Cellular Vesicles: New Insights in Engineering Methods, Interaction with Cells and Potential for Brain Targeting.

Cellular vesicles (CVs) have been proposed as alternatives to exosomes for targeted drug delivery. CVs, prepared from human embryonic kidney 293 cells (HEK-293), C57BL/6 mouse B16F10 skin melanoma cells (B16F10), and immortalized human cerebral microvascular endothelial cells (hCMEC/D3) by liposome technology methods, were characterized for morphology, cytotoxicity, and cell uptake properties. CV brain-targeting potential was evaluated in vitro on the hCMEC/D3 blood-brain barrier (BBB) model, and in vivo/ex vivo. CV sizes were between 135 and 285 nm, and the ζ-potential was negative. The dehydration-rehydration method conferred highest calcein loading and latency to CVs compared with other methods. The increased calcein leakage from CVs when compared with liposomes indicated their poor integrity, which was increased by pegylation. The in vivo results confirmed lower liver uptake by PEG-CVs (compared with nonpegylated) proving that the calcein integrity test is useful for prediction of CV biodistribution, as used for liposomes. The cell uptake of homologous origin CVs was not always higher compared with that of non-homologous. Nevertheless, CVs from hCMEC/D3 demonstrated the highest BBB permeability (in vitro) compared with OX-26 targeted liposomes, and brain localization (in vivo). CVs from hCMEC/D3 cells grown in different media demonstrated decreased interaction with brain cells and brain localization. Significant differences in proteome of the two latter CV types were identified by proteomics, suggesting a potential methodology for identification of organotropism-determining CV components.

Anti-interleukin 2 receptor monoclonal antibodies spare phenotypically distinct T suppressor cells in vivo and exert synergistic biological effects.

The therapeutic efficacies of ART-18, ART-65, and OX-39, mouse antibodies of IgG1 isotype recognizing distinct epitopes of the p55 beta chain of the rat IL-2-R molecule, were probed in LEW rat recipients of (LEW X BN)F1 heterotopic cardiac allografts (acute rejection in untreated hosts occurs within 8 d). A 10-d course with ART-18 prolongs graft survival to approximately 21 d (p less than 0.001). Therapy with ART-65, but not with OX-39, was effective (graft survival approximately 16 and 8 d, respectively). Anti-IL-2-R mAb treatment selectively spared T cells with donor-specific suppressor functions; the CD8+ (OX8+ W3/25-) fraction from ART-18-modified recipients, and primarily the CD4+ (W3/25+ OX8-) subset from ART-65-treated hosts conferred unresponsiveness to naive syngeneic rats after adoptive transfer, increasing test graft survival to approximately 16 and 45 d, respectively. Concomitant administration of ART-18 and ART-65 to recipient animals in relatively low doses exerted a strikingly synergistic effect, with 30% of the transplants surviving indefinitely and 50% undergoing late rejection over 50 d. These studies provide evidence that anti-IL-2-R mAbs selectively spare phenotypically distinct T cells with suppressor functions. The data also suggest that in vivo targeting of functionally different IL-2-R epitopes may produce synergistic biological effects.

T helper 1 (Th1)/Th2 cytokine expression shift of peripheral blood CD4+ and CD8+ T cells in patients at the post-acute phase of stroke.

Local humoral and cellular immune responses modulate the inflammatory processes involved in the development of atherosclerotic lesions, as well as in the evolution of brain infarcts in stroke patients. The role of systemic adaptive immunity on the progression of such disease manifestations is less clear. In the current study, we evaluated the percentages of T helper 1 (Th1) [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T cells in 23 patients with a history of ischaemic stroke (IS) at the chronic stable phase of the disease (median post-stroke time 34.5 months). Seven stroke-free individuals matched for age and vascular risk factors (matched controls, MC) were collected for comparison. To measure cytokine values at baseline and after stimulation, we used a flow cytometry method of intracellular cytokine staining. Intrinsic Th1 and Th2 cytokine production in unstimulated T cells was negligible in all study participants. Following mitogenic stimulation with phorbol 12-myristate13-acetate/ionomycin, both the IS and the MC groups exhibited a similarly strong Th1 response; IL-2 production predominated in the CD4+ T cells and IFN-gamma in the CD8+ T cells. However, when measuring the Th2 cytokine-production capacity post-stimulation, a significant increase in the percentage of IL-4-producing T cells was observed in the IS groups, compared with the MC group, resulting in a significantly lower ratio of IFN-gamma-/IL-4-producing T cells. No such Th2 enhancement could be confirmed for the case of IL-10. We propose that in IS patients there is a systemic shift of the immune system towards Th2 responses at the late post-acute phase of stroke.

