Nanoscale pattern formation on silicon surfaces bombarded with a krypton ion beam: experiments and simulations.

The nanoscale patterns produced by bombardment of the (100) surface of silicon with a 2 keV Kr ion beam are investigated both experimentally and theoretically. In our experiments, we find that the patterns observed at high ion fluences depend sensitively on the angle of incidence Θ. For Θ values between 74° and 85°, we observe five decidedly different kinds of morphologies, including triangular nanostructures traversed by parallel-mode ripples, long parallel ridges decorated by short-wavelength ripples, and a remarkable mesh-like morphology. In contrast, only parallel-mode ripples are present for low ion fluences except for Θ = 85°. Our simulations show that triangular nanostructures that closely resemble those in our experiments emerge if a linearly dispersive term and a conserved Kuramoto-Sivashinsky nonlinearity are appended to the usual equation of motion. We find ridges traversed by ripples, on the other hand, in simulations of the Harrison-Pearson-Bradley equation (Harrisonet al2017Phys. Rev.E96032804). For Θ = 85°, the solid surface is apparently stable and simulations of an anisotropic Edwards-Wilkinson equation yield surfaces similar to those seen in our experiments. Explaining the other two kinds of patterns we find in our experiments remains a challenge for future theoretical work.

Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning.

Donor-specific tolerance induced by bone marrow transplantation (BMT) would allow organ allografting without chronic immunosuppressive therapy. However, the toxicity of conditioning regimens used to achieve marrow engraftment has precluded the clinical use of BMT for tolerance induction. We have developed a BMT strategy that achieves alloengraftment without toxic or myelosuppressive host conditioning. B6 mice received depleting anti-CD4 and anti-CD8 monoclonal antibodies, local thymic irradiation, and a high-dose (174 x 10(6)) of major histocompatibility (MHC)-mismatched B10.A bone marrow cells (BMCs) divided over days 0 through 4. High levels of donor cells were observed among white blood cells (WBCs) of all lineages. Permanent, multilineage mixed chimerism; donor-specific skin-graft tolerance; and in vitro tolerance were observed in most animals. Large numbers of donor class II(high) cells were detected in thymuses of long-term chimeras, and their presence was associated with intrathymic deletion of donor-reactive host thymocytes. The treatment was not associated with significant myelosuppression, toxicity, or graft-versus-host disease (GVHD). Thus, high levels of allogeneic stem-cell engraftment can be achieved without myelosuppressive host conditioning. As stem-cell mobilization and in vitro culture techniques have increased the feasibility of administering high doses of hematopoietic cells to humans, this approach brings hematopoietic cell transplantation closer to clinical use for the induction of central deletional T-cell tolerance.

Hematopoietic cells and radioresistant host elements influence natural killer cell differentiation.

Radioresistant host elements mediate positive selection of developing thymocytes, whereas bone marrow-derived cells induce clonal deletion of T cells with receptors that are strongly autoreactive. In contrast to T cell development, little is known about the elements governing the natural killer (NK) cell repertoire, which, similar to the T cell repertoire, differs between individuals bearing different major histocompatibility complex (MHC) phenotypes. We have used murine bone marrow transplantation models to analyze the influence of donor and host MHC on an NK cell subset. We examined the expression of Ly-49, which is strongly expressed on a subpopulation of NK cells of H-2b mice, but not by NK cells of H-2a mice, probably because of a negative effect induced by the interaction of Ly-49 with Dd. To evaluate the effect of hematopoietic cell H-2a expression on Ly-49 expression of H-2b NK cells, we prepared mixed allogeneic chimeras by administering T cell-depleted allogeneic (B10.A, H-2a) and host-type (B10, H-2b) marrow to lethally irradiated B10 mice, or by administering B10. A marrow to B10 recipients conditioned by a nonmyeloablative regimen. Expression of H-2a on bone marrow-derived cells was sufficient to downregulate Ly-49 expression on both H-2a and H-2b NK cells. This downregulation was thymus independent. To examine the effect of H-2a expressed only on radioresistant host elements, we prepared fully allogeneic chimeras by administering B10 bone marrow to lethally irradiated B10.A recipients. B10 NK cells of these fully allogeneic chimeras also showed downregulation of Ly-49 expression. The lower level of H-2a expressed on H-2b x H-2a F1 cells induced more marked downregulation of Ly-49 expression on B10 NK cells when presented on donor marrow in mixed chimeras than when expressed only on radioresistant host cells. Our studies show that differentiation of NK cells is determined by interactions with MHC molecules expressed on bone marrow-derived cells and, to a lesser extent, by MHC antigens expressed on radioresistant host elements.

