Peptide-loaded dendritic cells prime and activate MHC-class I-restricted T cells more efficiently than protein-loaded cross-presenting DC

Undifferentiated and differentiated dendritic cells (uDC and dDC, respectively), derived from the bone marrow, were studied in vitro and in vivo. Ovalbumin (OVA) and two OVA-derived peptides binding to H-2K(b) and I-A(b), respectively, were used. Two IL-2 secreting T cell hybridomas specific for the OVA-derived epitopes were used in the in vitro read-out. The ability to cross-present the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL) was restricted to dDC, which express CD11c(+), CD86(+), and MHC-II(+). In vitro, the antigenicity of SIINFEKL-loaded DC declined at a slower rate than that of OVA-pulsed DC. Moreover, SIINFEKL-loaded DC were up to 50 times more efficient than DC-pulsed with OVA-protein for generation of an H-2K(b)-restricted response. Immunization of mice with SIINFEKL-loaded DC resulted in a much stronger H-2K(b)-restricted response than immunization with OVA-pulsed DC. These data might have important implications for the choice of antigen source in the design of DC-based vaccines.     

Floating the raft hypothesis for immune receptors: access to rafts controls receptor signaling and trafficking

The B cell antigen receptor (BCR) is a member of an important family of multichain immune recognition receptors, which are complexes composed of ligand-binding domains associated with signal-transduction complexes. The signaling components of these receptors have no inherent kinase activity but become tyrosine phosphorylated in their cytoplasmic domains by Src-family kinases upon oligomerization, thus initiating signaling cascades. The BCR is unique in this family in that, in addition to its signaling function, it also serves to deliver antigen to intracellular compartments where the antigen is processed and presented bound to major histocompatibility complex (MHC) class II molecules. Recent evidence indicates that both the signaling and antigen-trafficking functions of the BCR are regulated by cholesterol- and sphingolipid-rich plasma membrane microdomains termed rafts. Indeed, upon oligomerization, the BCR translocates into rafts that concentrate the Src-family kinase Lyn and is subsequently internalized directly from the rafts. Thus, translocation into rafts allows the association of the oligomerized BCR with Lyn and the initiation of both signaling and trafficking. Significantly, the access of the BCR to rafts appears to be controlled by a variety of B lymphocyte co-receptors, as well as factors including the developmental state of the B cell and viral infection. Thus, the translocation of the immune receptors into signaling-competent microdomains may represent a novel mechanism to initiate and regulate immune-cell activation.     

Thermal and Bioenergetics of Elasmobranchs: Bridging the Gap

Physiological telemetry is a powerful tool in studying the thermal biology and energetics of elasmobranchs in the laboratory and field. Controlled laboratory studies have increased our understanding of the physiology and behavior of many elasmobranchs, but have focused primarily on small, slow moving species. Extrapolating results from these laboratory studies to free-swimming animals in the field or to other unstudied species may be problematic, due to laboratory constraints or species specific differences. Some elasmobranchs are too large or logistically difficult to maintain in captivity, making them extremely difficult to study in the laboratory, and thus can only be studied in the field. Physiological telemetry offers a bridge between the laboratory and the field providing an opportunity to elucidate similarities and differences. Previous studies have coupled a variety of sensors with ultrasonic transmitters to relay information on epaxial muscle and stomach temperatures of free-swimming lamnid sharks. Even though these studies indicate lamnids exhibit elevated body temperatures, the degree to which these sharks may control body temperature is still not fully understood. Telemetry of heart rate, swimming speed, muscle contraction rate, and tail beat frequency has been used to estimate energy consumption of free-swimming elasmobranchs with varying success. Based on recent advances in technology, several hypotheses regarding thermoregulation, cardiac output, and obligate ram ventilation are discussed. Although many telemetry studies have been restricted by logistical difficulties in conducting long-term tracks, recent developments such as acoustic modems, underwater listening stations and satellite telemetry may significantly increase the amount and types of physiological data that can be collected. These improvements in technology and captive animal husbandry techniques will help to bridge the gap between the laboratory and the field.     

Dynamic changes in p27kip1 variant expression in activated lymphocytes

The p27Kip1 cell cycle inhibitor (p27) has emerged as a critical mediator of normal cellular growth control. We report the expression of a 24 kD C-terminal variant of p27 in normal peripheral blood lymphocytes. This variant is rapidly degraded in a proteasome-dependent manner when lymphocytes are activated by interleukin-2 or by superantigen. Whereas p24 degradation is complete within 16 h of mitogen addition, full-length p27 is decreased only modestly over 72 h of mitogen exposure and is present in activated and cycling lymphocytes. Persistent p27 is present in a complex with cyclin D3 in activated lymphocytes, and is localized both in the nucleus and cytoplasm. These results indicate that lymphocytes exiting from quiescence use several mechanisms to overcome the p27Kip1-enforced cell cycle checkpoint, and that elimination of p27 is not required for cell cycle entry.     

