Anti‐retroviral effects of type I IFN subtypes in vivo

Type I IFN play a very important role in immunity against viral infections. Murine type I IFN belongs to a multigene family including 14 IFN‐α subtypes but the biological functions of IFN‐α subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti‐viral effects of IFN‐α subtypes in vitro and in vivo. IFN‐α subtypes α1, α4, α6 or α9 suppressed Friend virus (FV) replication in vitro, but differed greatly in their anti‐viral efficacy in vivo. Treatment of FV‐infected mice with the IFN‐α subtypes α1, α4 or α9, but not α6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN‐α1 treatment correlated with an expansion of activated FV‐specific CD8⁺ T cells and NK cells into the spleen, whereas in IFN‐α4‐ and ‐α9‐treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti‐retroviral effects of different IFN‐α subtypes, which may be relevant for new therapeutic approaches.     

Neutralizing Monoclonal Antibodies Directed against Defined Linear Epitopes on Domain 4 of Anthrax Protective Antigen

The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA''s ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines.Bacillus anthracis has long been recognized as a serious public health threat, given the ease with which B. anthracis spores can be disseminated via aerosol and due to the high mortality rate that accompanies spore inhalation. These fears were realized in the fall of 2001, when B. anthracis spores were circulated through the U.S. postal system, resulting in five deaths, 22 known cases of infection, and the possible exposure of more than 30,000 people (7). Although routine vaccination of civilians against anthrax is neither necessary nor desirable, certain segments of the population, notably emergency first responders and research laboratory personnel, remain at risk of exposure and are in need of an effective preexposure vaccine. In the United States, the only licensed anthrax vaccine, Anthrax Vaccine Adsorbed (AVA) or Biothrax, has been subject to controversy for years, due to safety concerns (3), and is not considered an ideal vaccine due to its protracted vaccination schedule (six injections over 18 months). AVA, which consists of formalin-treated culture filtrate from an attenuated strain of B. anthracis adsorbed to aluminum hydroxide, is also inherently difficult to manufacture and to standardize. A recombinant anthrax vaccine, manufactured by VaxGen, was considered to be a leading candidate to replace AVA and to supply the Strategic National Stockpile with 75 million doses, but it was recently discontinued because of concerns regarding vaccine production and stability. For these reasons, there is now a concerted effort to develop a new anthrax vaccine based on a defined antigenic composition.Current anthrax vaccines are focused on protective antigen (PA), the primary determinant in AVA that is involved in immunity to anthrax infection (21). PA is the receptor-binding subunit common to both lethal factor (LF), and edema factor (EF), which, upon PA binding form lethal toxin (LT) and edema toxin (ET), respectively. The anthrax toxins are responsible for the high mortality rates associated with inhalational B. anthracis infection. PA is secreted by B. anthracis as an 83-kDa protein (PA₈₃) that binds two known anthrax toxin receptors (ATRs), namely, tumor endothelium marker 8 (TEM-8) and capillary morphogenesis protein 2 (CMG-2) (5, 6, 19). Following receptor engagement, the amino-terminal 20-kDa region of the PA (PA₂₀) is proteolytically cleaved by a furinlike protease, exposing a region of PA that allows for the formation of the holotoxin. The membrane-bound 63-kDa PA (PA₆₃) spontaneously oligomerizes into heptamers and then associates either with LF, a zinc-dependent protease specific for certain members of the mitogen-activated protein kinase kinase family (12, 13), or EF, a calmodulin-, Ca²⁺-dependent adenylate cyclase (20), to form LT and ET.It is well established that animals immunized with recombinant PA, or passively administered anti-PA antiserum, are protected against LT/ET exposure or spore challenge (reviewed in references 16 and 7). For example, we recently reported that LT-challenged mice treated with goat anti-PA antiserum (either alone or in combination with antibiotics) demonstrated increased survival rates compared to untreated mice (18). However, the antibody response to PA is complicated: total anti-PA titers, as measured by enzyme-linked immunosorbent assays (ELISAs), do not correlate well with protection (35). This is likely due to the fact that a large number of anti-PA antibodies are directed against non-neutralizing (or possibly even toxin-enhancing) epitopes (29, 41). Pioneering work by Little et al. (23) suggests that at least 20 antigenic determinants exist on PA. At present, only a few of these epitopes has been described in detail; several have been shown to be targets of monoclonal antibodies (MAbs) capable of effective neutralization in vivo or in vitro (1, 8, 10, 17, 44, 45). The fact that key neutralizing epitopes on PA remain widely unidentified poses a significant barrier to the evaluation of the efficacy of vaccines based on full-length and truncated PA subunits.PAs can be divided into four functional domains. Domain 1 (residues 1 to 258) contains the furin recognition site (¹⁶⁴RKKR¹⁶⁷), as well as the region of PA that is recognized by LF and EF (30). Domain 2 (residues 259 to 487) and part of domain 3 (residues 488 to 595) are implicated in heptamerization, pore formation, and translocation of EF/LF across endosomal membranes (4, 27, 28, 34). Domain 4 (residues 596 to 735) constitutes the region of the PA involved in receptor recognition and attachment (39). Antibodies against domain 4 are postulated to be the most effective in neutralizing LT and ET (1), since they are proposed to interfere with PA binding to ATR. However, the only neutralizing epitope that has been characterized in detail is the one recognized by MAb 14B7 (24, 37, 40). This MAb contacts the face of domain 4 that is involved in receptor recognition, and has been proposed to encompass residues 671 to 721. Certainly, additional neutralizing epitopes exist on domain 4 (1, 46). In particular, Abboud and Casadevall (1) suggested a linear epitope, immediately adjacent to or possibly overlapping the 14B7 binding site, as the target of neutralizing antibodies. The characterization of antibodies that are both linear epitope dependent and neutralizing will be useful in the evaluation specific correlates of immunity; such MAbs can be incorporated into functional screening assays of potential vaccine candidates.The goal of our study was to identify additional B-cell epitopes, within domain 4 of PA, that constitute the targets of neutralizing MAbs. Toward this end, we screened a collection of B-cell hybridomas, produced from PA₈₃-immunized mice, for MAbs capable of binding to a domain 4-specific peptide array. We identified two domain 4-reactive MAbs, 1-F1 and 2-B12. 1-F1 recognized a peptide spanning residues 692 to 703; this epitope maps to a region of domain 4 that overlaps with the binding site recognized by the well-characterized neutralizing antibody 14B7 (residues 671 to 721) (23, 40). 2-B12, on the other hand, recognized a peptide spanning residues 716 to 727; this epitope maps adjacent to the core 14B7 binding site and at a location not previously identified as being a target of neutralizing antibodies. Both MAbs neutralized LT in vitro and partially protected mice against LT challenge. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines.     

