Electroendocytosis: stimulation of adsorptive and fluid-phase uptake by pulsed low electric fields

We present a novel approach for stimulating uptake via endocytic pathways by exposing cells to a train of pulsed low electric fields (LEF) in the range of 2.5-20 V/cm. Electric field treatment of COS 5-7 and HaCaT cells in the presence of BSA-FITC augments the adsorption of the probe to plasma membranes with subsequent enhanced internalization. The uptake of BSA-FITC is maximal when the cells are exposed to LEF in the presence of the probe while uptake of a fluid-phase marker, propidium iodide (PI), is more effective when the probe is added immediately after termination of a 1-min exposure. LEF-stimulated uptake decays with a half-life of about 3 and 1 min for and BSA-FITC and PI, respectively. The uptake is inefficient at 4 degrees C but increases with temperature. The uptake proceeds via cell membrane vesiculation, showing a high extent of colocalization of BSA-FITC with plasma membrane vesicles labeled with a phospholipid fluorescent analogue. Unlike constitutive endocytosis where the BSA-FITC is exposed to acidic pH, in LEF-induced uptake the probe is exposed to the more alkaline pH of the cytosol. The staining kinetics of nuclear targets by PI reflects the release of the probe from the LEF-induced vesicles into the cytosol 1-3 h after exposure. The LEF-induced adsorptive pathway was approximately 2.5 more effective than the LEF-induced fluid-phase one. The observed 5- to 6-fold increase of BSA-FITC uptake induced by LEF may be partially attributed to a clathrin-dependent route (up to 25%), whereas the rest of the uptake may be assigned to macropinocytotic and clathrin/caveolin independent pathways or to a novel, yet unidentified, route driven by LEF. This study provides a basis for a general approach towards the efficient incorporation of a variety of molecules such as antibodies, enzymes or genes into cells.     

Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment

Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.     

Exposure of human peripheral blood lymphocytes to electromagnetic fields associated with cellular phones leads to chromosomal instability

Whether exposure to radiation emitted from cellular phones poses a health hazard is at the focus of current debate. We have examined whether in vitro exposure of human peripheral blood lymphocytes (PBL) to continuous 830 MHz electromagnetic fields causes losses and gains of chromosomes (aneuploidy), a major "somatic mutation" leading to genomic instability and thereby to cancer. PBL were irradiated at different average absorption rates (SAR) in the range of 1.6-8.8 W/kg for 72 hr in an exposure system based on a parallel plate resonator at temperatures ranging from 34.5-37.5 degrees C. The averaged SAR and its distribution in the exposed tissue culture flask were determined by combining measurements and numerical analysis based on a finite element simulation code. A linear increase in chromosome 17 aneuploidy was observed as a function of the SAR value, demonstrating that this radiation has a genotoxic effect. The SAR dependent aneuploidy was accompanied by an abnormal mode of replication of the chromosome 17 region engaged in segregation (repetitive DNA arrays associated with the centromere), suggesting that epigenetic alterations are involved in the SAR dependent genetic toxicity. Control experiments (i.e., without any RF radiation) carried out in the temperature range of 34.5-38.5 degrees C showed that elevated temperature is not associated with either the genetic or epigenetic alterations observed following RF radiation-the increased levels of aneuploidy and the modification in replication of the centromeric DNA arrays. These findings indicate that the genotoxic effect of the electromagnetic radiation is elicited via a non-thermal pathway. Moreover, the fact that aneuploidy is a phenomenon known to increase the risk for cancer, should be taken into consideration in future evaluation of exposure guidelines.     

Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.     

Differential sensitivity of naive and memory CD8+ T cells to apoptosis in vivo

Apoptosis is a critical regulator of homeostasis in the immune system. In this study we demonstrate that memory CD8(+) T cells are more resistant to apoptosis than naive cells. After whole body irradiation of mice, both naive and memory CD8(+) T cells decreased in number, but the reduction in the number of naive cells was 8-fold greater than that in memory CD8(+) T cells. In addition to examining radiation-induced apoptosis, we analyzed the expansion and contraction of naive and memory CD8(+) T cells in vivo following exposure to Ag. We found that memory CD8(+) T cells not only responded more quickly than naive cells after viral infection, but that secondary effector cells generated from memory cells underwent much less contraction compared with primary effectors generated from naive cells (3- to 5-fold vs 10- to 20-fold decrease). Increased numbers of secondary memory cells were observed in both lymphoid and non-lymphoid tissues. When naive and memory cells were transferred into the same animal, secondary effectors underwent less contraction than primary effector cells. These experiments analyzing apoptosis of primary and secondary effectors in the same animal show unequivocally that decreased downsizing of the secondary response reflects an intrinsic property of the memory T cells and is not simply due to environmental effects. These findings have implications for designing prime/boost vaccine strategies and also for optimizing immunotherapeutic regimens for treatment of chronic infections.     