人扁桃体中应激免疫抑制蛋白的初步观察

以前的实验证明,在应激条件下,外周淋巴组织中产生一种蛋白质,具有抑制某些免疫功能的作用,称为应激免疫抑制蛋白（immune suppressive protein of stress,ISPS）。本实验用人外周淋巴器官扁桃体进行了研究,证明扁桃体的提取物能抑制小鼠由Con A诱导的淋巴细胞转化,而且这种抑制作用可被ISPS单克隆抗体（2C4）部分翻转。间接ELISA法证明人扁桃体提取物能与2C4单克隆抗体相结合。以ISPS单克隆抗体（2C4）作免疫组织化学研究,证明人扁桃体中有很多染色呈阳性的细胞。这些结果从不同角度提示,人外周淋巴组织中存在一种与ISPS相类似的免疫抑制物质。     

Differential effects of iron deficiency on the expression of CD80 and CD86 co-stimulatory receptors in mitogen-treated and untreated murine spleen cells

The interaction of CD28 and its ligands (CD80, CD86) on antigen presenting cells and that of TCR/CD3-MHC are required for T lymphocyte activation. To determine whether impaired lymphocyte proliferation associated with iron deficiency is due to reduced expression of these ligands, spleen cells obtained from eight to nine C57BL/6 mice/group of iron deficient (ID), iron replete (R), control (C), pair-fed (PF), and high iron (HI) mice were labeled with anti-CD80-fluorescein isothiocyante (FITC) and anti-CD86-FITC. Diets differed only in iron concentration: 5, 50, and 125 mg/kg for the ID, C, and HI, respectively. Mean levels of hemoglobin and liver iron stores of ID and R mice were less than 50% those of C mice (P < 0.005). In non-activated and concanavalin A-treated cultures, significant differences were observed among groups in the percentage of CD80 + cells: ID>R > C = PF = HI (P < 0.05). The same trend was observed for CD86 + cells (P > 0.05). Fluorescence intensity (FI) of either marker did not significantly change by iron status. In vitro iron chelation by deferoxamine (20, 200 microg/ml) for 1, 2, and 24 h increased FI of both markers on unactivated B and T cells (P < 0.05). However, it had no effect on FI of either marker of mitogen-treated cells presumably because the maximum levels are achieved by the mitogen. Lymphocyte proliferative responses to mitogens positively and significantly correlated with CD80 and CD86 FI (r = 0.41-0.59) but negatively correlated with the percentages of CD80 + cells (r = -0.48) (P < 0.05). Data suggest that impaired lymphocyte proliferation associated with iron deficiency is not due to reduced CD80 and CD86 expression.     

Retention of antigen on follicular dendritic cells and B lymphocytes through complement-mediated multivalent ligand-receptor interactions: theory and application to HIV treatment

In HIV-infected patients, large quantities of HIV are associated with follicular dendritic cells (FDCs) in lymphoid tissue. During antiretroviral therapy, most of this virus disappears after six months of treatment, suggesting that FDC-associated virus has little influence on the eventual outcome of long-term therapy. However, a recent theoretical study using a stochastic model for the interaction of HIV with FDCs indicated that some virus may be retained on FDCs for years, where it can potentially reignite infection if treatment is interrupted. In that study, an approximate expression was used to estimate the time an individual virion remains on FDCs during therapy. Here, we determine the conditions under which this approximation is valid, and we develop expressions for the time a virion spends in any bound state and for the effect of rebinding on retention. We find that rebinding, which is influenced by diffusion, may play a major role in retention of HIV on FDCs. We also consider the possibility that HIV is retained on B cells during therapy, which like FDCs also interact with HIV. We find that virus associated with B cells is unlikely to persist during therapy.     

Effects of zinc supplementation to ration on some hematological parameters in broiler chicks

The aim of the study was to examine the effects of zinc supplementation on some hematological parameters. Sixty newborn male broiler chicks were utilized in the study. Zinc (Zn) was added into drinking water at levels of 0, 125, 500, and 1000 mg/kg. In the study, there was no significant difference between control and Zn-supplemented groups in erythrocyte count, hemoglobin amount, hematocrit levels, total leukocyte count, and differential leukocyte % levels, but the α-naphthyl acetate esterase ANAE(+) lymphocyte rate significantly (p<0.05) increased in the 125-ppm Zn-supplemented group compared with the control group. In conclusion, the data obtained may be beneficial in demonstrating the effects of zinc on, at least, these parameters.     

Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

Measurement of CTL-induced cytotoxicity: The caspase 3 assay

Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the ⁵¹Cr release assay. However, measurement of target cell lysis by ⁵¹Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the ⁵¹Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.