Intratumoral interleukin-2 immunotherapy: Activation of tumor-infiltrating and splenic lymphocytes in vivo

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 × 10⁴ Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 × 10³–5 × 10⁴ units) or diluent twice daily, either by i. p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 × 10⁴ units IL-2 significantly reduced tumor volume (P <0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/cnu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P <0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.Supported by the Tobacco-Related Disease Research Program of the University of California, the Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation, and Veterans Administration Medical Research Funds     

Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be locatedin the outer leaflet of the plasma membrane, recent evidenceindicates that GSLs are also associated with intracellular organelles.We now report that the subcellular localization of GSLs variesdepending on the GSL structure and cell type. GSL localizationwas determined by indirect immunofluorescence microscopy offixed permeabilized cells. A single GSL exhibited variable subcellularlocalization in different cells. For example, antibody to GalCeris localized primarily to the plasma membrane of HaCaT II-3keratinocytes, but to intracellular organelies in other epithelialcells. GalCer is localized to small vesicles and tubulovesicularstructures in MDCK cells, and to the surface of phase-denselipid droplets in HepG2 hepatoma cells. Furthermore, withina single cell type, individual GSLs were found to exhibit differentpatterns of subcellular localization. In HepG2 cells, LacCerwas associated with small vesicles, which differed from thephase-dense vesicles stained by anti-GalCer, and Gb4Cer wasassociated with the intermediate filaments of the cytoskeleton.Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides,localized to mitochondria. The distinct subcellular localizationpatterns of GSLs raise interesting questions about their functionsin different organelles. Together with published data on theenrichment of GSLs in specific organelles and in apical plasmamembrane, these findings indicate the existence of specificsorting mechanisms that regulate the intracellular transportand localization of GSLs. cytoskeleton glycosphingolipid intracellular organelles mitochondria subcellular localization     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

The challenge of estimating kelp production in a turbid marine environment

Coastal kelp forests produce substantial marine carbon due to high annual net primary production (NPP) rates, but upscaling of NPP estimates over time and space remains difficult. We investigated the impact of variable underwater photosynthetically active radiation (PAR) and photosynthetic parameters on photosynthetic oxygen production of Laminaria hyperborea, the dominant NE-Atlantic kelp species, throughout summer 2014. Collection depth of kelp had no effect on chlorophyll a content, pointing to a high photoacclimation potential of L. hyperborea towards incident light. However, chlorophyll a and photosynthesis versus irradiance parameters differed significantly along the blade gradient when normalized to fresh mass, potentially introducing large uncertainties in NPP upscaling to whole thalli. Therefore, we recommend a normalization to kelp tissue area, which is stable over the blade gradient. Continuous PAR measurements revealed a highly variable underwater light climate at our study site (Helgoland, North Sea) in summer 2014, reflected by PAR attenuation coefficients (K_d) between 0.28 and 0.87 m⁻¹. Our data highlight the importance of continuous underwater light measurements or representative average values using a weighted K_d to account for large PAR variability in NPP calculations. Strong winds in August increased turbidity, resulting in a negative carbon balance at depths >3–4 m over several weeks, considerably impacting kelp productivity. Estimated daily summer NPP over all four depths was 1.48 ± 0.97 g C · m⁻² seafloor · d⁻¹ for the Helgolandic kelp forest, which is in the range of other kelp forests along European coastlines.     

Parasitism and decreased response to sex pheromones in malePeriplaneta americana (Dictyoptera: Blattidae)

Parasites are known to alter the behavior of their hosts, but little is known about their effects on responses to sexual stimuli. Periplaneta americanainfected with the acanthocephalan Moniliformis moniliformiswere compared to uninfected animals in their behavioral and electroantennogram responses to a synthetic P. americanapheromone component, periplanone-B, and a pheromone mimic, bornyl acetate. In a t-maze there was no significant difference between infected animals' responses to periplanone-B and a random binomial distribution; the responses of uninfected animals were significantly nonrandom. The electroan-tennogram responses of infected and uninfected animals to bornyl acetate or periplanone-B did not differ significantly, however, indicating that the alteration probably does not occur at the peripheral level but at a central nervous system level.     

The α2(XI) collagen gene lies within 8 kb of Pb in the proximal portion of the murine major histocompatibility complex

A number of serious hereditary disorders are now known to be associated with defective expression of collagen genes, and these findings have underscored the important and varied roles that the collagen family of genes must play during normal mammalian development. Although the activities of genes encoding the quantitatively major types of collagen are fairly well characterized, functions of the many minor types of collagen remain a matter of speculation. As a first step toward a functional analysis of type XI collagen, a member of this class of poorly understand minor collagen proteins which is expressed primarily in hyaline cartilage, we have used human probes for the gene encoding the protein's 2-subunit (COL11A2) to isolate and map homologous murine DNA sequences. Our results demonstrate that Col11a-2 is embedded within the major histocompatibility complex (MHC), within 8.4 kb of the class II pseudogene locus, Pb, and confirm that human and murine 2(XI) collagen genes are located in very similar genomic environments. The conserved location of these genes raises the possibility that type XI collagen genes may contribute to one or more of the diverse hereditary disorders known to be linked to the MHC in mouse and human.     