Donor-derived interferon gamma is required for inhibition of acute graft-versus-host disease by interleukin 12.

We have demonstrated that a single injection of interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits acute graft-versus-host disease (GVHD) in mice. This effect of IL-12 can be diminished by anti-interferon (IFN)-gamma mAb. To determine the mechanism by which IFN-gamma affects IL-12-mediated GVHD protection, we have compared the effect of IL-12 on GVHD in C57BL/6 wild-type (WT) or IFN-gamma gene knockout (GKO) recipients of fully major histocompatibility complex plus minor antigen-mismatched allogeneic BMT from WT or GKO BALB/c mice. Lethal acute GVHD was readily induced in the absence of IFN-gamma. IL-12 inhibited GVHD mortality to a similar extent in WT and GKO recipients of WT allogeneic BMT. However, neither WT nor GKO recipients were protected by IL-12 from GVHD induced by GKO allogeneic BMT. Moreover, the effective inhibition of host-reactive donor T cell activation and expansion that is associated with IL-12-mediated GVHD protection was dependent on the ability of BALB/c donors to produce IFN-gamma. These results demonstrate that (a) acute GVHD can be induced in the absence of IFN-gamma, (b) host IFN-gamma does not play a critical role in IL-12-induced GVHD protection, and (c) the protective effect of IL-12 against GVHD is dependent on the ability of the donor to produce IFN-gamma.

Separate regulation of peripheral hematopoietic and thymic engraftment.

Long-term multilineage chimerism, indicating pluripotent hematopoietic stem cell engraftment, was achieved in an Ly5-congenic strain combination without irradiation or other host conditioning when a large number of donor marrow cells (1.4-2x10(8)) was administered. However, the initial (2-4 weeks posttransplantation) percentages of T and B lymphocytes of donor origin were markedly lower than those of myeloid lineages. Steady-state levels of donor and host repopulation of all lineages were reached by 7 to 15 weeks posttransplantation and remained relatively constant for at least 41 weeks. B cell chimerism was similar to that seen in myeloid lineages at steady state. In contrast, long-term donor representation in the T cell lineage was much lower than in the B cell or myeloid lineages. Host treatment with depleting anti-T cell monoclonal antibodies increased the donor contribution to early T cell repopulation, but long-term T cell chimerism was still significantly lower in all lymphohematopoietic tissues, including the thymus, than B cell or myeloid cell chimerism. Pretreatment of hosts with 3.5 Gy of local irradiation to the thymic region further increased the donor contribution to initial T cell repopulation, which equaled that of other lineages at 4 to 7 weeks. However, donor representation in the T cell lineage declined by the time steady-state chimerism was attained and was lower than donor representation in the B cell or myeloid lineages. A higher dose of thymic irradiation (7 Gy) led to a reduction in this discrepancy, so that levels of donor thymopoiesis and hematopoiesis in other lineages were similar by 23 to 27 weeks posttransplantation. The differential contribution of adult donor marrow to long-term, steady-state thymopoiesis vs. hematopoiesis in other lineages under certain conditions in this competitive repopulation assay suggests that functionally distinguishable progenitors are responsible for these activities.

Cocoa inhibits platelet activation and function.

BACKGROUND: Epidemiologic studies have shown inverse associations between dietary polyphenols and mortality from coronary heart disease. However, the basis for this protective association is uncertain. Food polyphenols reportedly have antioxidant properties and decrease platelet function in vitro. OBJECTIVE: This study sought to evaluate whether consumption of a polyphenol-rich cocoa beverage modulates human platelet activation and primary hemostasis. DESIGN: Peripheral blood was obtained from 30 healthy subjects before and 2 and 6 h after ingestion of a cocoa beverage (n = 10), a caffeine-containing control beverage (n = 10), or water (n = 10). Platelet activation was measured in terms of expression of activation-dependent platelet antigens and platelet microparticle formation by using fluorescent-labeled monoclonal antibodies and flow cytometry. Primary platelet-related hemostasis was measured with a platelet function analyzer. RESULTS: Ex vivo epinephrine- or ADP-stimulated expression of the fibrinogen-binding conformation of glycoprotein IIb-IIIa was lower 2 and 6 h after consumption of cocoa than before consumption. Cocoa consumption also decreased ADP-stimulated P-selectin expression. In contrast, epinephrine-induced platelet glycoprotein IIb-IIIa expression increased after consumption of the caffeine-containing beverage but not after water consumption. Platelet microparticle formation decreased 2 and 6 h after cocoa consumption but increased after caffeine and water consumption. Primary hemostasis in response to epinephrine in vitro was inhibited 6 h after cocoa consumption. The caffeine-containing beverage inhibited ADP-induced primary hemostasis 2 and 6 h after consumption. CONCLUSIONS: Cocoa consumption suppressed ADP- or epinephrine-stimulated platelet activation and platelet microparticle formation. Cocoa consumption had an aspirin-like effect on primary hemostasis.

