Multiple-antibiotic resistance of Enterococcus spp. isolated from commercial poultry production environments

The potential impact of food animals in the production environment on the bacterial population as a result of antimicrobial drug use for growth enhancement continues to be a cause for concern. Enterococci from 82 farms within a poultry production region on the eastern seaboard were isolated to establish a baseline of susceptibility profiles for a number of antimicrobials used in production as well as clinical environments. Of the 541 isolates recovered, Enterococcus faecalis (53%) and E. faecium (31%) were the predominant species, while multiresistant antimicrobial phenotypes were observed among all species. The prevalence of resistance among isolates of E. faecalis was comparatively higher among lincosamide, macrolide, and tetracycline antimicrobials, while isolates of E. faecium were observed to be more frequently resistant to fluoroquinolones and penicillins. Notably, 63% of the E. faecium isolates were resistant to the streptogramin quinupristin-dalfopristin, while high-level gentamicin resistance was observed only among the E. faecalis population, of which 7% of the isolates were resistant. The primary observations are that enterococci can be frequently isolated from the poultry production environment and can be multiresistant to antimicrobials used in human medicine. The high frequency with which resistant enterococci are isolated from this environment suggests that these organisms might be useful as sentinels to monitor the development of resistance resulting from the usage of antimicrobial agents in animal production.     

Evaluation of parameters of oxidative stress after in vitro exposure to FMCW- and CDMA-modulated radiofrequency radiation fields

The goal of this study was to determine whether radiofrequency (RF) radiation is capable of inducing oxidative stress or affecting the response to oxidative stress in cultured mammalian cells. The two types of RF radiation investigated were frequency-modulated continuous-wave with a carrier frequency of 835.62 MHz (FMCW) and code division multiple access centered on 847.74 MHz (CDMA). To evaluate the effect of RF radiation on oxidative stress, J774.16 mouse macrophage cells were stimulated with gamma-interferon (IFN) and bacterial lipopolysaccharide (LPS) prior to exposure. Cell cultures were exposed for 20-22 h to a specific absorption rate of 0.8 W/kg at a temperature of 37.0 +/- 0.3 degrees C. Oxidative stress was evaluated by measuring oxidant levels, antioxidant levels, oxidative damage and nitric oxide production. Oxidation of thiols was measured by monitoring the accumulation of glutathione disulfide (GSSG). Cellular antioxidant defenses were evaluated by measuring superoxide dismutase activity (CuZnSOD and MnSOD) as well as catalase and glutathione peroxidase activity. The trypan blue dye exclusion assay was used to measure any changes in viability. The results of these studies indicated that FMCW- and CDMA-modulated RF radiation did not alter parameters indicative of oxidative stress in J774.16 cells. FMCW- and CDMA-modulated fields did not alter the level of intracellular oxidants, accumulation of GSSG or induction of antioxidant defenses in IFN/LPS-stimulated cells. Consistent with the lack of an effect on oxidative stress parameters, no change in toxicity was observed in J774.16 cells after either optimal (with or without inhibitors of nitric oxide synthase) or suboptimal stimulation.     

Multiple V1/V2 env variants are frequently present during primary infection with human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.     

Alteration of the Magnetic Properties of Aquaspirillum magnetotacticum by a Pulse Magnetization Technique

The presence of a narrow shape and size distribution for magnetite crystals within magnetotactic organisms suggests strongly that there are species-specific mechanisms that control the process of biomineralization. In order to explore the extent of this control, cultures of Aquaspirillum magnetotacticum in the exponential growth phase were exposed to increasing magnetic pulses with the aim of separating cell populations on the basis of their magnetic coercivities. Isothermal remanent magnetization and anhysteretic remanent magnetization studies were performed with freeze-dried magnetic cells after the remagnetization treatment. Subpopulations of A. magnetotacticum that showed an increase in coercivity correlated with the intensity of the magnetic pulses were isolated. After successive subcultures of the remaining north-seeking cells, a maximum bulk coercivity (H_b^max) of 40 mT was obtained after treatment with a 55-mT pulse. Although we obtained A. magnetotacticum variants displaying higher coercivities than the wild-type strain, changes in crystal size or shape of the magnetite crystals were below reliable detection limits with transmission electron microscopy. Attempts to shift the coercivity towards higher values caused it to decrease, a change which was accompanied by an increase in magnetostatic interactions of the magnetosome chains as well as an increase in the cell population displaying an abnormal distribution of the magnetosome chains. Ultrastructural analyses of cells and magnetosomes revealed the appearance of cystlike bodies which occasionally contained magnetosomes. The increase in cystlike cells and abnormal magnetosome chains when higher magnetic pulses were used suggested that magnetosomes were collapsing because of stronger interparticle magnetostatic forces.     

