Tc Precursors Research Articles

Synthesis, biological evaluation and preclinical study of a novel 99mTc-peptide: A targeting probe of amyloid-β plaques as a possible diagnostic agent for Alzheimer's disease.

With respect to the main role of amyloid-β (Aβ) plaques as one of the pathological hallmarks in the brain of Alzheimer's patients, the development of new imaging probes for targeted detection of Aβ plaques has attracted considerable interests. In this study, a novel cyclopentadienyl tricarbonyl Technetium-99m (99mTc) agent with peptide scaffold, 99mTc-Cp-GABA-D-(FPLIAIMA)-NH2, for binding to the Aβ plaques was designed and successfully synthesized using the Fmoc solid-phase peptide synthesis method. This radiopeptide revealed a good affinity for Aβ42 aggregations (Kd=20µM) in binding affinity study and this result was confirmed by binding to Aβ plaques in brain sections of human Alzheimer's disease (AD) and rat models using in vitro autoradiography, fluorescent staining, and planar scintigraphy. Biodistribution studies of radiopeptide in AD and normal rats demonstrated a moderate initial brain uptake about 0.38 and 0.35% (ID/g) 2min post-injection, respectively. Whereas, AD rats showed a notable retention time in the brain (0.23% ID/g at 30min) in comparison with fast clearance in normal rat brains. Normal rats following treatment with cyclosporine A as a p-glycoprotein inhibitor showed a significant increase in the radiopeptide brain accumulation compared to non-treated ones. There was a good correlation between data gathered from single-photon emission computed tomography/computed tomography (SPECT/CT) imaging and biodistribution studies. Therefore, these findings showed that this novel radiopeptide could be a potential SPECT imaging agent for early detection of Aβ plaques in the brain of patients with AD.

Self-Assembled Multinuclear Complexes Incorporating 99m Tc.

Multinuclear complexes or clusters are rarely investigated in medicinal inorganic chemistry although they represent structural intermediates between molecules and nanomaterials. We present in this report two strategies towards 99m Tc-containing clusters. In a pre-assembly approach, the preformed but incomplete cluster fragment [Re3 (μ2 -OH)3 (μ3 -OH)(CO)9 ]- reacts with [99m Tc(CO)3 ]+ to the highly stable [99m TcRe3 (μ3 -OH)4 (CO)12 ] cube. The same structure self-assembles when reacting the mononuclear Re and 99m Tc precursors in one pot. Integrating the coordinating OH groups from Schiff bases in this concept leads straight to dinuclear, mixed-metal complexes of the type [99m TcRe(μ2 -O^N-R1 )2 (CO)6 ] in quantitative yields. Both strategies are unprecedented and open a future path towards clusters, incorporating a 99m Tc radiolabel while being decorated with targeting or cytotoxic moieties.

A Solid-Phase Labeling Strategy for the Preparation of Technetium and Rhenium Bifunctional Chelate Complexes and Associated Peptide Conjugates

A new solid-phase labeling strategy for the preparation of (99m)Tc and Re chelate complexes and associated peptide derivatives, was developed. Resin-bound monoamide monoamine (MAMA) chelates were prepared in such a manner that upon the addition of a suitable Re(V) and Tc(V) precursor the target metal complexes were selectively released from the resin. The desired products were isolated from unreacted ligand by a simple filtration/solid-phase extraction procedure. In addition to the preparation of a series of functionalized ligands, a peptide conjugate was constructed from one of the resin-bound chelates using a conventional automated peptide synthesizer. The yields of the Re chelate complexes were typically greater than 70%, while the maximum yield for reactions run at the tracer level using (99m)Tc was 50%. The reported approach has a number of attractive features, including the opportunity to prepare libraries of novel agents, the ability to isolate macroscopic amounts of Re complexes for use in in vitro screening studies and as well-characterized standards for tracer level work, and the ability to produce (99m)Tc complexes that are free of any unreacted starting material without having to employ preparative HPLC.