Synthesis and bacterial metabolism of cis- and trans-2-alkyl analogues of sodium cyclamate.

Sodium cyclamate is an effective artificial sweetner, which has been banned from the U.SD. market because of alleged carcinogenic properties. It appears that cyclohexylamine, liberated from cyclamate as a result of bacterial mtabolism, is the proximate carcinogen. In an effort to elucidate the extent to which analogues of cyclamate would enter into the bacterial metabolic pathway, as well as any stereochemical requirements which might exist, several 2-alkaly analogues of sodium cyclamate were prepared. It was found that trans-N-(2-methylcyclohexyl)sulfamate (trans-2a) and trans-N-(2-ethylcyclohexyl)sulfamate were hydrolyzed by freshly collected fecal suspensions from rats fed cyclamate, but not from control rats, at the same rate as cyclamate itself. trans-N-(2-Isopropylcyclohexyl)sulfamate (trans-2c) was not hydrolyzed at all. Surprisingly, two of the analogous cis compounds (cis-2a and cis-2c, respectively) were hydrolyzed by fecal suspensions from control, as well as from cyclamate-fed, rats. Moreover, cis-2a was hydrolyzed by incubating it in medium only. Thus, it is apparent that stereochemical influences on the chemical properties of these compounds are substantial. These results do not appear to point the way toward a safe, nonmetabolizable sweetening agent.

Thrombus imaging using technetium-99m-labeled high-potency GPIIb/IIIa receptor antagonists. Chemistry and initial biological studies.

Platelet-specific compounds which are radiolabeled with gamma-emitting radionuclides may be particularly useful for the noninvasive in vivo detection of thrombi. The synthesis of peptides which are potent inhibitors of platelet aggregation and which contain a chelator for the radionuclide technetium-99m are described. The target compounds were designed such that stable, oxotechnetium(V) species could be prepared where the site of metal coordination was well defined. A strategy was employed where the pharmacophore-Arg-Gly-Asp-(RGD), or RGD mimetic, was constrained in a ring which was formed by the S-alkylation of a cysteine residue with an N-terminal chloroacetyl group. Binding affinities were enhanced by the replacement of arginine with the arginine mimetics S-(3-aminopropyl)cysteine and 4-amidinophenylalanine. Further enhancements could be obtained by the synthesis of oligomers which contained two or more rings containing receptor binding regions. The increase in binding affinity seen was more than that expected from a simple stoichiometric increase of pharmacophore. The most potent compounds described had IC50s of approximately 0.03 microM for the inhibition of human platelet aggregation. Two of the more potent peptides (P280 and P748) were labeled with technetium-99m and assessed in a canine thrombosis model. The 99m Tc complexes of the peptides prepared in this work hold promise as thrombus imaging agents due to their high receptor binding affinity, ease of preparation, and expected rapid pharmacokinetics.

Somatostatin receptor-binding peptides labeled with technetium-99m: chemistry and initial biological studies.

