Lipopolysaccharide induces DNA-synthesis in a sub-population of hemocytes from the swimming crab, Liocarcinus depurator.

The swimming crab, Liocarinus depurator, contains a small proportion of circulating blood cells which enter S-phase in vitro, as revealed by BrdU assay. These cells are enriched within the semigranular cell band produced by density gradient centrifugation on Percoll and their proportion is significantly higher in blood samples taken from crabs injected 3h previously with lipopolysaccharide (LPS). The occurrence of these cells does not persist as blood samples taken 12h post-injection show similar numbers of these cells to those from saline-treated and untreated controls. However, their appearance appears to be dose related; hemolymph from animals given 15 microg LPS per animal contains greater numbers than that from animals given LPS doses of 0.15 or 0.015 microg per animal. Estimation of the probable number of these cells per ml of the hemolymph, taking into account cell viability and changes in the size of the semigranular cell pool, indicates that they are very rare, comprising merely ca 0.06% of the total cell count. Notwithstanding, this still represents a baseline population in the region of 1.25 x 10(4) ml-1. More importantly, their number rises, approximately 11-fold to ca 14 x 10(4) ml-1 of hemolymph by 3h of injection of LPS. These results show that certain hemocytes from a brachyuran crab can synthesise DNA in vitro, and offers evidence that these cells constitute a rare but distinct sub-population of hemocytes that co-migrate with the semigranular cells during density gradient centrifugation. Since the cells received the stimulus to enter S-phase in vivo, the response must represent one of the mechanisms used by decapods to restore the hemocyte number in the circulation after non-self challenge.     

System for inducible expression of cre-recombinase from the Foxa2 locus in endoderm, notochord, and floor plate.

We targeted the reverse tetracycline controlled transactivator (rtTA) to the Foxa2 locus (Foxa2(ITA)) to generate a system for regulating Cre-recombinase activity within Foxa2 expression domains, including the endoderm, notochord, and floor plate of early mouse embryos. The use of an internal ribosomal entry site to obtain rtTA expression preserves Foxa2 function of the targeted allele. Cre activity with this system reflects the level of endogenous Foxa2 activity and is also tightly controlled by doxycycline. The location of Cre activity within the broader Foxa2 expression domain can be restricted by altering the timing of doxycycline administration. Isolated floor plate expression can be obtained in this manner. This system will provide a useful tool for manipulating gene expression in endoderm, notochord, and floor plate, all of which are tissues with important structural and patterning functions during embryogenesis.     

Stimulus dependence of ocular dominance of complex cells in area 17 of the feline visual cortex

Summary Stimulus dependence of ocular dominance of 31 deep-layer complex cells was assessed from detailed monocular directional tuning curves for motion of bar stimuli or fields of static visual noise, in area 17 of normal adult cats, lightly anaesthetised with N₂O/O₂ supplemented with pentobarbitone. Virtually all cells were binocularly driven, with the anticipated ocular dominance distribution. Interocular differences in directional bias and sharpness of directional tuning for noise were observed in eleven cells, whereas preferred direction and sharpness of tuning for bar stimuli were similar for each eye. In the majority of cells (20/31), any differences between noise and bar tuning in one eye were replicated in the other. Ocular dominance of about half the cells (17/31) for noise and for bar motion was similar, or marginally shifted by up to one ocular dominance group. Substantial shifts in ocular dominance were seen in 14 cells — by up to two ocular dominance groups in 12 cells and by up to three ocular dominance groups in two cells. In three cases these shifts involved a reversal of eye dominance. Notwithstanding these changes, there were no obvious trends in shifts of ocular dominance in favour of the ipsilateral or contralateral eye, nor was there any tendency towards increased binocularity for noise.     

Chromatic sensitivity and spatial organization of cat visual cortical cells: cone-rod interaction

1. Colour sensitivity and spatial organization were determined for the dominant-eye receptive fields of thirty-eight simple or complex cells in cat primary visual vortex. Receptive fields were all from the cortical area associated with central vision. Each cell was investigated with threshold or suprathreshold monochromatic stimuli, under scotopic, low and high mesopic adaptation.2. The Purkinje shift, well defined for all units, was consistent with dual input from each of only two receptor mechanisms, viz. 556 nm cones and 500 nm rods. With change of adaptation level there was a systematic change in the peak sensitivity of spectral response curves to suprathreshold monochromatic stimuli, equated for quantum flux but of different wave-length. Equally with change of adaptation, the relative shift in threshold between wave-lengths selective for cone or rod activation was in close agreement with the change predicted from the Dartnall nomogram curves for visual pigments 556 and 507 respectively.3. For ganglion cells with concentric fields rod input derives from a spatially larger area than cone input. Rod field centre and rod field surround are substantially larger than the corresponding centre and surround for cones (Andrews & Hammond, 1970b). For cortical cells a conclusive comparable change could only be demonstrated for one simple unit. Its receptive field consisted of a horizontal excitatory stripe with asymmetric inhibitory flanks. When light-adapted the weaker, upper flank was functionally undetectable, indicative of purely rod input to this sideband, and the preference for upward movement was enhanced.4. No difference in receptive field configuration, or in spatial extent of input mediated by cones or by rods, was detected for any other unit. The discrepancy between retinal and cortical findings is discussed. It is inferred that cortical fields are compounded essentially by convergent input from geniculate cell field-centres.     

