Role of theHLA complex in the antibody response to malaria under natural conditions

Antibodies toP. falciparum antigens and to the EB virus antigens, VCA and EBNA, were determined soon after the end of the major rainy season in 140 Africans living in 3 villages at varying altitudes in northeastern Tanzania. Also, their peripheral blood mononuclear cells were stored in liquid nitrogen and subsequently used for HLA phenotype determination of serologically determined antigens coded by genes at theA andB loci and for cell culture with mitogens (PHA, Con-A PWM) and with allogeneic cells in the mixed leukocyte reaction. A strong correlation was found between the presence of high titres of immunofluorescent antibody to falciparum antigens and the combination of A2 with AW30 in the same individuals. Individuals having one or the other of these specificities, but not both, did not have unusually high titres (P=0.0005). Individuals having the combination A2 and BW17 also tended to have higher than average antibody titres to falciparum antigens, but the difference was not statistically significant (P=0.09). The data suggests that individuals with A2 and AW30 may haveHLA-associated genes having the function ofIr genes and that these genes interact from the trans position to affect the capacity to make antibodies to malarial antigens. Thus, these genes may confer a survival advantage for individuals exposed to malaria. In the cell culture studies there were no correlations between responses and with IFA titres toP. falciparum, except for an inverse association between responses to PWM and level of IFA titre. This suggests that the B cell response to mitogens is impaired in individuals with strong responses to malarial antigens. There was no association between any of the cell culture responses and the HLA phenotypes of the cell donors.Abbreviations used in this paper P. Plasmodium - EB Epstein-Barr - VCA virus capsid antigen - EBNA Epstein-Barr nuclear antigen - PHA phytohemagglutinin - Con-A concanavalin-A - PWM pokeweed mitogen - IFA immunofluorescent antibody - Ir immune response - EA early antigen - MLR mixed leukocyte reaction - EAIMVBD East African Institute for Malaria and Vector-Borne Diseases - MEM minimal essential medium - EBV Epstein-Barr virus - AMMN alpha medium minus nucleosides - equal to or less than - equal to or greater than - RGM reciprocal geometric mean     

Monofermentation of grass silage under mesophilic conditions: measurements and mathematical modeling with ADM 1

In this paper experimental data from grass fermentation and simulation results with the Anaerobic Digestion Model (ADM) No. 1 are described. Two laboratory reactors were operated under mesophilic conditions with volumetric loading rates in between 0.3 and 2.5 kg(VS)/(m(3) x d). Two different kinds of grass silage were used as substrates, resulting in an average specific biogas production of 600 L/kg(VS). The ADM 1 was calibrated both manually and with the help of a Genetic Algorithm in Matlab/Simulink. Results from calibration indicate that the NH3 inhibition constant used to model the inhibition of acetate uptake is three to five times higher compared with digested activated sludge. The hydrogen inhibition constants applied for propionate and valerate/butyrate uptake are around two orders of magnitude lower than for sludge digestion.     

Using a surrogate marker for early testing of a treatment effect

The development of methods to identify, validate, and use surrogate markers to test for a treatment effect has been an area of intense research interest given the potential for valid surrogate markers to reduce the required costs and follow‐up times of future studies. Several quantities and procedures have been proposed to assess the utility of a surrogate marker. However, few methods have been proposed to address how one might use the surrogate marker information to test for a treatment effect at an earlier time point, especially in settings where the primary outcome and the surrogate marker are subject to censoring. In this paper, we propose a novel test statistic to test for a treatment effect using surrogate marker information measured prior to the end of the study in a time‐to‐event outcome setting. We propose a robust nonparametric estimation procedure and propose inference procedures. In addition, we evaluate the power for the design of a future study based on surrogate marker information. We illustrate the proposed procedure and relative power of the proposed test compared to a test performed at the end of the study using simulation studies and an application to data from the Diabetes Prevention Program.     