The synthesis of peptides which possess a high affinity for the somatostatin receptor and contain a chelator for the radionuclide technetium-99m is described. The target compounds were designed such that they would form stable, oxotechnetium(V) chelate complexes in which the Oxorhenium(V) chelate complexes of these peptides were prepared as nonradioactive surrogates for the technetium complexes. Peptide oxorhenium complexes and Tc-99m complexes eluted closely upon HPLC analysis. The receptor-binding affinities of both the free and rhenium-coordinated species were measured in vitro. The binding affinities of the free peptides (Ki's in the 0.25 - 10 nM range) compared favorably with [DTPA]octreotide (Ki = 1.6 nM), which, as the indium-111 complex, is already approved for somatostatin receptor (SSTR)-expressing tumor imaging in the United States and Europe. Furthermore, the rhenium-coordinated peptides had binding affinities which, in many cases, were higher than those of the corresponding free peptides, with several complexes having a Ki's of 0.1 nM. Some of the more potent SSTR-binding peptides were labeled with technetium-99m and assessed in an in vivo study with tumor-bearing rats. The 99m Tc-labeled peptides prepared in this study should be useful as SSTR-expressing tumor-imaging agents due to their high SSTR-binding affinities, ease of preparation, and, because they are low molecular weight peptides, expected pharmacokinetics characterized by rapid tracer excretion from the body resulting in high-contrast images.

IL-2 inhibits early increases in serum gamma interferon levels associated with graft-versus-host-disease.

We have recently demonstrated that a short course of high-dose IL-2 administered to lethally irradiated mice leads to marked protection from early and late GVHD mortality, especially when T cell-depleted (TCD) host-type bone marrow cells (BMC) are also administered. IL-2 inhibits the GVHD-inducing activity of donor CD4+ cells without inhibiting their graft-vs.-leukemia effects. Since CD4+ T-lymphocytes produce a variety of cytokines, some of which have recently been implicated in the pathogenesis of GVHD, we have studied the possible effect of IL-2 administration on serum levels of various cytokines. Acute GVHD was induced in lethally irradiated B10 mice by bone marrow transplantation (BMT) with MHC-mismatched allogeneic (A/J) BMC and splenocytes. TCD B10 (host-type) BMC were coadministered to maximize the protective effect of IL-2. Serum cytokine levels were compared in recipients of these inocula with or without a protective course of IL-2 treatment. A marked increase in serum IFN-gamma levels was noted on days 3 through 5 post-BMT in GVHD mice compared with syngeneic BMT control recipients. This GVHD-induced rise in serum IFN-gamma was markedly inhibited in IL-2-protected mice. Murine IL-2 levels were only slightly increased in sera of GVHD mice, and were not influenced by treatment with human recombinant IL-2. Serum levels of the monokines TNF-alpha and IL-1 alpha showed variable early elevations in GVHD mice with or without IL-2 treatment, and were not different from levels observed in syngeneic controls. Serum levels of IFN-gamma, IL-1 alpha, and TNF-alpha all declined markedly by day 7 to 8 post-BMT, when GVHD mortality begins. Administration of neutralizing anti-IFN-gamma mAb did not attenuate and tended to accelerate GVHD mortality, and administration of exogenous IFN-gamma did not overcome the protective effect of IL-2 against GVHD. Together, our results indicate that GVHD is associated with high serum levels of several proinflammatory cytokines in the first week post-BMT, but that these levels decline by the time when GVHD mortality begins. IL-2 specifically inhibits the GVHD-associated production of IFN-gamma, but this inhibition in itself does not explain and may even mitigate the protective effect of IL-2 against early GVHD mortality. However, the demonstration that IL-2 markedly inhibits the production of a GVHD-associated cytokine raises the possibility that alterations in the production of as yet undefined cytokines may be responsible for IL-2-induced GVHD protection.

Discordant xenogeneic neonatal thymic transplantation can induce donor-specific tolerance.

The limited supply of human organs for transplantation necessitates the development of methods leading to acceptance of xenografts. To avoid the hazards of the high-dose chronic immunosuppressive pharmacotherapy which would otherwise be required for successful xenografting, it would be desirable to induce permanent tolerance to xenogeneic donors. We have recently demonstrated that xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymic and hematopoietic tissue into thymectomized, T cell-depleted, and natural killer-cell-depleted mice, or into natural killer cell-depleted nude mice. We have now extended these studies by replacing fetal tissue with neonatal pig thymic and hematopoietic tissue, and by examining the in vivo responses of reconstituted mice to pig skin grafts. Neonatal tissue was studied because it might be more practicable than fetal tissue for the purpose of transplantation to primates. BALB/c nu/nu mice transplanted with neonatal (<24-hr-old) pig thymus and spleen fragments developed circulating mouse CD4+ cells. The pig thymus grafts were necessary for mouse T-cell development, as CD4 recovery did not occur in recipients of neonatal pig splenic tissue alone. The CD4+ cells that developed included Vbeta8.1/2+ T cells in similar proportions as in BALB/c mice, and Vbeta11+ and Vbeta5+ CD4 T cells were deleted almost as completely as in normal BALB/c mice. This deletion was detected among CD4 single-positive graft thymocytes. In 9 of 12 evaluable animals, mixed lymphocyte responses demonstrated tolerance to donor-type pig SLA antigens, with responsiveness to alloantigens and/or third-party pig xenoantigens. Furthermore, grafting of neonatal pig thymus conferred the ability to reject allogeneic mouse skin in 7 of 10 animals. In addition, 7 of 10 animals accepted paternal (donor SLA-matched) skin (median survival time [MST] > 100 days), whereas 4 of 4 animals rejected third-party SLA-mismatched pig skin (MST=40.5 days). We conclude that neonatal pig thymi transplanted to BALB/c nu/nu mice can support the development of mouse CD4+ cells that are functional and specifically tolerant to donor-type pig antigens.