Thermal stress exposure of pupal oriental fruit fly has strong and trait-specific consequences in adult flies

Global climate change is projected to increase the incidence of heat waves, their magnitude and duration resulting in insects experiencing increasing environmental stress in both natural and managed ecosystems. While studies on insect thermal tolerance are rapidly increasing, variation across developmental or juvenile stress cross-stage effects within and across generations remain largely unexplored. Yet in holometabolous insects, heat stress at an early developmental stage may influence performance and survival during later stages. Here, we investigated the effects of pupal mild heat stress on the performance of laboratory-reared adult Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) measured as longevity, critical thermal maximum (CT_max), critical thermal minima (CT_min), heat knockdown time (HKDT) and chill coma recovery time (CCRT). Pupal heat stress significantly influenced performance of B. dorsalis adults resulting in impaired longevity and heat tolerance (CT_max and HKDT) in both sexes with improved and compromised cold tolerance (CT_min and CCRT) in females and males, respectively. These findings highlight the role of juvenile stages in mediating stress responses at adult stages. For B. dorsalis, pupal heat stress largely compromised thermal tolerance implying that the species has limited potential to shift its geographic range in heat prone areas. Significant benefits in cold tolerance in females following heat stress may help in improving survival in the cold in the short-term despite restricted activity to the same traits in males. This study suggests that basal heat tolerance and not short-term compensatory thermal plasticity following heat stress may have aided the recent invasion of B. dorsalis in African landscapes.     

DIVERGENT AMBULATORY AND GROOMING BEHAVIOR IN SERIALLY BOTTLENECKED LINES OF THE HOUSEFLY

The extent of genome-wide restructuring predicted in bottleneck models of speciation is addressed in assays of non-reproductive behavior in lines of the housefly. After five serial founder-flush cycles of one of three sizes (1, 4, or 16 pairs), each bottleneck line showed significant differentiation from the outbred control in ambulatory levels and grooming sequences in videotaped records of precopulatory activity. Only one line (4-pair) showed overall lethargy which was associated to inbreeding depression in egg-to-adult viability, thus exemplifying a case of probable extinction due to bottlenecks. The two most hyperactive lines (1- and 16-pair) showed very similar directions of differentiation from the control in locomotor activity and grooming behavior, as well as in mating behavior evaluated from a separate study. This high congruence suggested that directional selection toward the phenotypic optima of the ancestor operated on the bottleneck populations and that a 10-fold difference in theoretical inbreeding coefficients did not affect the magnitude of response. The remaining two bottleneck lines showed some independence from these general trajectories, their divergence along minor axes of ancestral intercorrelation structure possibly being more important to the formation of new species. Significant perturbations of the thresholds for execution of grooming and locomotor movements suggested increased evolutionary potential for ritualization (i.e., sexual selection for adoption of non-reproductive behavior into courtship repertoire) due to bottlenecks.     

REPRODUCTIVE ATTRITION IN THE RARE CHAPARRAL SHRUB FREMONTODENDRON DECUMBENS LLOYD (STERCULIACEAE)

We examined reproductive attrition in Fremontodendron decumbens to characterize sexual reproduction in this rare California shrub. Reproductive individuals produced an average of 2,900 flower buds in a season, with no significant difference in bud production between two seasons. Because of intense insect predation, <;2% of initiated flower buds became mature fruits. A threefold decrease in predation of flower buds between seasons resulted in an increase in seed output the second season, indicating that seed production was partially predator-limited. Most seeds (97.8%) were dormant due to an impermeable seed coat. Breaking of the coat, mechanically or by heat, allowed high levels of germination. Chamise charate and ash added to the potting medium resulted in the highest level of germination and emergence. Rodents were more important than birds as seed predators, destroying 90% of seeds under parent shrub canopies within 8–10 months. Seeds already integrated into the seed bank were comparatively safe from predation, relative to newly added seeds. If predation was prevented, seeds were long-lived under field conditions (>;80% survived after 5.75 years). Most seedlings produced in unburned chaparral by planting heat-treated seeds in openings between shrubs were destroyed by predators (rodents and insects). All seedlings that escaped predation died during the summer drought. We concluded that sexual reproduction was limited by (in order of importance): 1) lack of fire, 2) predehiscence predation by insects, and 3) postdehiscence predation by rodents. Size distributions from two populations revealed that, despite the apparent absence of sexual reproduction in unbumed chaparral, two unbumed sites contained a large proportion of individuals in small size classes. Excavation of several small individuals demonstrated they were sprouts from the roots of nearby larger shrubs. Because asexual reproduction by rootsprouting circumvents the high attrition of sexual reproductive effort on unbumed sites, rootsprouting may be a significant reproductive strategy of some ‘sprouter’ species in chaparral.