Testing for heterogeneity in the utility of a surrogate marker

In studies that require long-term and/or costly follow-up of participants to evaluate a treatment, there is often interest in identifying and using a surrogate marker to evaluate the treatment effect. While several statistical methods have been proposed to evaluate potential surrogate markers, available methods generally do not account for or address the potential for a surrogate to vary in utility or strength by patient characteristics. Previous work examining surrogate markers has indicated that there may be such heterogeneity, that is, that a surrogate marker may be useful (with respect to capturing the treatment effect on the primary outcome) for some subgroups, but not for others. This heterogeneity is important to understand, particularly if the surrogate is to be used in a future trial to replace the primary outcome. In this paper, we propose an approach and estimation procedures to measure the surrogate strength as a function of a baseline covariate W and thus examine potential heterogeneity in the utility of the surrogate marker with respect to W. Within a potential outcome framework, we quantify the surrogate strength/utility using the proportion of treatment effect on the primary outcome that is explained by the treatment effect on the surrogate. We propose testing procedures to test for evidence of heterogeneity, examine finite sample performance of these methods via simulation, and illustrate the methods using AIDS clinical trial data.     

Semiparametric modeling of longitudinal measurements and time-to-event data--a two-stage regression calibration approach

SUMMARY: In this article we investigate regression calibration methods to jointly model longitudinal and survival data using a semiparametric longitudinal model and a proportional hazards model. In the longitudinal model, a biomarker is assumed to follow a semiparametric mixed model where covariate effects are modeled parametrically and subject-specific time profiles are modeled nonparametrially using a population smoothing spline and subject-specific random stochastic processes. The Cox model is assumed for survival data by including both the current measure and the rate of change of the underlying longitudinal trajectories as covariates, as motivated by a prostate cancer study application. We develop a two-stage semiparametric regression calibration (RC) method. Two variations of the RC method are considered, risk set regression calibration and a computationally simpler ordinary regression calibration. Simulation results show that the two-stage RC approach performs well in practice and effectively corrects the bias from the naive method. We apply the proposed methods to the analysis of a dataset for evaluating the effects of the longitudinal biomarker PSA on the recurrence of prostate cancer.     

CD54 is a surrogate marker of antigen presenting cell activation

There is no single universally accepted hallmark of antigen presenting cell (APC) activation. Instead a variety of methods are used to identify APCs and assess their activation state. These activation measures include phenotypic methods [e.g., assessing the increased expression of surface markers such as major histocompatability (MHC) class II] and functional assays (e.g., evaluating the enhanced ability to take up and process antigen, or stimulate naïve T cells). Sipuleucel-T is an investigational autologous active cellular immunotherapy product designed to stimulate a T cell immune response against human prostatic acid phosphatase (PAP), an antigen highly expressed in prostate tissue. Sipuleucel-T consists of peripheral blood mononuclear cells (PBMCs), including activated APCs displaying epitopes of PAP. In order to develop a robust reproducible potency assay that is not hampered by MHC restriction we have developed a method to simply assess the biological activation of antigen presenting cells (APCs). In the course of sipuleucel-T characterization, we analyzed various phenotypic and functional parameters to define the activation state of APCs obtained from peripheral blood. Flow cytometric assays revealed that CD54+ cells are responsible for antigen uptake, and that expression of CD54 predominantly localizes to APCs. Costimulation, as measured by an allogeneic mixed lymphocytic reaction (alloMLR) assay, showed that activity was restricted to the CD54+ cell population. Similarly, CD54+ cells harbor all of the PAP-specific antigen presentation activity, as assayed using a PAP-specific HLA-DRβ1-restricted T cell hybridoma. Finally we show that CD54 expression is substantially and consistently upregulated on APCs during culture with a GM-CSF fusion protein, and that this upregulation activity can be quantified. Thus these data support the use of CD54 upregulation as a surrogate for assessing human APC activation and validates its utility as a potency measure of sipuleucel-T.     