Procleix Ultrio transcription-mediated amplification vs. serological blood screening in south-western Greece.

We present here our overall experience after 27 months of performance of the Procleix Ultrio [HIV-1, hepatitis C virus (HCV), hepatitis B virus (HBV)] transcription-mediated amplification (TMA) assay. The aim of this report is to assess the impact of nucleic acid testing (NAT) implementation in blood screening of south-western Greek blood donors. We processed 38,264 units of blood as neat samples with the Procleix Ultrio TMA assay (Chiron/GenProbe, Emeryville/San Diego, CA, USA) between 1 January 2005 and 31 March 2007. NAT results were compared to those obtained from routine serology tests and quantitative polymerase chain reaction (PCR) assays. Overall, 52 units of blood tested positive for HBV (1.4 per thousand), 8 for HCV (0.2 per thousand) and none for HIV or multiple infections. The yield of TMA was 0.183 per thousand for HBV (7/38 264) and 0 for HCV. The TMA HBV-positive donations were tested for HBV DNA by a quantitative PCR assay and were found negative (below the detection limit of the method, 200 copies/mL). Follow-up testing showed that the TMA HBV-positive donations were positive for anti-hepatitis B core antigen immunoglobulin G antibodies. Implementation of the TMA assay in the individual donation configuration increased HBV detection compared to serological screening or a commonly used quantitative PCR assay. Follow-up studies will determine the impact of NAT implementation in HBV transmission in countries with an intermediate HBV incidence.

Effect of non-operative management (NOM) of splenic rupture versus splenectomy on the distribution of peripheral blood lymphocyte populations and cytokine production by T cells.

Post-traumatic splenectomy is associated with increased postoperative morbidity and mortality and long-term impairment of humoral and cellular immunity. Alternatives to surgery have been developed to minimize or avoid the immediate and/or long-term complications of splenectomy. Herein we investigated the long-term effect of non-operative management (NOM) of the traumatic rupture of the spleen on the distribution of peripheral blood (PB) lymphocyte populations and cytokine production by T cells. PB samples were drawn from six NOM patients, 13 age-matched adults who had undergone splenectomy after trauma (SP patients) and 31 age-matched controls. Cellular phenotypes and the intracellular production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-10 cytokines in T cells were determined in whole blood +/- mitogens by flow cytometry. NOM patients did not show any changes in the absolute numbers of lymphocytes or the distribution of their subsets, compared to the controls. In contrast, SP patients showed a sustained increase in the percentage and/or absolute numbers of lymphocytes, CD8 T cells, activated CD8 T cells, natural killer (NK) T cells, NK cells and gammadelta T cells, and a reduction in naive CD4 T cells. The constitutive or induced cytokine production by T cells of the NOM group was similar to the control group, whereas SP patients had increased percentages of constitutive IL-2- and IFN-gamma-producing CD8 T cells and IFN-gamma-producing CD4 T cells. Our findings indicate collectively that the healing process in NOM does not affect the architecture of the spleen to such an extent that it would lead to long-term alterations of the proportions of PB lymphocytes or the T cell cytokine profiles.

Regulation of interleukin-2 receptor expression and receptor release.