Mechanistic basis for the chemopreventive effects of black raspberries at a late stage of rat esophageal carcinogenesis

The present study used a postinitiation protocol to investigate molecular mechanisms by which black raspberries (BRBs) influence the late stages of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. F344 rats were injected with NMBA and then fed either control diet or a diet containing 5% BRB powder. Control rats were injected with DMSO/water (20:80), the vehicle for NMBA. Esophagi from control, NMBA- and NMBA?+?BRB-treated rats were collected at 35 wk for histopathological, molecular, and immunohistochemical analyses. Treatment with 5% BRBs reduced the number of dysplastic lesions and the number and size of esophageal papillomas in NMBA-treated rats. When compared to esophagi from control rats, NMBA treatment led to the differential expression of 4807 genes in preneoplastic esophagus (PE) and 17?846 genes in esophageal papillomas. Dietary BRBs modulated 626 of the 4807 differentially expressed genes in PE and 625 of the 17?846 differentially expressed genes in esophageal papillomas towards normal levels of expression. In both PE and in papillomas, BRBs modulated the mRNA expression of genes associated with carbohydrate and lipid metabolism, cell proliferation and death, and inflammation. In these same tissues, BRBs modulated the expression of proteins associated with proliferation, apoptosis, inflammation, angiogenesis, and both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Interestingly, matrix metalloproteinases involved in tissue invasion and metastasis, and proteins associated with cell-cell adhesion, were also modulated by BRBs. This is the first report of the effects of berries on the expression of genes associated with the late stages of rat esophageal carcinogenesis.     

