Determination of Sample Sizes for Demonstrating Efficacy of Radiation Countermeasures

Summary .  In response to the ever increasing threat of radiological and nuclear terrorism, active development of nontoxic new drugs and other countermeasures to protect against and/or mitigate adverse health effects of radiation is ongoing. Although the classical LD₅₀ study used for many decades as a first step in preclinical toxicity testing of new drugs has been largely replaced by experiments that use fewer animals, the need to evaluate the radioprotective efficacy of new drugs necessitates the conduct of traditional LD₅₀ comparative studies ( FDA, 2002 ,  Federal Register   67, 37988–37998). There is, however, no readily available method to determine the number of animals needed for establishing efficacy in these comparative potency studies. This article presents a sample-size formula based on Student's  t  for comparative potency testing. It is motivated by the U.S. Food and Drug Administration's (FDA's) requirements for robust efficacy data in the testing of response modifiers in total body irradiation experiments where human studies are not ethical or feasible. Monte Carlo simulation demonstrated the formula's performance for Student's  t , Wald, and likelihood ratio tests in both logistic and probit models. Importantly, the results showed clear potential for justifying the use of substantially fewer animals than are customarily used in these studies. The present article may thus initiate a dialogue among researchers who use animals for radioprotection survival studies, institutional animal care and use committees, and drug regulatory bodies to reach a consensus on the number of animals needed to achieve statistically robust results for demonstrating efficacy of radioprotective drugs.     

Comparative foraging behavior of Neotropical robber flies (Diptera: Asilidae)

Summary Robber fly species within a Panamanian rain forest comprised distinct shade-seeking (SS) and light-seeking (LS) groups. Thoracic temperatures of LS species averaged 9.2°C greater than ambient, whereas those of SS species averaged only 1.3°C above ambient. Among SS species, attack rate decreased with increasing body mass, whereas relocation rate and attack and relocation distances increased with increasing body mass. Attack and relocation distances of LS species were similar to those of SS species of similar size. Large (>100 mg) LS species, however, had much higher attack and relocation rates than large SS species. The potential costs and benefits of basking are briefly discussed.     

Quantifying the importance of geographic replication and representativeness when estimating demographic rates,using a coastal species as a case study

Demographic rates are rarely estimated over an entire species range, limiting empirical tests of ecological patterns and theories, and raising questions about the representativeness of studies that use data from a small part of a range. The uncertainty that results from using demographic rates from just a few sites is especially pervasive in population projections, which are critical for a wide range of questions in ecology and conservation. We developed a simple simulation to quantify how this lack of geographic representativeness can affect inferences about the global mean and variance of growth rates, which has implications for the robust design of a wide range of population studies. Using a coastal songbird, saltmarsh sparrow Ammodramus caudacutus, as a case study, we first estimated survival, fecundity, and population growth rates at 21 sites distributed across much of their breeding range. We then subsampled this large, representative dataset according to five sampling scenarios in order to simulate a variety of geographic biases in study design. We found spatial variation in demographic rates, but no large systematic patterns. Estimating the global mean and variance of growth rates using subsets of the data suggested that at least 10–15 sites were required for reasonably unbiased estimates, highlighting how relying on demographic data from just a few sites can lead to biased results when extrapolating across a species range. Sampling at the full 21 sites, however, offered diminishing returns, raising the possibility that for some species accepting some geographical bias in sampling can still allow for robust range‐wide inferences. The subsampling approach presented here, while conceptually simple, could be used with both new and existing data to encourage efficiency in the design of long‐term or large‐scale ecological studies.     

Enhanced PD-1 Expression by T Cells in Cerebrospinal Fluid Does Not Reflect Functional Exhaustion during Chronic Human Immunodeficiency Virus Type 1 Infection