Reproducibility of anthropometric measurements in children: a longitudinal study

The purpose of this study was to establish the reproducibility of a series of anthropometric measures performed twice during one week during a three year period in boys and girls. The subjects of this investigation were 39 children (21 boys and 18 girls), 9-10 year of age at the beginning of the study. Children were measured three times with one year interval. Children were classified by Tanner stage 1-2 during the first measurements, stage 1-3 during the second measurements and stage 1-4 during the third measurements. Body height and weight were measured and BMI calculated. All anthropometric parameters were measured according to the protocol recommended by the International Society for the Advancement of Kinanthropometry (Norton & Olds 1996). Nine skinfolds, 13 girths, eight lengths and eight breadths/lengths were measured. The reproducibility of body height (r = 0.995-0.999), body weight (r = 0.990-0.999) and BMI (r = 0.969-0.999) was very high in boys and girls. The intraclass correlations (ICC), technical errors (TE) and coefficients of variation (CV) were quite different depending on the measurement site of the skinfold thickness. It was surprising that the ICCs were highest and TEs and CVs were lowest during the second year of the measurement. The computed ICC was high, and TE and CV values were quite similar and relatively low in girth, length and breadth/length measurements. It was concluded that the reproducibility of girths, lengths and breadths/lengths in children is very high and the reproducibility of skinfolds is high. Specifically, the reproducibility is very high immediately before puberty in boys and girls.     

The transmissibility of a potyvirus by an aphid vector: reliability of measurements under controlled conditions

Reproducibility of measurements of the transmission rate of a given potyvirus by a given aphid vector was assessed using a standardised transmission procedure. The plant-virus-vector system studied was: Cucurbita pepo - Papaya ringspot virus - Aphis gossypii. Several elements concerning the rearing and handling of the aphids were analysed. One of these, the climatic conditions under which the aphids were reared, influenced the transmission rate of the virus. Six trials were carried out to measure the rate of transmission of the virus from a defined leaf surface area. The measurements appeared to be reliable. A two year experiment was carried out to compare transmission rates at different dates. Although strong variations were observed in some source plants, the transmission rates obtained for samples of 10 source plants were stable, with a 10% coefficient of variation. The results show that transmissibility of the virus is a stable property which is independent of the trial date. They are discussed in relation to standardised elements of the transmission process.     

The joint modeling of a longitudinal disease progression marker and the failure time process in the presence of cure

In this paper we present an extension of cure models: to incorporate a longitudinal disease progression marker. The model is motivated by studies of patients with prostate cancer undergoing radiation therapy. The patients are followed until recurrence of the prostate cancer or censoring, with the PSA marker measured intermittently. Some patients are cured by the treatment and are immune from recurrence. A joint-cure model is developed for this type of data, in which the longitudinal marker and the failure time process are modeled jointly, with a fraction of patients assumed to be immune from the endpoint. A hierarchical nonlinear mixed-effects model is assumed for the marker and a time-dependent Cox proportional hazards model is used to model the time to endpoint. The probability of cure is modeled by a logistic link. The parameters are estimated using a Monte Carlo EM algorithm. Importance sampling with an adaptively chosen t-distribution and variable Monte Carlo sample size is used. We apply the method to data from prostate cancer and perform a simulation study. We show that by incorporating the longitudinal disease progression marker into the cure model, we obtain parameter estimates with better statistical properties. The classification of the censored patients into the cure group and the susceptible group based on the estimated conditional recurrence probability from the joint-cure model has a higher sensitivity and specificity, and a lower misclassification probability compared with the standard cure model. The addition of the longitudinal data has the effect of reducing the impact of the identifiability problems in a standard cure model and can help overcome biases due to informative censoring.     

A measure of the proportion of treatment effect explained by a surrogate marker

Randomized clinical trials with rare primary endpoints or long duration times are costly. Because of this, there has been increasing interest in replacing the true endpoint with an earlier measured marker. However, surrogate markers must be appropriately validated. A quantitative measure for the proportion of treatment effect explained by the marker in a specific trial is a useful concept. Freedman, Graubard, and Schatzkin (1992, Statistics in Medicine 11, 167-178) suggested such a measure of surrogacy by the ratio of regression coefficients for the treatment indicator from two separate models with or without adjusting for the surrogate marker. However, it has been shown that this measure is very variable and there is no guarantee that the two models both fit. In this article, we propose alternative measures of the proportion explained that adapts an idea in Tsiatis, DeGruttola, and Wulfsohn (1995, Journal of the American Statistical Association 90, 27-37). The new measures require fewer assumptions in estimation and allow more flexibility in modeling. The estimates of these different measures are compared using data from an ophthalmology clinical trial and a series of simulation studies. The results suggest that the new measures are less variable.     

