Weight of evidence evaluation for chemical-induced immunotoxicity for PFOA and PFOS: findings from an independent panel of experts.

Immunotoxicity is the critical endpoint used by some regulatory agencies to establish toxicity values for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). However, the hypothesis that exposure to certain per- and polyfluoroalkyl substances (PFAS) causes immune dysregulation is subject to much debate. An independent, international expert panel was engaged utilizing methods to reduce bias and "groupthink". The panel concluded there is moderate evidence that PFOS and PFOA are immunotoxic, based primarily on evidence from animal data. However, species concordance and human relevance cannot be well established due to data limitations. The panel recommended additional testing that includes longer-term exposures, evaluates both genders, includes other species of animals, tests lower dose levels, assesses more complete measures of immune responses, and elucidates the mechanism of action. Panel members agreed that the Faroe Islands cohort data should not be used as the primary basis for deriving PFAS risk assessment values. The panel agreed that vaccine antibody titer is not useful as a stand-alone metric for risk assessment. Instead, PFOA and PFOS toxicity values should rely on multiple high-quality studies, which are currently not available for immune suppression. The panel concluded that the available PFAS immune epidemiology studies suffer from weaknesses in study design that preclude their use, whereas available animal toxicity studies provide comprehensive dataset to derive points of departure (PODs) for non-immune endpoints. The panel recommends accounting for potential PFAS immunotoxicity by applying a database uncertainty factor to POD values derived from animal studies for other more robustly supported critical effects.

Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals: recommendations of the Innovative Medicines Initiative ABIRISK consortium.

Biopharmaceuticals (BPs) represent a rapidly growing class of approved and investigational drug therapies that is contributing significantly to advancing treatment in multiple disease areas, including inflammatory and autoimmune diseases, genetic deficiencies and cancer. Unfortunately, unwanted immunogenic responses to BPs, in particular those affecting clinical safety or efficacy, remain among the most common negative effects associated with this important class of drugs. To manage and reduce risk of unwanted immunogenicity, diverse communities of clinicians, pharmaceutical industry and academic scientists are involved in: interpretation and management of clinical and biological outcomes of BP immunogenicity, improvement of methods for describing, predicting and mitigating immunogenicity risk and elucidation of underlying causes. Collaboration and alignment of efforts across these communities is made difficult due to lack of agreement on concepts, practices and standardized terms and definitions related to immunogenicity. The Innovative Medicines Initiative (IMI; www.imi-europe.org), ABIRISK consortium [Anti-Biopharmaceutical (BP) Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu] was formed by leading clinicians, academic scientists and EFPIA (European Federation of Pharmaceutical Industries and Associations) members to elucidate underlying causes, improve methods for immunogenicity prediction and mitigation and establish common definitions around terms and concepts related to immunogenicity. These efforts are expected to facilitate broader collaborations and lead to new guidelines for managing immunogenicity. To support alignment, an overview of concepts behind the set of key terms and definitions adopted to date by ABIRISK is provided herein along with a link to access and download the ABIRISK terms and definitions and provide comments (http://www.abirisk.eu/index_t_and_d.asp).

First-in-human trial of the safety, pharmacokinetics and immunogenicity of a PEGylated anti-CD40L antibody fragment (CDP7657) in healthy individuals and patients with systemic lupus erythematosus.

OBJECTIVE: The objective of this paper is to investigate the safety, pharmacokinetics (PK) and immunogenicity of CDP7657, a PEGylated anti-CD40L antibody fragment, in healthy individuals and patients with systemic lupus erythematosus (SLE). METHODS: This randomized, double-blind, single-dose, dose-escalation phase I study consisted of two parts. In part 1, 28 healthy individuals received CDP7657 IV (0.004-5 mg/kg) or placebo. In part 2, 17 patients with SLE received CDP7657 IV (5-60 mg/kg) or placebo. The CDP7657:placebo ratio was 3:1. RESULTS: Adverse events (AEs) were reported by 76% of healthy individuals and 100% of patients with SLE treated with CDP7657; most were mild or moderate in intensity. Two healthy individuals reported serious AEs (SAEs), one of which was considered treatment related (infusion-related reaction; 5 mg/kg cohort). One patient with SLE (60 mg/kg cohort) experienced three SAEs, one of which was considered treatment related (herpes zoster infection). No thromboembolic events were reported. CPD7657 exposure increased in a dose-proportional manner. Low anti-CDP7657 antibody titres were detected in the majority of CDP7657-treated participants with no apparent impact on the PK of CDP7657. CONCLUSION: Single doses of CDP7657 showed predictable PK in healthy individuals and patients with SLE and were well tolerated, with no safety signals of concern. These findings support further investigation of CDP7657 as a therapy for SLE.