Molecular and functional profiling of memory CD8 T cell differentiation

How and when memory T cells form during an immune response are long-standing questions. To better understand memory CD8 T cell development, a time course of gene expression and functional changes in antigen-specific T cells during viral infection was evaluated. The expression of many genes continued to change after viral clearance in accordance with changes in CD8 T cell functional properties. Even though memory cell precursors were present at the peak of the immune response, these cells did not display hallmark functional traits of memory T cells. However, these cells gradually acquired the memory cell qualities of self-renewal and rapid recall to antigen suggesting the model that antigen-specific CD8 T cells progressively differentiate into memory cells following viral infection.     

Immunohistochemical Analysis of 1,25-dihydroxyvitamin D3 Receptor in Cervical Carcinoma

The immunohistochemical localization and expression of 1,25-dihydroxyvitamin D₃ receptors (VDR) has been investigated in normal human cervical tissue (n = 15) and in cervical carcinomas (n = 23). VDR immunoreactivity (monoclonal antibody 9A7r354;) was compared with the staining patterns of transglutaminase K, cytokeratin 10 and Ki-67 in these tumours. Moderate to strong nuclear immunoreactivity for VDR was detected in almost all cervical carcinomas analysed. VDR staining was homogeneous, with no visual differences between individual tumour cells. Some 60% of normal cervical tissues revealed weak immunoreactivity for VDR. In normal cervical tissue, nuclear VDR staining was confined to the lower cervical layers, predominantly to the basal cell layer. Both the intensity of VDR immunostaining and the number of VDR-positive cells were up-regulated in cervical carcinomas compared with normal cervical tissue. No visual correlation wa s found for the coexpression of VDR with markers of proliferation and differentiation. Our findings indicate that: (1) cervical tissue may be a new target organ for therapeutically applied vitamin D analogues; (2) VDR is up-regulated at the protein level in cervical carcinomas compared with normal cervical tissue; (3) up-regulation of VDR in cervical carcinoma is induced not exclusively by alterations in epithelial differentiation or proliferation, but by different, unknown mechanisms; and (4) calcitriol and new vitamin D analogues exerting fewer calcaemic side-effects may be promising new drugs for the treatment or chemoprevention of metastasizing cervical carcinomas as well as of cervical precancerous lesions.     

Geographic variability of flower colour inCrocus scepusiensis (Iridaceae)

The distribution of four variants for flower colour ofCrocus scepusiensis in the northern part of the Western Carpathians is described. The frequency of white stigmata morphs declines from east to west. In the center of the area stigmata colour morphs show strikingly patchy distribution. White perianth morphs usually occur at low frequencies and their distribution with minor exceptions is restricted to the central part of the area, where patchy distribution of the morphs is a rule. These distribution patterns suggest that the founder effect has played a major role in determining the genetic composition of individual populations. The cline for stigmata colour may also be explained by the dynamics of population expansion. No influence of selection can be demonstrated, but the association between perianth and stigmata colour, and the excessively low frequency of white perianth morphs may imply that the polymorphisms are not selectively neutral.     

Chromosomal differentiation withinCrocus vernus agg. (Iridaceae) in the Carpathian Mts.

Western and eastern Carpathian populations ofCrocus heuffelianus s. lat. (incl.C. scepusiensis) have 2n = 18 but differ in karyotype. While western populations are chromosomally monomorphic, eastern populations exhibit geographical karyotypic differentiation.     

A contribution to the karyology ofCrocus vernus agg. (Iridaceae) from the Iserian Mts. (Sudety Mts., Poland)

The Iserian Mts. form ofCrocus vernus agg. has 2n = 16 and a karyotype clearly deviating fromC. heuffelianus s. lat.