The generation and cell surface expression of IL-2 receptors was monitored by: (i) an ELISA that permits quantitative determination of detergent-solubilized or soluble IL-2 receptors; and (ii) detection of the binding of 125I-labelled recombinant IL-2 and of anti-IL-2 receptor antibodies to receptor bearing cells. Upon lectin stimulation both high and low affinity IL-2 receptors became expressed in parallel at the cell surface. Both high and low affinity receptors were upregulated by IL-2. Upon lectin activation the amount of cell-associated receptors increased and on day 2 of the culture period IL-2 receptors were detectable in the culture supernatant. IL-2 upregulated both high and low affinity IL-2R expression on T-lymphoblasts. IL-2R bearing leukemic cells and T lymphoblasts released IL-2R when cultured in vitro. IL-2R release by T lymphoblasts was enhanced dramatically by IL-2. On the other hand, IL-2-receptor positive leukemic cells released receptors in an IL-2 independent manner. Release of receptors could also be detected in serum-free medium. At least a part of the released receptors could be specifically bound to immobilized pure recombinant IL-2 and to monoclonal anti-IL-2-receptor antibodies. Small but significant amounts of soluble IL-2 receptors were detectable in the sera of normal mice. In sera of mice inoculated with IL-2-receptor positive syngeneic leukemic cells, elevated levels of IL-2 receptors were detectable. Release of IL-2 receptors seems to represent one of the major routes by which the receptors are cleared from the cell surface.

An ELISA that detects cell-associated and released rat IL-2 receptors in a soluble form.

A mouse anti-rat interleukin-2 receptor (IL-2R) monoclonal antibody (mAb), ART-65, has recently been developed which recognizes the rat IL-2R but binds to an epitope distinct from that recognized by the mAb ART-18. The availability of two mAb against the rat IL-2R molecule permitted the development of an enzyme-linked immunosorbent assay (ELISA) which detects soluble and solubilized rat IL-2R. Using this ELISA, time course studies of ConA and LPS-activated rat splenocytes revealed that IL-2R are released into their environment during the culture period, and that IL-2R can be detected in rat sera. The significance of the results is discussed.

Synthetic peptides as putative therapeutic agents in transplantation medicine: application of PEPSCAN to the identification of functional sequences in the extracellular domain of the interleukin-2 receptor beta chain (IL-2Rbeta).

A desired treatment strategy in transplantation medicine is the selective targeting of alloreactive T cells without impairing antileukemic and antiviral activities. One approach is the synthesis of peptides that interfere with the binding of interleukin-2 (IL-2) to its high affinity receptor (IL-2R). This blocks the activation and proliferation of the antigen-activated T cells and the secretion of IL-2. The latter binds to its receptor, via the extracellular domain of the IL-2Rbeta chain, while its cytoplasmic domain is required for intracellular signal transduction. In this study, the PEPSCAN method was applied in order to identify antigenic sequences (epitopes) in the extracellular domain of the IL-2Rbeta. Based on the primary amino acid (aa) sequence of the IL-2Rbeta, a total of 239 overlapping dodecapeptides, spanning the entire sequence of IL-2Rbeta, were synthesized by PEPSCAN and their immunoreactivity was tested by ELISA using monoclonal antibodies (mAbs) specific for IL-2Rbeta such as TU11, Mikbeta1, HuMikbeta1 and TU27. TU11 recognized a linear epitope located in the region 85R-Q(96). None of the 239 synthetic peptides was recognized by TU27. Mikbeta1 (and HuMikbeta1) recognized a discontinuous epitope formed by aa located in the IL-2Rbeta domains L(106) to P(148) and E(170) to A(202). Subsequently, synthetic peptides corresponding to the identified putative epitopic sequences were prepared by solid phase synthesis and their immunogenicity in vivo was assessed by raising polyclonal antibodies. Given that Mikbeta1 and HuMikbeta1 inhibit binding of IL-2 on the IL-2Rbeta, we addressed the question of whether the identified antigenic sequences serve as putative IL-2 binding domains. Synthetic peptides corresponding to these sequences were tested for their ability to compete with IL-2 for binding and, thereby, inhibit IL-2-induced proliferation of mitogen-stimulated human peripheral blood T cells. Sequences 107M-E(118) and 178Y-Q(199) probably represent functional IL-2 binding domains on IL-2Rbeta, since these synthetic peptides significantly inhibited the proliferation of activated T cells and secretion of IL-2.

Interaction between C3 and IL-2; inhibition of C3b binding to CR1 by IL-2.