Synthesis and biological evaluation of sulfonyl acrylonitriles as novel inhibitors to peritoneal carcinomatosis

The vast majority of cancer patients die from metastasis, the process by which cancer cells spread to secondary tissues through body fluids. Peritoneal carcinomatosis is a type of metastasis in which cancer cells gain access to the intra-abdominal cavity and then implant in the peritoneum, the thin tissue that lines the abdominal wall and internal organs. Unfortunately, peritoneal carcinomatosis can occur following surgical resection of intra-abdominal malignancies. We previously reported proapoptotic activity of (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile (BAY 11-7085, 1) on colon and pancreatic cancer cells during adhesion and demonstrated that this compound could significantly inhibit peritoneal carcinomatosis in mice.(1,2) In order to determine the chemical basis of the anti-metastatic properties of BAY 11-7085, a series of analogs were synthesized and evaluated for their ability to induce apoptosis in pancreatic and ovarian cancer cells during adhesion to mesothelial cells, which line the surface of the peritoneum. The co-culture assay results were validated using a murine peritoneal carcinomatosis model. These analogs may greatly benefit patients undergoing surgical resections of colorectal, pancreatic, and ovarian cancers depending on their tolerability.     

Novel role for surfactant protein A in gastrointestinal graft-versus-host disease

Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A-deficient mice on a C57BL/6 background (H2b; SP-A(-/-)) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A-deficient recipient mice that have undergone an allogeneic BMT [SP-A(-/-)alloBMT] or SP-A-sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A-deficient recipient mice that have undergone a syngeneic BMT or SP-A-sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A(-/-) alloBMT or SP-A-sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A(-/-)alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A(-/-)alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.     

Human and rat brain lipofuscin proteome

The accumulation of an autofluorescent pigment called lipofuscin in neurons is an invariable hallmark of brain aging. So far, this material has been considered to be waste material without particular relevance for cellular pathology. However, two lines of evidence argue that lipofuscin may play a yet unidentified role for pathological cellular functions: (i) Genetic forms of premature accumulation of similar autofluorescent material in neuronal ceroid lipofuscinosis indicate a direct disease-associated link to lipofuscin; (ii) Retinal pigment epithelium cell lipofuscin is mechanistically linked to age-associated macular degeneration. Here, we purified autofluorescent material from the temporal and hippocampal cortices of three different human individuals by a two-step ultracentrifugation on sucrose gradients. For human brain lipofuscin, we could identify a common set of 49 (among > 200 total) proteins that are mainly derived from mitochondria, cytoskeleton, and cell membrane. This brain lipofuscin proteome was validated in an interspecies comparison with whole brain rat lipofuscin (total > 300 proteins), purified by the same procedure, yielding an overlap of 32 proteins (64%) between lipofuscins of both species. Our study is the first to characterize human and rat brain lipofuscin and identifies high homology, pointing to common cellular pathomechanisms of age-associated lipofuscin accumulation despite the huge (40-fold) difference in the lifespan of these species. Our identification of these distinct proteins will now allow research in disturbed molecular pathways during age-associated dysfunctional lysosomal degradation.     

Enhancing Blockade of Plasmodium falciparum Erythrocyte Invasion: Assessing Combinations of Antibodies against PfRH5 and Other Merozoite Antigens

No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC₅₀ values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.     

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

αA-Crystallin (αA) and αB-crystallin (αB), the two prominent members of the small heat shock family of proteins are considered to be two subunits of one multimeric protein, α-crystallin, within the ocular lens. Outside of the ocular lens, however, αA and αB are known to be two independent proteins, with mutually exclusive expression in many tissues. This dichotomous view is buoyed by the high expression of αA and αB in the lens and their co-fractionation from lens extracts as one multimeric entity, α-crystallin. To understand the biological function(s) of each of these two proteins, it is important to investigate the biological basis of this perceived dichotomy; in this report, we address the question whether αA and αB exist as independent proteins in the ocular lens. Discontinuous sucrose density gradient fractionation and immunoconfocal localization reveal that in early developing rat lens αA is a membrane-associated small heat shock protein similar to αB but with remarkable differences. Employing an established protocol, we demonstrate that αB predominantly sediments with rough endoplasmic reticulum, whereas αA fractionates with smooth membranes. These biochemical observations were corroborated with immunogold labeling and transmission electron microscopy. Importantly, in the rat heart also, which does not contain αA, αB fractionates with rough endoplasmic reticulum, suggesting that αA has no influence on the distribution of αB. These data demonstrate presence of αA and αB in two separate subcellular membrane compartments, pointing to their independent existence in the developing ocular lens.