During chronic viral infections, T cells are exhausted due to constant antigen exposure and are associated with enhanced programmed death 1 (PD-1) expression. Deficiencies in the PD-1/programmed death-ligand 1 (PD-L1) pathway are associated with autoimmune diseases, including those of the central nervous system (CNS). To understand the role of PD-1 expression in regulating T-cell immunity in the CNS during chronic infection, we characterized PD-1 expression in cerebrospinal fluid (CSF) and blood of individuals with chronic human immunodeficiency virus type 1 (HIV-1) infection. PD-1 expression was higher on HIV-specific CD8⁺ T cells than on total CD8⁺ T cells in both CSF and blood. PD-1 expression on CSF T cells correlated positively with CSF HIV-1 RNA and inversely with blood CD4⁺ T-cell counts, suggesting that HIV-1 infection drives higher PD-1 expression on CSF T cells. However, in every HIV-positive individual, PD-1 expression was higher on T cells in CSF than on those in blood, despite HIV-1 RNA levels being lower. Among healthy HIV-negative controls, PD-1 expression was higher in CSF than in blood. Furthermore, frequencies of the senescence marker CD57 were lower on CSF T cells than on blood T cells, consistent with our prior observation of enhanced ex vivo functional capacity of CSF T cells. The higher PD-1 expression level on CSF T cells therefore does not reflect cellular exhaustion but may be a mechanism to downregulate immune-mediated tissue damage in the CNS. As inhibition of the PD-1/PD-L1 pathway is pursued as a therapeutic option for viral infections, potential effects of such a blockade on development of autoimmune responses in the CNS should be considered.Programmed death 1 (PD-1; also called CD279) and its ligands, PD-L1 (also called B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD-273), regulate T-cell activation, peripheral tolerance, and autoimmunity (22, 43). PD-1 can be expressed on CD8⁺ and CD4⁺ T cells, B cells, natural killer T cells, and activated monocytes. PD-L1 is expressed on various cells, including T and B cells, dendritic cells, macrophages, mast cells, nonhematopoietic cell types (including vascular endothelial cells, pancreatic islet cells, astrocytes, keratinocytes, and microglial cells), and cells in immune privileged sites, including the placenta and the eye (22). PD-L2 expression is inducible and is restricted to dendritic cells, monocytes, macrophages, and mast cells (22). During chronic infections, the PD-1/PD-L1 pathway inhibits antigen-specific T-cell responses (7, 8, 35, 46). In human immunodeficiency virus type 1 (HIV-1)-infected individuals, PD-1 expression on HIV-specific T cells in peripheral blood is upregulated and correlates positively with plasma viremia and inversely with CD4⁺ T-cell counts (7, 46). PD-1 expression on HIV-specific T cells is also associated with T-cell exhaustion, as defined by a reduced ability to proliferate and produce cytokines (7, 46). Inhibition of the PD-1/PD-L1 pathway augments HIV-specific CD8⁺ and CD4⁺ T-cell function, and antiretroviral therapy is associated with a significant reduction of PD-1 expression on HIV-specific T cells in peripheral blood (8).The PD-1/PD-L1 pathway also limits immune-mediated tissue damage that may be caused by overreactive peripheral T cells, especially in immune privileged sites such as the central nervous system (CNS). In 1999, the importance of PD-1 for peripheral tolerance was first suggested by studies which showed that PD1^−/− mice develop lupus-like autoimmune diseases (32). In humans, polymorphisms in the PDCD1 gene, which encodes PD-1, have been associated with autoimmune diseases, including lupus, diabetes, rheumatoid arthritis, and multiple sclerosis (20, 21, 25). Upregulation of PD-L1 in multiple sclerosis lesions from human brain tissue suggests a role for the PD-1/PD-L1 pathway in regulating T-cell activation and controlling immunopathological damage (33).The CNS is involved by HIV-1 early during primary infection (6, 13), and approximately 40% of patients who develop advanced AIDS without receiving antiretroviral therapy develop cognitive impairment (6, 13, 38). While HIV-1 proteins gp120 (3, 16) and Tat (30) are directly neurotoxic and may contribute to HIV-associated dementia, detrimental neuropathogenic effects have also been postulated for inflammatory and innate immune cells, especially monocytes/macrophages and T cells (11, 19, 49, 50). Immune responses cause neuropathogenesis during other viral infections, and cytotoxic T lymphocytes can worsen the disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (14). However, we recently described higher frequencies of functional HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) than in blood among asymptomatic HIV-positive individuals with little or no HIV-1 RNA in CSF, suggesting that HIV-1-specific CD8⁺ T cells help to control intrathecal viral replication (40).To understand the role of the PD-1/PD-L1 pathway in regulating T-cell responses during viral infection of the CNS, we characterized PD-1 expression on T cells in CSF and peripheral blood among asymptomatic HIV-positive individuals. We hypothesized that T-cell PD1 expression would be lower in CSF than in blood, since HIV-1 RNA concentrations are lower in CSF than in plasma and the magnitude and breadth of IFN-γ-secreting HIV-specific T cells are greater in CSF than in blood (40). We show that, in CSF, HIV-1 RNA correlates directly with PD-1 expression on CD4⁺, CD8⁺, and HIV-specific CD8⁺ T cells. Unexpectedly, PD-1 expression on all T cells is higher in CSF than in blood in HIV-positive patients and healthy HIV-negative controls. In contrast, expression of the senescence marker CD57 is lower in CSF than in blood. These data suggest that higher PD-1 expression on T cells in CSF may be a mechanism to regulate T-cell immunity in the CNS, rather than indicating T-cell exhaustion, and that this regulation is increased by HIV-1 replication.     

The LHbeta gene of several mammals embeds a carboxyl-terminal peptide-like sequence revealing a critical role for mucin oligosaccharides in the evolution of lutropin to chorionic gonadotropin in the animal phyla

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) beta to the beta-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHbeta gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHbeta gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHbeta gene (boCTP) would be sufficient to generate the LHbeta species of a ruminant with properties typical to the CGbeta subunit. The mutated bovine LHbeta-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGbeta CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGbeta subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGbeta-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGbeta subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHbeta to CGbeta evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHbeta gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.