We previously reported that C3 has a role in the enhancement of the IL-2 dependent proliferation of helper T cells. Because the IL-2R has a structural homology with the complement proteins, such as CR1 and CR2, we studied the possible ligand crossreactions on CR1 and IL-2-receptor, and the direct interaction between C3 and IL-2. While C3 has an enhancing effect on the IL-2 dependent proliferation of HT-2, a CR1-positive mouse T-cell line, the growth of the CTLL-16 line (CR1-negative) is not affected by C3. It has been proven that neither the insolubilized C3 nor the soluble C3b-like C3 react with the IL-2 binding epitope of the IL-2 receptor. However, using human RBC we have demonstrated that the binding of aggregated C3 to CR1 is inhibited by rIL-2, in a dose-dependent manner. When RBC were incubated with rIL-2 and FITC-labelled Fab-anti-CR1 simultaneously, there was no inhibition in the fluorescence intensity. As detected by ELISA, rIL-2 was bound to the same extent by insolubilized C3, C3b, and C3c, while C3d coat had lower binding capacity. The receptor-binding epitope of IL-2 is intact in the complex of complement proteins and rIL-2, as demonstrated by the binding of DMS1, a monoclonal antibody reacting with the receptor site of IL-2. It is strongly suggested that C3b may play a role in the growth of CR1 positive T cells.

Age-related changes in regional brain activation during phonological decoding and printed word recognition.

Using magnetic source imaging, age-related changes in spatiotemporal brain activation profiles associated with printed word recognition and phonological decoding (pseudoword reading) were examined in 27 adults and 22 children without reading problems. Adults showed a distinct spatiotemporal profile during reading of both types of print consisting of bilateral activation of occipital cortices, followed by strongly left-predominant activation of basal temporal regions, and, finally, left hemisphere temporoparietal (including the angular gyrus) and inferior frontal activation. Children lacked the clear temporal distinction in the engagement of basal and temporoparietal areas and displayed significantly weaker activation of the left inferior frontal gyrus. In addition, the consistent hemispheric asymmetries in the degree of activation of basal temporal regions that were present in the adult readers were not apparent in the children. In contrast, the strong left hemisphere preponderance in the degree of activation of temporoparietal areas was present in children as well as adults, regardless of the type of print they were asked to read. The data suggest that the degree of specialization of cortical regions for reading, as well as the pattern of regional interactions that supports this specialization, may change with age.

Serum transforming growth factor-beta 1 is related to the degree of immunoparesis in patients with multiple myeloma.

The expansion of myeloma cells is regulated by cytokines, among which IL-6 is a major growth factor. It has been recently suggested that serum transforming growth factor beta 1 (TGF beta 1), a cytokine found in large amounts in alpha-granules of platelets, might play a role in multiple myeloma (MM). It was the purpose of this study to determine serum TGF beta 1 levels in MM patients and to seek a correlation with disease parameters. Measurements were done by ELISA. We studied 35 MM patients (19 stage II, 16 stage III, 20 IgG, 8 IgA and 6 BJ, 1 IgD) in different phases of the disease, 27 healthy individuals and 17 thrombocytopenic patients with other haematological diseases (three MDS, three congenital thrombocytopenia, 11 ITP). Overall samples from MM patients were included: 10 at diagnosis, 18 in remission and 32 in relapse. In normal controls TGF beta 1 serum levels ranged from 1 to 33 ng/ml (median 16.5 ng/ml). In both thrombocytopenic controls with other diseases and thrombocytopenic MM patients (seven samples), TGF beta 1 serum levels were very low (median 3.2 and 4.5 ng/ml respectively). In MM patients with PLT > 100 x 10(9)/L (53 samples), TGF beta 1 serum levels were in the normal range in patients without immunoparesis (1 to 27 ng/ml, median 16.6 ng/ml), whereas they were higher in patients with immunoparesis (polyclonal immunoglobulins (Igs) below lower normal reference values) ranging from 10.2 to 45 ng/ml (median 26.8 ng/ml) (P < 0.01). Serum TGF beta 1 levels fluctuated in the same patient at different times but not according to relapse or remission. Correlation was found only between serum TGF beta 1 levels and immunoparesis and not between serum TGF beta 1 levels and disease stage or Ig subtype nor with prognostic factors for MM (serum CRP, beta 2M or IL-6). This finding suggests that the remaining normal plasma cells are sensitive to the inhibitory action of TGF beta 1 on Ig production. In conclusion TGF beta 1 serum levels are very low in thrombocytopenic patients confirming that platelets are the major source of this cytokine. Furthermore, a strong correlation was found between TGF beta 1 serum levels and immunoparesis in MM patients.