Arsenite exposure compromises early embryonic development in the Golden hamster

The toxicity of arsenite to 8-cell stage hamster embryos was evaluated. Females were superovulated and mated; embryos were collected and grown for 72 h in culture medium containing vehicle control, 25, 50, 250, 500, or 750 nM arsenite. Morphological observations were taken at 0 and 24 h increments. A TUNEL assay was used for determining DNA damage. Survival was expressed by the ability to undergo zona escape. The control group had 78% survival and no evidence of deformities. Embryos in the 25, 50 and 250 nM groups had survival rates of 63%, 55% and 27%, respectively. Arsenite exposure caused total embryo lethality, major deformities, complete failure to undergo zona lysis, and significantly higher number of cells with fragmented DNA in embryos at the 500 and 750 nM concentrations. The study underscores the sensitivity of preimplantation stage embryos to the presence of even relatively small amounts of arsenic in luminal fluid.     

Bladder cancer cell lines adapt their aggressiveness profile to oxygen tension

During the process of tumor growth, cancer cells will be subjected to intermittent hypoxia. This results from the delay in the development of the vascular network in relation to the proliferation of cancer cells. The hypoxic nature of a tumor has been demonstrated as a negative factor for patient survival. To evaluate the impact of hypoxia on the survival and migration properties of low and high-grade bladder cancer cell lines, two low-grade (MGHU-3 and SW-780) and two high-grade (SW-1710 and T24) bladder cancer cell lines were cultured in normoxic (20% O₂) or hypoxic atmospheric conditions (2% O₂). The response of bladder cancer cell lines to hypoxic atmospheric cell culture conditions was examined under several parameters, including epithelial-mesenchymal transition, doubling time and metabolic activities, thrombospondin-1 expression, whole Matrix Metallo-Proteinase activity, migration and resistance to oxidative stress. The low-grade cell line response to hypoxia was heterogeneous even if it tended to adopt a more aggressive profile. Hypoxia enhanced migration and pro-survival properties of MGHU-3 cells, whereas these features were reduced for the SW-780 cell line cultured under low oxygen tension. The responses of tested high-grade cell lines were more homogeneous and tended to adopt a less aggressive profile. Hypoxia drastically changed some of the bladder cancer cell line properties, for example matrix metalloproteinases expression for all cancer cells but also switch in glycolytic metabolism of low grade cancer cells. Overall, studying bladder cancer cells in hypoxic environments are relevant for the translation from in vitro findings to in vivo context.     

The sensitivity of carpal bone indices to rotational malpositioning

We investigated the dependence of 20 radiographic carpal measurements (carpal indices) on rotational positioning errors in posteroanterior and lateral radiographs. The measurements were made from "true perspective" digitally reconstructed radiographs created from computed tomography data. Most posteroanterior indices were not affected by rotation. Carpal height, carpal height ratio, revised carpal height ratio, capitate-radius distance, and carpal ulnar translocation were particularly robust. Lateral-view indices involving the scaphoid were the most sensitive to simulated malpositioning: radioscaphoid, scapholunate, and scaphocapitate angles were reduced from 58 degrees, 48 degrees, and 56 degrees at true lateral to 30 degrees, 24 degrees, and 34 degrees, respectively, at 20 degrees external rotation. Observers were unable to estimate the degree of malpositioning accurately in either view. Our results support use of the "scaphopisocapitate" criterion for assessing correct positioning in lateral plain radiographs.