Viral hemagglutinin augments peptide-specific cytotoxic T cell responses

In attempt to increase the induction of peptide-specific cytolytic T cells (CTL) we investigated the effect of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene product on the activation of peptide-specific CTL. Spleen cells of CH3 mice immunized against the influenza nucleoprotein peptide 50–63 (NP 50–63) were restimulated in vitro (i) with peptide-pulsed syngeneic fibroblast cells (Ltk^?) as antigen-presenting cells, which were in addition (ii) infected with NDV or (iii) stably transfected with the HN cDN A of NDV. A greater than sixfold increase in peptide-specific CTL responses was observed in cultures restimulated with peptide-pulsed Ltk^? cells which co-expressed viral hemagglutinin due to either infection or transfection. A similar augmentation was seen in CTL responses against other types of antigen (major histocompatibility complex alloantigens, minor histocompatibility antigens or tumor antigens) when suboptimal cultures were stimulated with the respective antigen-presenting cells modified by NDV infection. These findings suggest that NDV or viral HN expressed on antigen-presenting cells or tumor cells can exert a T cell co-stimulatory function.     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

CD25 has become widely used as a marker for a subset of regulatory CD4(+) T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4(+)CD25(+) regulatory T cells in rats. In the thymus, CD4(+)CD8(-)CD25(+) cells were found to have a more mature phenotype than the corresponding CD4(+)CD8(-)CD25(-) cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4(+)CD25(+) cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4(+)CD25(+) T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4(+) T cells in rats and no further subdivision of the regulatory component of CD4(+)CD25(-)CD45RC(low) T cells has yet been achieved.     

Thermoluminescent dosimetry of the SourceTech Medical model STM1251 125I seed

Many new models of 125I seeds are being introduced, mainly due to the increase in prostate seed implants. We have evaluated the SourceTech Medical (STM), model STM1251, 125I seed using thermoluminescent dosimeters (TLDs) in a solid water phantom. TLD cubes, LiF TLD-100, with dimension 1 mm on each edge, were irradiated at various distances, 1, 2, 3, and 5 cm, at angles ranging from 0 degrees to 90 degrees in 10 degrees increments. Sensitivity calibration of the TLDs was achieved by irradiation to 10 cGy with 6 MV x rays from a clinical linear accelerator, Clinac 600C. Concurrent with the 125I seed exposures, several TLDs were also exposed to 10 cGy with the 600C as a control set. Dose rates per unit air kerma strength were determined based on the 1999 NIST traceable standard for the STM1251 seed. They are presented as a function of distance r and angle theta. The TG-43 parameters, including the dose rate constant, lambda, anisotropy function, F(r,theta), radial dose function, g(r), anisotropy factor, phian(r), and anisotropy constant, phi, were obtained for use in radiation treatment planning software. The value of lambda was determined as 1.07 +/- 5.5% cGy U(-1) h(-1), which is comparable to model 6702 and to the value determined using the point extrapolation method by Kirov and Williamson. We also find agreement between our TLD data and their Monte Carlo results for g(r), F(r,theta), phian(r), and phi. Additionally, agreement is found with the TLD data of Li and Williamson for lambda and g(r).     

Plasma metabolites after 2-deoxy-D-glucose in rats with brain dopamine-depleting lesions

Rats with selective brain dopamine-depleting lesions produced by 6-hydroxydopamine failed to increase their food intake after 2-deoxy-D-glucose administration. Their hyperglycemic response to 2DG was identical to that of intact controls. Neither group showed significant mobilization of free fatty acids or production of ketones.     

Quinine drinking: More regulatory puzzles

Rats were permanently hypodipsic when offered a quinine adulterated fluid on a chronic basis. Plasma osmolarity and Na concentration were normal, but the quinine drinkers showed a slight hyperkalemia compared to water drinking controls. The quinine-drinking rats maintained hydromineral equilibrium through the excretion of a small amount of concentrated urine. The quinine intake was closely matched to need, and fell to near zero when food was removed or water was supplied intravenously. This harmony of intake and output was disrupted after acute hypertonic NaCl load: while the obligatory salt diuresis was no different between water and quinine drinkers, the latter did not drink (except at the lowest level of adulteration) within several hours. However, by 24 hr all had shown a delayed drinking response. This delay in drinking of quinine was also evident after non-painful intravenous NaCl infusions, but no drinking occurred after nephrectomy. Quinine drinkers were also unresponsive to isoproterenol and intracranial dipsogens. These data are discussed in terms of their implication for definitions of regulatory drinking behavior.     

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier

The effect of forskolin on voltage-activated Na⁺ and K⁺ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltageclamp technique. Peak Na⁺ currents (I _Na) were reduced by 35% and the rate of decline of Na⁺ current during continuous depolarization was accelerated following treatment with 450 M forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1–10 Hz, a further inhibition of I _Na (60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV ( 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na⁺ channel in the blocking process. Inhibition of the delayed K⁺ current (I _K) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC₅₀ 80 M) and a concentration-independent acceleration of current inactivation. A similar inhibition of I _K was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition of I _K and perhaps I _Na follows directly from drug binding and is not a consequence of AC activation.     

A sensitive assay for detecting low-affinity interactions at the cell surface reveals no additional ligands for the adhesion pair rat CD2 and CD48

The ligand for the T cell antigen CD2 is CD48 in rodents, but CD58 in humans. The extracelluar parts of these three antigens are structurally related in that all contain two immunoglobulin superfamily (IgSF) domains. There have been reports of alternative ligands for CD2 in the human, but not so far in rodents. We describe the analysis of ligands for rat CD2 and CD48 using fluorescent beads capable of displaying a high ligand density and detecting low-affinity interactions like that of CD2 with CD48 (Kd = 60 ? 90 μM). Monovalent chimeric proteins containing the two IgSF domains of rat CD48 or CD2 and domains 3 and 4 of rat CD4 (CD4d3+4) were anchored to fluorescent covaspheres^TM via a CD4 monoclonal antibody (mAb) with the CD48 or CD2 domains available for ligand binding. Multivalent CD48-CD4d3 + 4 covaspheres^TM gave strong specific binding to rat CD2 expressed on the surface of transfected Jurkat cells. CD48-CD4d3+4 was compared with CD48-IgG and CD48-IgM as tools for detecting binding at the cell surface. Soluble divalent CD48-IgG and decavalent CD48-IgM bound to soluble CD2 with a K_off of around 10^?3 s^?1 as determined using a BIAcore^TM biosensor. However, binding to cells by CD48-IgG and CD48-IgM was only detectable when they were immobilized on covaspheres^TM and represented no increase in sensitivity over CD48-CD4 covaspheres^TM when tested for binding to cells expressing high and low levels of CD2. CD48-CD4d3 + 4 covaspheres^TM only bound to rat cells expressing CD2. In the reverse orientation, binding of CD2-CD4d3 + 4 covaspheres^TM was dependent on expression of CD48. Pre-incubation of cells with CD2 or CD48 mAb abolished all binding of CD48-CD4d3 + 4 or CD2-CD4d3 + 4, respectively. The data provide no evidence for an alternative ligand for rat CD2 or CD48.