Synthetic peptides mapping to epitopes of the extracellular domain of the interleukin-2 receptor beta-chain to inhibit T-cell activation.

Expression of the high-affinity IL-2 receptor (IL-2R) on the surface of activated T cells makes it an attractive target for selective inhibition of alloreactive T cells in organ transplantation. IL-2 binds to its receptor via the extracellular domain of the beta-chain. In this study we synthesized synthetic peptides that map to epitopes of this domain and tested their ability to inhibit the activation and proliferation of mitogen-stimulated peripheral-blood T cells. Solid-phase synthesis was applied to create three oligopeptides of primary structures corresponding to the epitopes M(107)-E(118), Y(178)-Q(199), and E(190)-Q(199) of the extracellular domain of the IL-2Rbeta-chain. A nonhomologous peptide served as control. Peripheral-blood mononuclear cells isolated from 16 healthy volunteers (median age 41 years, range 26 to 56 years) were cultured with various concentrations of peptides and the mitogen phytohemagglutinin (PHA). The inhibitory effect of the peptides on cell proliferation was evaluated by automated cell counting and colorimetric proliferation assays. Cell activation was assessed by immunophenotyping using antibodies directed toward CD4, CD25, or CD69. The amount of IL-2 in culture supernates was measured by enzyme-linked immunoassay. Cultures in the presence of the peptide M(107)-E(118) (500 nmol/L) inhibited PHA-induced T-cell proliferation by 38%, and IL-2 secretion by 57%. Immunophenotyping confirmed suppression of activated T cells. Peptides Y(178)-Q(199) and E(190)-Q(199) inhibited proliferation, but failed to significantly affect IL-2 secretion. The control peptide showed no effect on the activation parameters. Our data indicate that the M(107)-E(118) peptide has promise for organ transplantation therapy.

Properties of transcription factors regulating interleukin-2 gene transcription through the NFAT binding site in untreated or drug-treated naive and memory T-helper cells.

Combining in vitro DNA binding studies and functional transcription assays in the Xenopus oocyte, we have tested the presence and functional state of transcription factors controlling the interleukin-2 (IL-2) promoter through the NFAT binding site. In naive T-helper cells, the IL-2 gene is repressed by a silencer. After first mitogenic stimulation, this silencer becomes undetectable while an activator is newly synthesized. In resting memory cells, the activator has low DNA-binding affinity and is located in the cytoplasm. However, no silencer is formed. Upon renewed cellular activation, this pre-existing activator is again targeted to the nucleus and regains function in promoting transcription. Cyclosporin A and FK506 act on two distinct levels of the IL-2 control mechanism. They prevent nuclear transport and reactivation of the performed activator in memory cells and, in naive cells, they render the silencer resistant to displacement by the activator. DNA-binding of silencer and activator from T-helper, and NFAT-1 from Jurkat cells, requires the same three G residues, but cross-linking analyses show differences in their constituent subunits. Supershift experiments show that the activator contains fra-2 and junD, whereas the silencer reacts with none of the antibodies tested.

Four epitopes on the rat 55-kDa subunit of the interleukin 2 receptor as defined by newly developed mouse anti-rat interleukin 2 receptor monoclonal antibodies.

Four new mouse monoclonal antibodies (mAb), ART-38, ART-35, ART-75 and ART-94, directed against the rat interleukin 2 receptor (IL 2R) have been developed. As shown by immunoprecipitation studies they all recognize specifically the 55-kDa subunit of the rat IL 2R. These mAb were compared to three previously characterized mouse mAb directed against the 55-kDa molecule of the rat IL 2R, namely the ART-18, ART-65 and OX-39 mAb. Out of all seven mAb, only ART-18 and OX-39 were found to inhibit the IL 2 binding to activated T cells, while IL 2R inhibited the binding of ART-18 alone. ART-18 was the only mAb found to inhibit the IL 2-dependent proliferation of cells carrying the IL 2R. Scatchard plot analyses showed gross differences in the numbers of mAb-binding sites ranging between 30,000 (ART-65) and 165,000 (ART-75) as well as in their affinities which ranged between 2.5 X 10(-9) M (ART-38) and 8.3 X 10(-10) M (OX-39). Cross inhibition studies revealed that the mAb recognize four different epitopes on the 55-kDa rat IL 2R subunit.