Direct Synthesis of Tricarbonyl(cyclopentadienyl)rhenium and Tricarbonyl(cyclopentadienyl)technetium Units from Ferrocenyl Moieties − Preparation of 17α‐Ethynylestradiol Derivatives Bearing a Tricarbonyl(cyclopentadienyl)technetium Group

AbstractThe preparation of new tricarbonyl(cyclopentadienyl)rhenium and tricarbonyl(cyclopentadienyl)technetium derivatives is described. The approach used was applied to a cold target model, 17α‐ethynylestradiol substituted at the 17α position by a ketotricarbonyl(cyclopentadienyl)rhenium group. The novel organometallic reaction applied here, when extended to potential radiopharmaceuticals, consists of a cyclopentadienyl‐ligand transfer between ketoferrocenes and fac‐[99mTc(H2O)3(CO)3]+ in a water/DMSO 1:1 mixture. This reaction lead to the synthesis of a new estradiol derivative, labeled with 99mTc. The synthesis of another product was performed by using [99TcCl3(CO)3]2− as the Tc precursor and its X‐ray crystal structure was determined. In each case high yields were obtained. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

`Maturization' of high- Tc precursor powders for use in superconducting tapes

In recent years, the focus of research on high- T c superconductors has shifted towards the use of these materials in practical applications. Superconducting tapes and wires have the potential for use in power transmission. After fabrication, the tapes are subjected to mechanical and thermal treatments, causing changes in the phase assemblage to occur within. It is therefore of paramount importance to understand the behaviour of the precursor powders under different heat treatment schedules. In this paper, we present our studies on precursor powders with the nominal starting composition Bi 1.7Pb 0.4Sr 1.8Ca 2.2Cu 3.2O y synthesized by spray pyrolysis. By a combination of X-ray diffraction and magnetization studies, we have identified a `mature' heating schedule, by which we can control the Pb content in the (Bi,Pb)-2212 precursor powders, which we take as the schedule for optimum conversion to the (Bi,Pb)-2223 phase.

INITIAL IFN-γ SECRETION BY NATURAL KILLER CELL AUGMENTS T CELL IMMUNITY IN HUMAN ANTI-PORCINE XENORESPONSE.

518 The success of pig to human transplantation is hurdled by the vigorous humoral and cellular responses toward the xenograft. Extensive studies have shown that the humoral response is mainly caused by the preformed natural antibodies which recognize the α-gal epitopes on the xenograft, however, the cause of a vigorous cellular response is still unclear. In this study we demonstrated that human xenoresponse against cultured porcine endothelial cells was more vigorous than the response against human allogeneic endothelial cells (poliferation index of 63.5 vs. 28.5 in a 7 day culture). We hypothesized that this vigorous xenogeneic T cell response could be due to the higher precursor frequency of reactive T cell in xenoresponse than in alloresponse. To our surprise, limiting dilution analysis of IL-2 secreting Th cells and cytotoxic Tc cells showed a lower Th and Tc precursor frequency against the xemogeneic porcine endothelial cells (Th=1/21761, Tc=1/24347) in comparison to the allogeneic human endothelial cells (Th=1/14036, Tc=1/15665). By separating the CD3− and CD3+ cells, we demonstrate that the CD3− cell population which was mainly composed of natural killer cells secrete high amount IFN-γ in the anti-porcine xenoresponse but not in the anti-human alloresponse. This initial IFN-γ secretion augments T cell IL-2 secretion and proliferation in the xenoresponse and lead to a Th1 like T cell response against the porcine endothelial cells. These results agree with reported high frequency of the NK cell in the xenograft infiltrating population. This also provides evidence that the innate immune system could augment the T cell mediated specific immunity in xenograft rejection. In summary, human NK cells that recognize porcine endothelial cells secrete high IFN-γ upon initial contact with xenoantigens. This high initial IFN-γ secretion found in xenoresponse but not in alloresponse augments T cell immune response by recruiting and providing the necessary cytokine for T cell proliferation.

Alloreactive cytotoxic T cells recognize MHC class I antigen without peptide specificity.