Telomere length analysis on cord blood cells by the flow-FISH method

Telomerase is the enzyme responsible for synthesizing telomeric repeats at the ends of chromosomes to maintain telomere length. Recent studies have suggested that telomere shortening may serve as a surrogate marker of the progression of malignant disorders and seems to be accelerated in allogeneic bone marrow transplant recipients. In this study, the results of the telomere length of nine cord blood mononuclear cell samples are presented. Telomere length was measured by the flow-FISH method, using a peptide nucleic acid probe. The proportion of cord blood cell subsets (CD19/CD34/CD3) was also evaluated. The telomere length of the internal control 1301 cell line was estimated to be 100%. The mean telomere length of cord blood cells was 18.5 +/- 3.9%, compared with the internal control. The progenitor CD34+ cells were detected as 2.6 +/- 0.7% in the lymphoid gate measured. Linear correlation analysis did not find any connection between the cell subsets (CD3+, CD34+, CD19+) and the telomere length. The findings confirm that the telomere flow-FISH method is sufficient for estimation of the telomere length. Assessment of the current procedures of collection, manipulation, and ex vivo expansion of cord blood cells in terms of their effect on telomere shortening might be important.     

Serum deprivation improves seeding and repopulation of acellular matrices with valvular interstitial cells

Cell-extracted valvular tissues (acellular scaffolds, or aScaffolds) offer unique advantages over synthetic polymers for cardiac valve engineering applications in that they retain extracellular matrix molecules to support cellular ingrowth. The extracellular matrix is important in directing many cellular pathways, such as adhesion, proliferation, migration, differentiation, and survival. However, repopulating this type of scaffold often requires high seeding densities or recurrent cell delivery. The optimization of valvular interstitial cell (VIC) seeding onto aScaffolds is reported herein. VICs (the most prevalent cell type in valve leaflets) have maximal growth in 15-20% serum concentrations on tissue-culture polystyrene. Interestingly, after VIC seeding onto aScaffolds, a reduction of serum content, from 15% serum to 5% or less, was found to increase significantly the number of adherent cells, as well as induce transfer of VICs from a tissue-culture polystyrene surface to the aScaffold. aScaffolds seeded and cultured with periods of reduced serum levels were shown to support and enhance VIC viability and attachment, as well as accelerate VIC migration into the aScaffold, leading to a uniformly repopulated valve leaflet construct after 4 weeks of static culture.     

Diminished neo-antigen response to keyhole limpet hemocyanin (KLH) vaccines in patients after treatment with chemotherapy or hematopoietic cell transplantation

Relapse is the most common cause of treatment failure for advanced cancer, even those treated with autologous hematopoietic cell transplantation (HCT). Effective tumor-specific immunotherapy may decrease relapse, however, this will fail if the immune system is unable to respond. We developed a strategy to test immune responses with a single injection of the bona fide neo-antigen KLH. The model was first tested in 37 normal volunteers using three KLH vaccines: Intracel KLH, Biosyn KLH, and Biosyn KLH + adjuvant. Despite finding the immunogenic epitope conserved in both products, intact Intracel KLH induced a better response compared to a purified 350/390 kDA subunit of KLH contained in the Biosyn KLH product. Addition of a synthetic oil adjuvant (Montanide ISA51) restored the response to a single injection of Biosyn KLH. A quantitative readout measured by a KLH-specific cellular and humoral response with isotype switching 1 month after KLH vaccination was established. To test the integrity of the adaptive immune response in cancer patients, we vaccinated 14 patients post-HCT and 19 patients with advanced cancer with KLH vaccines that elicited a 100% response rate in normal volunteers. In marked contrast to normal subjects, both responses were significantly impaired up to 16 months after autologous HCT with an intermediate response in advanced cancer patients. KLH vaccines are safe and require only a single injection to test neo-antigen responses providing an optimal platform for definitive testing of strategies to improve diminished immune recovery after chemotherapy or post-HCT.     

Traumatic Memories: Empirical Foundations, Forensic and Clinical Implications

This article reviews empirical research on memories for negative personal experiences among adults. It examines basic concepts (including neural underpinnings), theoretical models of the affect-memory relationship, and data from three sources: victims or witnesses to crimes and atrocities, "flashbulb memories" for traumatic events, and laboratory simulations of shocking experiences. Evidence suggests that memories for traumatic experiences contain more central than peripheral detail, are reasonably accurate and well-retained for very long periods, but are not completely indelible. Assertions of eyewitness memory's vulnerability to change through suggestion have overstated the evidence. Forensic and clinical implications are discussed and a plea issued for more study of the memory phenomena that characterize posttraumatic stress disorder (PTSD) and are the focus of trauma survivors' treatment.