Electromagnetic absorption in a multilayered slab model of tissue under near-field exposure conditions

The electromagnetic energy deposited in a semi-infinite slab model consisting of skin, fat, and muscle layers is calculated for both plane-wave and near-field exposures. The plane-wave spectrum (PWS) approach is used to calculate the energy deposited in the model by fields present due to leakage from equipment using electromagnetic energy. This analysis applies to near-field exposures where coupling of the target to the leakage source can be neglected. Calculations were made for 2,450 MHz, at which frequency the layered slab adequately models flat regions of the human body. Resonant absorption due to layering is examined as a function of the skin and fat thicknesses for plane-wave exposure and as a function of the physical extent of the near-field distribution. Calculations show that for fields that are nearly constant over at least a free-space wavelength, the energy deposition (for the skin, fat, and muscle combination that gives resonant absorption) is equal to or less than that resulting from plane-wave exposure, but is appreciably greater than that obtained for a homogeneous muscle slab model.     

Counterfactual links to the proportion of treatment effect explained by a surrogate marker

In a randomized clinical trial, a statistic that measures the proportion of treatment effect on the primary clinical outcome that is explained by the treatment effect on a surrogate outcome is a useful concept. We investigate whether a statistic proposed to estimate this proportion can be given a causal interpretation as defined by models of counterfactual variables. For the situation of binary surrogate and outcome variables, two counterfactual models are considered, both of which include the concept of the proportion of the treatment effect, which acts through the surrogate. In general, the statistic does not equal either of the two proportions from the counterfactual models, and can be substantially different. Conditions are given for which the statistic does equal the counterfactual model proportions. A randomized clinical trial with potential surrogate endpoints is undertaken in a scientific context; this context will naturally place constraints on the parameters of the counterfactual model. We conducted a simulation experiment to investigate what impact these constraints had on the relationship between the proportion explained (PE) statistic and the counterfactual model proportions. We found that observable constraints had very little impact on the agreement between the statistic and the counterfactual model proportions, whereas unobservable constraints could lead to more agreement.     

Toward optimized antibody microarrays: a comparison of current microarray support materials

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.     

Impact of quarantine on the 2003 SARS outbreak: a retrospective modeling study

During the 2003 Severe Acute Respiratory Syndrome (SARS) outbreak, traditional intervention measures such as quarantine and border control were found to be useful in containing the outbreak. We used laboratory verified SARS case data and the detailed quarantine data in Taiwan, where over 150,000 people were quarantined during the 2003 outbreak, to formulate a mathematical model which incorporates Level A quarantine (of potentially exposed contacts of suspected SARS patients) and Level B quarantine (of travelers arriving at borders from SARS affected areas) implemented in Taiwan during the outbreak. We obtain the average case fatality ratio and the daily quarantine rate for the Taiwan outbreak. Model simulations is utilized to show that Level A quarantine prevented approximately 461 additional SARS cases and 62 additional deaths, while the effect of Level B quarantine was comparatively minor, yielding only around 5% reduction of cases and deaths. The combined impact of the two levels of quarantine had reduced the case number and deaths by almost a half. The results demonstrate how modeling can be useful in qualitative evaluation of the impact of traditional intervention measures for newly emerging infectious diseases outbreak when there is inadequate information on the characteristics and clinical features of the new disease-measures which could become particularly important with the looming threat of global flu pandemic possibly caused by a novel mutating flu strain, including that of avian variety.     

Characterization of a surrogate murine antibody to model anti-human CD3 therapies

Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.