Role of intrathymic rat class II+ cells in maintaining deletional tolerance in xenogeneic rat-->mouse bone marrow chimeras.

BACKGROUND: Mixed xenogeneic bone marrow chimerism and tolerance can be induced in mice conditioned with a nonmyeloablative regimen followed by injection of T cell-depleted rat bone marrow cells. We hypothesized that, despite a gradual decline in rat hematopoiesis observed in these chimeras, as long as rat class II+ antigen-presenting cells remain in their thymi, tolerance will persist as a result of deletion of donor-reactive thymocytes. METHODS: The level of chimerism and of mouse Vbeta5 and Vbeta11 T-cell deletion was followed over time. These results were correlated with the presence of rat class II+ cells in the thymus by immunohistochemistry and the presence of tolerance in long-term chimeras by in vivo and in vitro assays. RESULTS: (1) Proliferation and cytotoxicity assays, as well as skin graft survival, demonstrated the presence of specific tolerance to host and to donor rat, with normal reactivity to third-party rat and mouse stimulators, even as late as 85 weeks after bone marrow transplantation. (2) The absence of mature Vbeta5+ and Vbeta11+ host T cells in the thymus and periphery was always associated with the presence of rat class II+ cells in the thymus, and incomplete deletion of T cells expressing these Vbeta families was observed in thymi in which rat class II+ cells were not detectable. CONCLUSIONS: Donor-specific T-cell tolerance is maintained during the period when donor-type reconstitution declines, and is most likely mediated by intrathymic clonal deletion of T cells that recognize antigens expressed on class II+ rat cells.

Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen.

BACKGROUND: Mixed hematopoietic chimerism induced with a nonmyeloablative conditioning regimen leads to donor-specific transplantation tolerance. Analyses of specific Vbeta-bearing T-cell families that recognize endogenous superantigens demonstrated that donor-specific tolerance is due mainly to an intrathymic deletional mechanism in these mixed chimeras. However, superantigens are not known to behave as classical transplantation antigens. We therefore used T-cell receptor (TCR) transgenic (Tg) recipients expressing a clonotypic TCR specific for an allogeneic major histocompatibility complex antigen to further assess deletional tolerance. METHODS: 2C TCR Tg mice (H2b), whose Tg TCR recognizes major histocompatibility complex class I Ld, were used as recipients of Ld+ bone marrow cells after conditioning with depleting anti-CD4 and CD8 monoclonal antibodies, 3 Gy whole-body irradiation, and 7 Gy thymic irradiation. Chimerism and deletion of CD8+ 2C recipient T cells was evaluated by flow cytometry and by immunohistochemical staining. Tolerance was tested with in vitro cell-mediated lympholysis assays and in vivo by grafting with donor skin. RESULTS: Intrathymic and peripheral deletion of 2C+ CD8-single-positive T cells was evident in mixed chimeras, and deletion correlated with the presence of donor-type cells with dendritic morphology in the thymus, and with chimerism in lymphohematopoietic tissues. Chimeras showed tolerance to the donor in cell-mediated lympholysis assays and specifically accepted donor skin grafts. CONCLUSIONS: Tolerance to transplantation antigens is achieved through intrathymic deletion of donor-reactive T cells in mixed chimeras prepared with a nonmyeloablative conditioning regimen and allogeneic bone marrow transplantation.

Inhibition of graft-versus-host disease by interleukin-2 treatment is associated with altered cytokine production by expanded graft-versus-host-reactive CD4+ helper cells.