In this report, experiments are described to differentiate between three potential models of class I MHC allorecognition, namely 1) recognition of peptide-free MHC, 2) peptide-MHC-specific recognition, and 3) peptide-MHC-nonspecific recognition. Using a nucleoprotein peptide (NPP) with a sequence derived from influenza virus nucleoprotein with high affinity for Kd class I MHC molecules, it is shown that target cells rapidly become lysable by Kd-NPP self-restricted cytotoxic T (Tc) cells, and retain sufficient Kd-NPP complexes for at least 72 h. Kd-specific alloreactive Tc cells at the clonal and polyclonal level do not show decreased lysis of Kd-bearing targets in the continuous long term (48 h) presence of NPP. Kd-stimulator cells modified with NPP are able to induce potent Kd-NPP-specific self-restricted Tc cells, however Kd-NPP stimulator cells do not generate Kd-NPP specific alloreactive Tc cells from CBA and B10.A (5R) mouse strains as tested by limiting dilution split clone experiments. Human cells infected with the vaccinia virus recombinant coding for the murine Kd class I MHC Ag can be lysed by murine Kd-specific alloreactive Tc cells. In addition the rate of reemergence of alloreactive and self-restricted Tc cell epitopes on virally infected target cells that had their cell-surface class I MHC Ag removed is identical. These results are consistent with model 3 namely that the majority of Tc precursor and effector cells recognize class I MHC Ag without peptide specificity.

Lipophilic technetium complexes. VI. Neutral oxotechnetium(V) complexes with monothiole/tridentate dithiole coordination

Reaction of tridentate dithiole ligands HSCH 2CH 2XCH 2CH 2SH (HSXSH, X= O, S) and monothioles RSH (thiophenoles, aliphatic thioles, mercaptoacetic acid ethylester) with Tc(V) gluconate (or other starting materials containing the oxotechnetium(V) core such as tetrachlorooxotechnetate) leads to a series of mononuclear, neutral complexes of the general formula TcO(SXS)(SR). In the absence of the monodentate thioles, dinuclear complexes of the general formula (TcO) 2(SXS) 3 are formed. Infrared, NMR, and UV-Vis data of the resulting compounds are reported. A concept for designing smallsized, neutral Tc complexes, involving the principle of common reaction of both a tridentate and a monodentate ligand with an appropriate Tc precursor, is proposed.

Varicella-zoster virus-specific cytotoxic T lymphocytes (Tc): detection and frequency analysis of HLA class I-restricted Tc in human peripheral blood.

The cytotoxic T-cell (Tc) response to varicella-zoster virus (VZV) is incompletely characterized. We investigated whether VZV-specific Tc restricted by class I products of the major histocompatibility complex can be generated from the peripheral blood of VZV-immune donors. Cell lines were established from peripheral blood lymphocytes (PBL) of seropositive donors by secondary in vitro restimulation. If cell-free VZV was used as the stimulating antigen, the resulting lines were predominantly CD4+ and did not show class I-restricted cytotoxicity; when autologous infected fibroblasts were used for in vitro stimulation, the resultant lines were usually cytotoxic, although in only 4 of 11 subjects tested was this cytotoxicity HLA restricted and virus specific. PBL were also tested for Tc activity without prior restimulation; VZV-specific Tc activity was only demonstrable in the PBL of a subject convalescent following zoster but not from subjects with recent varicella infection or from normal subjects. VZV-specific Tc precursor frequencies were then determined in six selected subjects by limiting-dilution analysis. A measurable frequency was detectable in four of the six seropositive subjects, ranging from 11/10(6) T cells in an asymptomatic carrier, to 63/10(6) T cells in a subject with recent zoster. We conclude that virus-specific major histocompatibility complex class I-restricted Tc precursors may be present in the peripheral blood of normal individuals seropositive for VZV but at a frequency lower than that for other herpesviruses with nonneuronal sites of latency.