Generation of a minimal alpha5beta1 integrin-Fc fragment.

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377-390), a series of recombinant soluble human alpha(5)beta(1) integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional alpha(5)-subunit truncation contained the N-terminal 613 residues, whereas the shortest beta(1)-subunit was a fragment containing residues 121-455. Each of these minimally truncated integrins displayed the antibody binding characteristics of alpha(5)beta(1) purified from human placenta and bound ligand with the same apparent affinity as the native receptor.

The effect of energy supplementation on nitrogen utilization in lactating dairy cows fed grass silage diets.

An experiment was conducted to examine the effect that various isoenergetic diets, containing different quantities of soluble carbohydrate and fiber and different types of starch, have on nitrogen (N) balances. Six lactating dairy cows in early to midlactation consuming grass silage diets with not less than 600 g/kg total DMI as forage were used in the experiment. Four concentrates were prepared that had higher amounts of either fiber, soluble sugars, corn (low degradable starch source), or barley (high degradable starch source). Overall N utilization by the cows was poor, rarely exceeding 0.30 g milk N/g of dietary N intake. Fecal N outputs accounted for more than half of total N excreted in all treatments except for diets supplemented with high degradable starch, in which urinary N excretion was significantly higher compared with the other treatments. Milk yield was unaffected by concentrate type, averaging 19.9 kg/d, but milk protein content decreased from 32.9 for starch-based diets to 30.9 and 30.0 g/kg for the soluble sugar- and fiber-based diets, respectively. The efficiency of N utilization improved in the low degradable starch treatment, which had lower N excretion (65%) and higher protein concentration in milk. Furthermore, feeding cows corn-based concentrates reduced urinary N excretion by almost 30% compared with barley-based concentrates; therefore, feeding corn-based diets is recommended for the reduction of nitrogen pollution in lactating dairy cows.

The effect of protein supplementation on nitrogen utilization in lactating dairy cows fed grass silage diets.

The study set out to examine the effects of supplementing grass silage with various levels of protein concentration and degradability on dietary nitrogen (N) excretion in lactating dairy cows consuming at least 60% forage. Six Holstein/Friesian cows in early to midlactation were offered six diets comprising two levels of crude protein (210 and 290 g/kg DM) and three levels of protein degradability in the concentrate achieved using different amounts of untreated or formaldehyde-treated soybean meal. Despite a difference of almost 100 g/d in N intake, apparent fecal and milk N outputs were not significantly affected. Protein degradability also had no effect on N outputs in feces and milk. However, there was a major effect of both level and degradability of CP on urinary N output. Moreover, an interaction between level and degradability of CP was detected, such that the rate at which urinary N increases with increasing CP degradability was higher on the high-CP than on the low-CP diet. A low level of protein (150 g/kg DM in the diet) and medium to low rumen-degradable protein supplements provided a significant reduction in N excretion without compromising lactational performance (mean 24.8 kg/d), in terms of both milk yield and composition. This study also demonstrated that a high efficiency of N utilization could be achieved on low-CP diets (supplying less than 400 g N/d), with feces being the main route of N excretion, whereas an exponential excretion of urinary N was observed as N intake exceeded 400 g N/d.

The Epstein-Barr virus promoter initiating B-cell transformation is activated by RFX proteins and the B-cell-specific activator protein BSAP/Pax5.

