explored.

The laboratory diagnosis of syphilis remains a challenge to physicians. The organism cannot be subcultured and dark-field microscopy requires a skilled operator. Current routine assays rely on serological-based techniques, which cannot distinguish syphilis from other treponemal infections such as yaws and pinta. False-positive and false-negative results can occur, as well as difficulties between differentiating between acute and chronic infection. Concomitant infection with human immunodeficiency virus (HIV) can also skew results. Molecular analysis with polymerase chain reaction (PCR) is promising but is still in its infancy and standardisation is required. Therefore in order to diagnose syphilis correlation with the history and clinical examination findings remain of paramount importance. Key Concepts: Models of the cyclical epidemics of syphilis, which have been proposed and the factors that influence their periodicity and size of recurrence. Comparison with the Treponema species that infect man with those from primates points to the origin of the disease, whereas study of their molecular structure throws light on treponemal biology. The role of serological tests in the diagnosis of syphilis is analysed. Rapid screening tests are contrasted with more sophisticated laboratory tests. An example of the latter is the place of confirmatory tests. The reasons for the gradual introduction of molecular tests like PCR into the diagnostic armoury and their current shortcomings are evaluated. The practical difficulties of manufacturing and introducing a syphilis ‘vaccine’ are addressed, despite the theoretical and experimental support for this approach. Keywords: syphilis; serology; treponema; PCR; immunisation

Although the genetic variability of hepatitis B virus (HBV) in HBV-infected patients has been extensively studied, reports on genotypes, subtypes and mutations in the S region of HBV strains from Chinese blood donors are limited. In this study, 245 blood samples from HBsAg-positive blood donors were collected from five geographically diverse blood centers in China. The S region of HBV was amplified, and the HBV genotype and subtype were determined. The amino acid sequences of the S region were aligned, and mutations related to the failure of immunization and HBsAg detection were determined. Of the 245 samples, 228 (93 %) were genotyped successfully. We found that genotypes B, C, D and A accounted for 58.8 %, 21.9 %, 6.6 % and 3.95 % of the isolates, respectively. The distribution of HBV antigen subtypes was as follows: adw (67.6 %), adr (23.3 %) and ayw (8.7 %). Mutations were present in 39 (17.1 %) of 228 samples in the major hydrophilic region (MHR) of the S region. This study demonstrated that HBV genotype/subtype B/adw was the most frequent strain circulating in HBV-infected Chinese blood donors, followed by C/adr. The occurrence of MHR mutants in HBV-infected blood donors and the potential failure to detect some of them in collected units poses a threat to transfusion safety.

BACKGROUND: To determine if the fetus was affected by maternal antibodies to BMP-2, the antibody response and developmental effects in fetuses from does immunized against recombinant human BMP-2 were evaluated. METHODS: Female New Zealand White rabbits received four intramuscular injections (on premating days 1, 8, 22, and 43 [3 days before mating]) of saline and adjuvant (TiterMax® Gold [control]) or recombinant human BMP-2 (2 mg/dose) and adjuvant (treated). On GD 29, fetuses were examined, and maternal and fetal anti-BMP-2 titer levels and neutralizing activity were assessed. RESULTS: Anti-BMP-2 antibodies were detected in 17 of 18 treated does (127 of 151 fetuses), and low levels were detected in 2 of 16 control does (no fetal exposure observed). In general, levels of fetal anti-BMP-2 antibodies were similar to those in the does, and pregnancy did not boost the immune response to BMP-2. There were no effects of immunization or anti-BMP-2 antibody titer levels on embryo–fetal viability, fetal weight, or fetal external, visceral, or skeletal development. Only a small number of fetuses (n = 4) displayed detectable neutralizing anti-BMP-2 antibodies, but there were no treatment-related effects in those fetuses. CONCLUSIONS: The lack of embryo–fetal effects may be due to dosage effects of neutralizing anti-BMP-2 antibodies, timing of exposure (stage and duration) to neutralizing anti-BMP-2 antibodies, and/or redundancy of effects of the various BMPs. Birth Defects Res (Part B) 92:543–552, 2011. © 2011 Wiley Periodicals, Inc.