Three‐cell‐type clusters of T cells with antigen‐presenting cells best explain the epitope linkage and noncognate requirements of the in vivo cytolytic response

Using an established i.p. adoptive transfer system in which primed precursors of cytolytic (Tc) cells are combined with primed helper (Th) cells and alloantigen, the three-cell-type hypothesis of T-T cooperation has been tested. This hypothesis assumes that cooperation takes place through the formation of clusters, consisting of one or more Tc precursors plus one or more Th cells binding to antigen on the surface of antigen-presenting cells (APC). By using antigens in which the H-2Db epitopes recognized by the Tc precursors are presented either on the same cell as the BALB minor histocompatibility epitopes recognized by the Th cells, or a different one, the value of epitope-linkage has been explored. Epitope-linkage is found to enhance the response at low concentrations of antigen but not at high ones, in accordance with the prediction that at low concentrations individual APC will usually take up either one antigen or the other but not both from an unlinked mixture, while at high concentrations most APC will take up both whether they are linked or not. In a further test of this hypothesis, no requirement for a cognate T-T interaction could be detected. With the three-cell-type interaction thus better established, its implications in terms of efficiency compared with linked-cognate interactions and its evolution are discussed.

Clonal analysis of helper and effector T-cell function in neonatal transplantation tolerance: clonal deletion of helper cells determines lack of in vitro responsiveness.

Mice rendered tolerant at birth of H-2 alloantigens display concordant in vivo and in vitro phenotypes: they fail to reject skin grafts bearing the tolerated antigens, and their lymphoid cells fail to participate in tolerogenspecific mixed lymphocyte reactions (MLRs) and cell-mediated lympholysis (CML). Tolerant animals normally reject third-party skin allografts and develop positive MLRs and CML to third-party antigens. It has been suggested that clonal deletion of antigen reactive cells is the basis for this spectrum of responses. To investigate further the basis for the lack of in vitro alloreactivity, we conducted limiting dilution studies with lymph node cells from adult mice tolerant of various H-2 disparities. When the frequencies of (a) cells responding to the tolerogen in MLR and (b) interleukin-2-producing cells against the tolerogen were determined, it appeared that both types of cells were functionally deleted, that is, the frequency of cells responding to tolerogen-bearing stimulator cells was identical with that of cells stimulated with syngeneic cells. On the assumption that cells from H-2 tolerant mice are deficient in helper cell activity toward the tolerogen, we performed CML cultures under conditions in which exogenous help was provided in the form of supernatants derived from concanavalin A stimulated rat spleen cell cultures. Lymphoid cells from H-2 tolerant mice generated significant cytotoxicity toward the tolerogen under these conditions, although the absolute level of killing was reduced compared with that of cells from normal mice. Limiting dilution assays confirmed that Tc precursors were present in tolerant mice, and that they were reduced to less than 10% of normal numbers; however, tolerogen-specific Tc precursors were present in frequencies significantly greater than self-reactive Tc precursors. These data indicate that a deletion mechanism operates in neonatal transplantation tolerance to reduce the clone size of all three categories of functional T cells assayed, but that its efficiency is greatest among cells destined to provide specific T-cell help. The absolute functional deletion of helper cells determines the in vitro CML unresponsiveness of lymphoid cells from tolerant mice, and may be a crucial factor in promoting the in vivo phenotype of skin allograft tolerance.

Human cytomegalovirus-specific cytotoxic T lymphocytes: requirements for in vitro generation and specificity.

The nature of any virus-specific T cells involved in controlling human cytomegalovirus (HCMV) infection in normal subjects harboring latent virus is unknown. As an approach to this problem, peripheral blood mononuclear cells (PBM) from normal seropositive subjects were cocultured with HCMV and responding T cells expanded in interleukin 2 (IL2)-dependent culture, determining in particular whether HCMV-specific cytotoxic T cells (Tc) were generated. Coculture of PBM with free HCMV resulted in the generation of short-term T cell lines of predominantly helper phenotype (Leu 3a+), expressing no cytotoxicity. However, when PBM were cocultured on HCMV-infected fibroblasts (autologous to the donor in these experiments) predominantly Leu 2a+ lines were generated, which lysed HCMV-infected cells. The cytotoxicity of these short-term IL 2-dependent lines was HCMV-specific and human HLA-restricted; HCMV-infected target cells expressing only early viral antigens were lysed. It is concluded that HCMV-specific Tc precursors are present in peripheral blood of latently infected individuals without preceding overt infection and that effector Tc may be capable of lysing infected cells prior to viral replication.

Selective suppression of the cytotoxic T cell response to influenza virus in mice.