Epstein-Barr virus (EBV)-induced B-cell growth transformation, a central feature of the virus' strategy for colonizing the human B-cell system, requires full virus latent gene expression and is initiated by transcription from the viral promoter Wp. Interestingly, when EBV accesses other cell types, this growth-transforming program is not activated. The present work focuses on a region of Wp which in reporter assays confers B-cell-specific activity. Bandshift studies indicate that this region contains three factor binding sites, termed sites B, C, and D, in addition to a previously characterized CREB site. Here we show that site C binds members of the ubiquitously expressed RFX family of proteins, notably RFX1, RFX3, and the associated factor MIBP1, whereas sites B and D both bind the B-cell-specific activator protein BSAP/Pax5. In reporter assays with mutant Wp constructs, the loss of factor binding to any one of these sites severely impaired promoter activity in B cells, while the wild-type promoter could be activated in non-B cells by ectopic BSAP expression. We suggest that Wp regulation by BSAP helps to ensure the B-cell specificity of EBV's growth-transforming function.

Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation.

Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

Expression of a soluble functional form of the integrin alpha4beta1 in mammalian cells.

The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.

The activity of the Epstein-Barr virus BamHI W promoter in B cells is dependent on the binding of CREB/ATF factors.

The programme of Epstein-Barr virus (EBV) gene expression that leads to virus-induced growth transformation of resting B lymphocytes is initiated through activation of the BamHI W promoter, Wp. The factors regulating Wp, and the basis of its preferential activity in B cells, remain poorly understood. Previous work has identified a B cell-specific enhancer region which is critical for Wp function and which contains three binding sites for cellular factors. Here we focus on one of these sites and show, using bandshift assays, that it interacts with three members of the CREB/ATF family of cell transcription factors, CREB1, ATF1 and ATFa. A mutation which abrogates the binding of these factors reduces Wp reporter activity specifically in B cell lines, whereas a mutation which converts the site to a consensus CREB-binding sequence maintains wild-type promoter function. Furthermore Wp activity in B cell, but not in non-B cell, lines could be inhibited by cotransfection of expression plasmids expressing dominant negative forms of CREB1 and ATF1. Increasing the basal activity of CREB/ATF proteins in cells by treatment with protein kinase A or protein kinase C agonists led to small increases in Wp activity in B cell lines, but did not restore promoter activity in non-B cell lines up to B cell levels. We conclude that CREB/ATF factors are important activators of Wp in a B cell environment but require additional B cell-specific factors in order to mediate their effects.

Characterisation of regulatory sequences at the Epstein-Barr virus BamHI W promoter.

Epstein-Barr virus, a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this growth transforming infection depends on a viral promoter in BamHI W (Wp) whose regulation is poorly understood. Using Wp reporter constructs in in vitro transfection assays, we found that Wp was 11- to 190-fold more active in B cell than in non-B cell lines and that three regions of the promoter (termed UAS1, UAS2, and UAS3) contributed to transcriptional activation. The upstream regions UAS3 (-1168 to -440) and UAS2 (-352 to -264) both functioned in a cell lineage-independent manner and were together responsible for the bulk of Wp activity in non-B cells; mutational analysis indicated the importance of a YY1 binding site in UAS2 in that context. By contrast, UAS1 (-140 to -87) was B cell specific and was the key determinant of the promoter's increased activity in B cell lines. Mutational analysis of UAS1 sequences combined with in vitro bandshift assays revealed the presence of three binding sites for cellular factors in this region. When mutations that abolished factor binding in bandshift assays were introduced into a Wp reporter construct, the loss of any one of the three UAS1 binding sites was sufficient to reduce promoter activity by 10- to 30-fold in B cells. From sequence analysis, two of these appear to be novel transcription factor binding sites, whereas the third was identified as a cyclic AMP response element (CRE). Our data indicate that this CRE interacts with CREB and ATF1 proteins present in B cell nuclear extracts and that this interaction is important for Wp activity.

Evaluation of an Aeroponics System to Screen Maize Genotypes for Resistance to Fusarium graminearum Seedling Blight.

A noncirculating aeroponics system was evaluated as a method for rapid screening of maize genotypes for resistance to Fusarium graminearum seedling blight/root rot. The system allows for nondestructive, repetitive sampling of seedlings for assessing disease progress and seedling growth. Shoot growth and root rot were assessed at 3-day intervals, and final shoot and root dry weight were determined 15 days after inoculation. The nine hybrids screened differed in severity of root rot as early as 6 days after inoculation, indicating differences in resistance to F. graminearum. Inoculation did not always significantly affect shoot growth, root dry weight, or shoot dry weight, but differences in these agronomic traits were observed among hybrids.LH119 × LH51 and Pioneer Brand 3379 showed the greatest resistance to root rot. Area under-disease progress curve and a critical stage of disease assessment (9 days after inoculation) gave similar rankings of hybrids for root rot resistance, indicating that a single disease assessment (versus multiple assessments) may be adequate in screening for resistance with this aeroponics system.