[Regulation of transcription of the interleukin-2 gene in B-lymphocytes]. / Régulation de la transcription du gène de l'interleukin-2 dans les lymphocytes B.

Since most B cell clones immortalized with EBV virus can be induced to produce interleukin-2, a typical T cell cytokine, we studied the role of different elements of the IL-2 promoter in such clones by transfection. It was found, in particular, that the element TCEd, which binds the transcription factor NF-kB, is very active in all three B clones tested. This element has no activity in T cells of the Jurkat line. The NFATd element, which binds the transcription factor NFAT-1 and is very active in T cells, is only weakly active in one B clone and not at all in another. Different elements thus contribute to IL-2 promoter activity in different cells.

Trans-active factors controlling the IL-2 gene in adult human T-cell subsets.

IL-2 secretion in total or subsets of PHA/PMA-stimulated PBMC-derived human T-lymphocytes was monitored and found to be largely due to CD4(+)CD8(-) cells. The presence and functional state of transcription factors (TF) was assessed by protein-DNA interaction assays and functional transactivation experiments in the Xenopts oocyte system, modulating IL-2 transcription by injection of proteins. The results reveal that CD4(+)CD8(-) cells contain both, functional silencer in their resting, and positive TF in their activated states while the CD4(+)CD8(-) group contains only non-functional positive TF. This demonstrates that the on/off switch of IL-2 transcription is based on the same mechanism in primary T-lymphocytes of mouse spleen and in peripheral human CD4(+)CD8(-) cells.

Blocking of interleukin 2 (IL 2) binding to the IL 2 receptor is not required for the in vivo action of anti-IL 2 receptor monoclonal antibody (mAb). I. The production, characterization and in vivo properties of a new mouse anti-rat IL 2 receptor mAb that reacts with an epitope different to the one that binds to IL 2 and the mAb ART-18.

A mouse anti-rat interleukin 2 (IL 2) receptor (IL 2R) monoclonal antibody (mAb), ART-65, has been developed. As shown by fluorescence-activated cell sorter analysis and immunoprecipitation studies, ART-65 recognizes in a species-specific manner the same molecule as does ART-18, a mAb which has been shown previously to recognize the rat receptor for IL 2. ART-65 and ART-18 do not competitively inhibit the binding of each other to activated T cells. ART-65, in contrast to ART-18, does not inhibit the binding of IL 2 to cells nor does it have any inhibitory effect in vitro on IL 2-driven proliferation of rat T lymphoblasts. Therefore, ART-65 is another mAb recognizing the rat IL 2 receptor, but binding to an epitope distinct from that recognized by either IL 2 or ART-18. We compared the in vivo activity of the mAb ART-65 and ART-18 with that of the W3/25 mAb in a local graft-vs-host reaction (GVHR). Similar to the anti-W3/25 treatment, ART-65 and ART-18 inhibited GVHR. The results demonstrate that GVHR depends on a small subpopulation of IL 2R+ cells present in the W3/25+ T cell population because IL 2R-targeted therapy was as effective as the treatment with W3/25 mAb which reacts with the entire T helper cell population. Moreover, the results argue against the possibility that anti-IL 2R mAb act via blockade of the IL 2 binding to IL 2R+ cells and/or by inhibiting the IL 2-driven expansion of the antigen-activated clones. The results support the view that IL 2R-targeted therapy results in the elimination of the IL 2R+ cells.

Demonstration of two distinct forms of released low affinity-type rat IL-2 receptors.

The release of IL-2 binding proteins, derived from the 55,000 MW low affinity IL-2R (L chain), has been observed for virtually all L chain-bearing cells in either humans, the mouse or the rat. Based on the characterization of the released human L chain as a molecule 10,000 MW smaller than the cell surface receptor, either proteolytic cleavage or differential splicing of the L chain encoding mRNA have been suggested as mechanisms underlying the receptor release. Combining affinity labelling of the L chain with [125I]IL-2 and immunoprecipitation with L chain-specific monoclonal antibody (mAb) applied for the detection of soluble rat IL-2R revealed the existence of two classes of soluble receptors, one being of the same size as cell surface-expressed L chain, the other of 40,000 apparent molecular mass. These findings raise the possibility of other mechanisms of receptor release than those discussed for human L chain.