Mice injected with inactivated (UV light-irradiated) influenza virus produce specific antibody, become sensitized for a delayed-type hypersensitivity reaction, but do not generate specific cytotoxic T (Tc) cells. If injected 4-5 days later with infectious virus, the formation of Tc cells is suppressed by > 90%. If A strain viruses are used, the suppression observed is cross-reactive within A strain viruses but does not extend to B/LEE or to Sendai virus. Serum from mice injected with UV-irradiated virus contains antibodies which on adoptive transfer can inhibit Tc cell formation when infectious homologous virus is used to challenge the recipients. Spleen cells from the same mice, upon adoptive transfer, also inhibit (50-70%) Tc cell formation if transferred within 24 h of injection of infectious virus, and the specificity pattern observed is cross-reactive within A strains. The activity of the cells mediating suppression is destroyed by monospecific anti-Thy-1.2 antibody and complement. The immune cells require I region sharing between donor and recipient mice for their suppressor activity to be effective. (There is also a partial requirement for K, D region sharing, but the possible rejection of transferred cells is not excluded.) Dilution assays in which clonal expansion of Tc precursors is used to estimate their frequency and the presence of T helper (Th) cells indicate that suppressed mice possess Tc precursors and primed cells which, upon restimulation, act as Th cells. Furthermore, injection of irradiated Th cells with inactivated virus does not significantly reduce the ensuing suppression.

Specific depletion of alloreactive cytotoxic lymphocyte precursors

THE major barrier to replacement therapy in various clinical immunodeficiency states is the likelihood that the transferred immunocompetent T lymphocytes would react against alloantigens of the recipient and produce graft-versus-host (GVH) disease1. The logical resolution of this problem would be to delete or inactivate those clones of T cells which are specifically coded to react against the histocompatibility antigens of the recipient tissues. The remaining cell population could then fully reconstitute the recipient without danger of GVH reactions. Specific clonal deletion of this kind has been obtained in experimental animal situations by pre-circulation through a third-party host2,3 or by induction of specific anti-idiotype immunity4. These studies show the validity of this approach but are technically complicated and have limited application. A simpler method of obtaining this result has been sought based on the following strategy. To reconstitute a strain A mouse by transfer of lymphocytes from an allogeneic strain B, the strain B lymphocytes are first incubated on a monolayer of cells of strain A. Those clones which recognise the strain A alloantigens should adhere to the monolayer; the non-adherent populations of cells should now be unable to react against alloantigens of strain A but should still respond to alloantigens of other haplotypes. Although this kind of technique has been widely effective in removing fully activated cytotoxic lymphocytes (Tc)5–9, its use to deplete alloreactive Tc precursors has given variable results8,10–16. Significant depletion of precursors has been reported primarily when the non-adherent cells have been assayed in vivo13–16, but this has been claimed to be due to change in the responder cells, not depletion, as the same population assayed by in vitro generation of Tc shows no evidence of depletion16. We present here a simple depletion method developed from our finding that when splenic lymphocytes are incubated for a few hours before being formed into a monolayer, they can effectively absorb alloreactive Tc precursors even when assayed by the sensitive in vitro assays.

Different rules govern help for cytotoxic T cells and B cells.

RECENT experiments with radiation chimaeras1,2 indicate that T cells differentiating in an H–2D compatible, but H–2I different, thymic environment are completely unresponsive to vaccinia virus. The explanation favoured2 for this surprising finding is that participation of helper T cells3 (TH) is an absolute requirement for the generation of virus-immune cytotoxic T cells (Tc). No response is seen because the TH populations are ‘locked in’ to recognising the spectrum of H–2I antigens presented on radiation-resistant thymic cells2. They cannot, therefore, interact with the Tc precursors which are genetically different in the I region. The result is a total failure of responsiveness. We report here that we have tested this concept further by stimulating acutely tolerised4,5 T-cell populations in H–2I different, but H–2D compatible virus-infected recipients. Our results indicate that there is apparently no need for participation of TH populations restricted to the H–2I region specificities expressed in the recipient environment. This differs from the situation for TH–B-cell collaboration4,5, and suggests that help for the Tc response operates directly between T-cell subsets.