Harold Kirby's symbionts of termites: karyomastigont reproduction and calonymphid taxonomy.

Harold Kirby's brilliant principle of mastigont multiplicity is published here posthumously more than 40 years after it was written. He applies this principle to large multinucleate protist symbionts of termites in establishing the taxonomy of Calonymphids (Family Calonymphidae in Phylum Zoomastigina, Kingdom Protoctista). The nuclei and kinetosomes in these heterotrophic cells are organized into trichomonad-style mastigont units which reproduce independently of cytokinesis to generate nine new Calonympha and nineteen new Stephanonympha species. The total of six genera (Calonympha, Coronympha, Diplonympha, Metacoronympha, Snyderella and Stephanonympha, all symbionts of dry-wood-eating termites, Kalotermitidae) are recognized. With the aid of Michael Yamin, the distribution of all twenty-eight of Kirby's Calonympha and Stephanonympha species are tabulated. In italic type I have annotated this paper to be comprehensible to a wide readership of cell biologists, protistologists and those interested in insect symbionts. Although this extremely original and careful work was not finished when Kirby died suddenly in 1952, I deemed it important and complete enough to finally publish it so that it would not be lost to scientific posterity.

Brief neuropsychologic differentiation of demented versus depressed elderly inpatients.

Recent investigations have suggested the utility of brief, psychometric screening batteries in the early detection of abnormal mental decline. This study extended the investigation of one of these batteries, comprised of three tests (Controlled Oral Word Association, Visual Retention, Temporal Orientation), to the difficult issue of differentiating dementia from depression in a hospitalized sample composed of a group of depressed only patients (N = 50) vs an age-matched demented group (N = 50), some of whom presented mixed dementia/depression syndromes. Demented patients consistently performed more poorly as a group than depressed patients on each of the three measures. This was the case even when three-group (demented only, mixed demented/depressed, depressed only) comparisons were conducted. Impairment was more common on one or more tests with demented vs depressed patients. However, limitations for screening purposes and for the definitive detection of dementia were noted in view of only moderate predictive power of the tests with discriminant function analysis. Nevertheless, the potential clinical utility of the three tests in the general hospital and other primary care settings was apparent.

Comparison of two cognitive screening measures for efficacy in differentiating dementia from depression in a geriatric inpatient population.

The Folstein Mini-Mental State Examination (MMSE) and dementia screening measures developed by Benton (temporal orientation, oral word association, and visual reconstruction tasks) were compared for diagnostic efficacy with geriatric inpatients manifesting depression without dementia or mild or moderate dementias complicated by depression. Both instruments showed generally acceptable differentiation between dementia and depression-only cases overall. The MMSE was less sensitive in identifying mild and moderate multi-infarct dementias but showed better specificity than the Benton measures. The degree and type of dementia and the associated risk of classification error were found to be important factors in the choice of screening instruments.

Psychometric assessment of depression in an elderly general medical population. Over- or underassessment?

A total of 247 consecutively evaluated geriatric medical patients was administered a battery of neuropsychological and psychological tests as part of their diagnostic workup for unexplained deterioration in their functioning. Depression was assessed with a short form of the MMPI, the Brief Symptom Inventory, and the Geriatric Depression Scale. By Research Diagnostic Criteria, most suffered from major (59%) or minor (21%) depressions; some degree of cognitive impairment was seen in 80% of the patients, defining a population of "vulnerable" geriatric patients typical of referrals to a general medical hospital setting. Using both conventional score cutoff criteria and discriminant analyses, false-negative rates up to 53% for major depression and 100% for minor depression were found. Psychometric misrecognition of depression was not related to degree of dementia or education but on some measures was positively associated with verbal intelligence level and patient age. Contrary to previous suggestions that psychometric measures overestimate depression in the elderly, these findings suggest that there may be a subgroup of elderly in which treatable affective distress is not appreciated.