In a fully MHC plus multiple minor antigen-mismatched murine bone marrow transplantation (BMT) model, we have demonstrated that a short course of high dose IL-2, begun on the day of BMT, protects against graft-versus-host disease (GVHD). This inhibitory effect is directed against donor CD4+ cells. To determine whether the mechanism of IL-2-induced GVHD protection involves clonal deletion or anergy of host-reactive donor T helper cells (Th), we performed limiting dilution analyses to measure the frequency of activated Th that reacted to donor, host, and third-party antigens in GVHD control and IL-2-protected mice. Marked and specific expansion of host-reactive Th was observed to a similar extent in GVHD control and IL-2-protected mice by day 5 after BMT, and the number of these cells in the spleen increased by several orders of magnitude between days 3 and 5 after BMT, which suggests that recirculation from other tissues occurred in this period. A high proportion (approximately 80%) of donor T cells expressed CD25 in both GVHD control and IL-2-protected mice on day 4 after BMT, which suggests a high level of bystander T cell activation. Since marked quantitative differences in the GVH response were not observed between GVHD control and IL-2-protected mice, we assessed both groups for qualitative differences in the Th response. Spleen cells isolated in the first 8 days after BMT were cultured with host-type, donor-type, or third-party stimulators or without stimulators, and cytokines were measured in supernatants harvested at 24 hr. GVHD was associated with marked increases in supernatant IFN-gamma levels from day 3 to day 6 after BMT, and with increases in IL-2 levels compared with naive A/J controls or syngeneic BMT controls stimulated with host antigens. Production of these cytokines was specifically induced by host-type antigens. Supernatants from spleens of IL-2-treated mice showed delayed kinetics of IFN-gamma production, and tended to contain higher levels of IL-4 in response to host antigen compared with GVHD controls on days 2 and 4 after BMT. Both IL-4 and IFN-gamma were produced almost exclusively by CD4+ cells in spleens of GVHD control and IL-2-protected mice on day 4. However, no consistent difference was observed between the groups in supernatant IL-2 or IL-10 levels, ruling out a simple Th1 to Th2 switch.(ABSTRACT TRUNCATED AT 400 WORDS)

Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance.

BACKGROUND: Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them. METHODS: C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells. RESULTS: Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance. CONCLUSIONS: One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.

Specific prolongation of skin graft survival following retroviral transduction of bone marrow with an allogeneic major histocompatibility complex gene.

Engrafted allogeneic hematopoietic cells have a unique capacity to induce a state of donor-specific transplantation tolerance across major histocompatibility complex barriers. This state allows permanent acceptance of donor-type organ grafts, with otherwise normal immunocompetence. We hypothesized that introduction of allogeneic MHC genes into autologous bone marrow which is then returned to recipient mice might similarly induce specific tolerance to products of the introduced MHC genes, without the risk of graft-vs-host disease. We demonstrate here that the introduction of MHC class I Kb cDNA by retrovirus-mediated gene transfer into B10.AKM (Kk) hematopoietic cells confers specific hyporesponsiveness to allogeneic skin grafts expressing Kb.

Pre-clinical evaluation of technetium-99m platelet receptor-binding peptide.

UNLABELLED: P748 is a dimeric peptide which incorporates two high affinity GPIIb/IIIa receptor-binding domains and a novel 99mTc binding sequence, which provides the platelet imaging agent 99mTc-P748. The aim of this study was to evaluate 99mTc-P748 preclinically for use as a hot spot scintigraphic thrombus imaging agent. METHODS: Technetium-99m-P748 was prepared by either a ligand exchange or a one-vial kit. The oxorhenium congener, [ReO]P748, was prepared by ligand exchange from Bu4NReOBr4. The binding of P748 peptide and [ReO]P748 to GPIIb/IIIa receptors on activated platelets was assessed by their inhibition of ADP stimulated human platelet aggregation in platelet rich plasma (PRP). The localization of 99mTc-P748 in deep vein and pulmonary thrombi was assessed in a canine thrombosis model and the biodistribution of 99mTc-P748 was determined in rats. RESULTS: P748 peptide inhibited the aggregation of human platelets in PRP by 50% at a concentration (IC50) of 28 nM and [ReO]P748 had an IC50 of 36 nM showing the high in vitro receptor binding affinity of both the peptide and its rhenium complex (and by analogy its technetium complex). Technetium-99m-P748 was readily prepared at room temperature in 15 min in > or = 90% radiochemical yield and purity and provided definitive images of femoral vein thrombi within 20 min and pulmonary thrombi, within 1 hr in the canine model. Femoral vein thrombus-to-blood and thrombus-to-muscle ratios at 4 hr averaged 6.7 and 46, respectively. Pulmonary thrombus-to-blood and thrombus-to-normal lung ratios at 4 hr averaged 29 and 27, respectively. Dog and rat studies both showed rapid clearance of the radiotracer from the blood and with no significant hepatobiliary excretion but with notable early kidney retention. CONCLUSION: The combination of high in vitro receptor-binding affinity, high thrombus uptake and definitive in vivo images of both femoral vein and pulmonary thrombi show that 99mTc-P748 has considerable potential as a clinical imaging agent for the detection of venous thromboembolism.

Alloengraftment in IL-2-treated mice.

Despite major advances, graft-versus-host disease (GVHD) remains a major obstacle to clinical bone marrow transplantation. Prophylaxis by T cell depletion is associated with increased rates of engraftment failure and leukemic relapse. Treatment with high-dose IL-2 can markedly protect lethally irradiated mice from GVHD-related mortality, especially when T cell-depleted (TCD) syngeneic bone marrow cells (BMC) are co-administered. In these IL-2-protected animals, allogeneic reconstitution is observed, and a graft-versus-leukemia effect of allogeneic T cells is preserved. To determine whether IL-2 might increase alloresistance under conditions in which alloengraftment is more difficult to achieve, we have now evaluated the possible effect of IL-2 on: (1) competitive repopulation of lethally irradiated mice by mixtures of TCD allogeneic and TCD syngeneic BMC; (2) radiation protection by TCD allogeneic BMC; (3) timing of hematologic recovery; and (4) allogeneic engraftment in sublethally irradiated recipients. The results show that IL-2 has only a limited and strain-restricted effect on alloengraftment. This effect may reflect activation of alloresistant host natural killer cells, a cell population which is not essential for the protective effect of IL-2 against GVHD.

IL-2-induced GVHD protection is not inhibited by cyclosporine and is maximal when IL-2 is given over a 25 h period beginning on the day following bone marrow transplantation.

We have recently demonstrated, in a fully MHC-mis-matched murine bone marrow transplantation (BMT) model, that administration of a short course of high-dose interleukin 2 (IL-2) markedly delays the onset of graft-versus-host disease (GVHD) without compromising alloengraftment or the graft-versus-leukemia (GVL) effect of allogeneic T cells. Early timing of IL-2 administration and high dose were shown to be critical to achieve this protective effect. Although a 2.5 day course of IL-2, begun on the day of BMT, was found to confer marked protection without observable toxicity, shorter courses and a higher dose of IL-2 than 5 x 10(4) Cetus units per treatment have not been previously evaluated in this model. We now demonstrate that administration of a three-treatment course of IL-2 over a 25 h period beginning 15 h following BMT is sufficient to provide maximal GVHD protection, that increasing the IL-2 dose beyond 5 x 10(4) units per treatment does not further improve the level of GVHD protection, and that further division of IL-2 treatments to achieve more constant tissue levels does not result in improved GVHD protection. We also demonstrate that IL-2 is still protective when administered in combination with cyclosporine. These results suggest that IL-2 administered in a sufficient short course to avoid toxicity might have the potential to achieve effective GVHD prophylaxis in humans, even if given in combination with cyclosporine.

Infection control practitioners and committees in skilled nursing facilities in Connecticut.

All skilled nursing facilities (SNFs) in Connecticut were surveyed and more than 71% responded to a Centers for Disease Control-funded project, a component of which is reported herein. The study describes the infection control practitioner (ICP), assistance provided ICPs from external sources, and infection control committees. Almost all ICPs received some training in infection control and worked in the field for an average of 3 1/2 years. Both the number of hours devoted to infection control and the percentage of time spent by the ICP on infection control activities increased with the size of the facility. More than one half of the ICPs in SNFs have relationships with hospital ICPs. The majority of SNF infection control committees met quarterly. The chairperson most often was a physician, although ICPs held this office in almost one third of the reporting SNFs. We conclude that ICPs in Connecticut SNFs have increased in number and that they devote more time and effort to infection control than in previous years.