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.

An fMRI study of executive functioning after severe diffuse TBI.

PRIMARY OBJECTIVE: Preliminary study of whether severe diffuse traumatic brain injury (TBI) increases extent of frontal tissue recruited by cognitive control tasks. RESEARCH DESIGN: Functional magnetic resonance imaging (fMRI) on N-back working memory (WM)and arrows inhibition tasks in a 46 year old man who had severe diffuse TBI 1 year earlier, a 44 year old man (inhibition task) and three women (working memory task), age 20-26 years. Images were acquired by 1.5 T magnet with BOLD method and PRESTO pulse sequence and analysed using SPM. MAIN OUTCOMES AND RESULTS: Frontal activation increased under 2-back relative to 1-back condition of working memory in all participants with more extensive activation in the TBI patient relative to controls. Frontal activation increased with inhibition on the arrows task, but was greater in the TBI patient. CONCLUSION: Severe diffuse TBI results in recruitment of additional neural resources for cognitive control.

Judgments of social appropriateness by children and adolescents with autism.

Children and adolescents with autism (autism group, n = 19) and those without autism (Nonautism group, n = 19) of similar age and IQ were asked to make judgments of the social appropriateness of 24 videotaped, staged scenes with adult actors. Each scene depicted an appropriate or an inappropriate interaction. Half contained verbalizations, and half did not. After each scene, the participant was asked: (1) Was that o.k. or was something wrong with it? If the participant judged the scene was wrong, she or he was asked: (2) What was wrong with it?; and (3) Why was that wrong? Both groups correctly identified inappropriate behaviors most of the time, and correct behaviors almost all of the time. However, the Nonautism group detected inappropriate behaviors significantly more often than the Autism group, for verbal but not nonverbal scenes. It was also significantly easier for both groups to identify inappropriate behaviors in the nonverbal than in the verbal scenes. Ratings of the explanations given for Question 3 differed significantly between the groups for verbal but not for nonverbal scenes, with Nonautism participants more likely to give explanations involving social norms and principles, and the Autism group more likely to give explanations that were irrelevant or idiosyncratic.

The brief psychiatric rating scale for children (BPRS-C): validity and reliability of an anchored version.

OBJECTIVE: Because the accuracy of problems reported by referred children may be compromised by their academic, cognitive, or motivational limitations, clinician rating forms may contribute to the accurate assessment of youth adjustment. One such measure, the 21-item Brief Psychiatric Rating Scale for Children (BPRS-C), received psychometric study to estimate its potential contribution to the measurement of symptom dimensions. BPRS-C reliability and concurrent validity were calculated for youths who were receiving psychiatric services within a medical school department. METHOD: Five hundred forty-seven children aged 3 to 18 years were rated by faculty or trainees; a subsample of 90 was concurrently rated by two observers. BPRS-C psychometric performance was demonstrated through interrater agreement, factor analysis, and multivariate analyses of variance across seven diagnosis-based groups. RESULTS: Although items and scales demonstrated substantial reliability and concurrent validity, item factor analysis revealed a few apparent errors in item-to-scale assignment. These errors were minimized by the use of three new second-order factor-derived scales: Internalization, Developmental Maladjustment, and Externalization. CONCLUSIONS: The BPRS-C can be easily integrated into academic clinical practice and is a reliable and valid method of child description. Additional study of three new BPRS-C factor scales and the application of the BPRS-C to the quantification of clinician observation of child symptomatic status are warranted.

Cocoa and wine polyphenols modulate platelet activation and function.

There is speculation that dietary polyphenols can provide cardioprotective effects due to direct antioxidant or antithrombotic mechanisms. We report in vitro and postingestion ex vivo effects of cocoa procyanidins, a procyanidin-rich cocoa beverage and dealcoholized red wine (DRW) on human platelet activation. In a series of in vitro studies, cocoa procyanidin trimers, pentamers or DRW (3 and 10 micromol/L) were incubated with citrated peripheral whole blood in the presence and absence of platelet agonists. Platelet activation was detected using fluorescent-labeled monoclonal antibodies recognizing the fibrinogen binding conformation of GPIIb-IIIa (referred to herein as PAC-1 binding) and the activation-dependent platelet epitope CD62P (P-selectin). The percentage of CD42a-positive platelets coexpressing PAC-1 binding and/or CD62P was determined by multiparameter flow cytometry. Procyanidin trimers, pentamers and DRW added to whole blood in vitro increased PAC-1 binding and P-selectin expression. In contrast, procyanidin trimers, pentamers and DRW inhibited the platelet activation in response to epinephrine. The effects on platelet activation of cocoa beverage and DRW consumption were also studied in healthy subjects. Citrated blood was obtained before and 2 and 6 h after the ingestion of a cocoa beverage, a caffeine-containing beverage, DRW or water. Platelet activation was measured by flow cytometry. The consumption of DRW did not affect the expression of activation-dependent platelet antigens, either unstimulated or after ex vivo activation with epinephrine. However, the consumption of DRW increased PAC-1 binding in response to 100 micromol/L ADP ex vivo. Cocoa consumption reduced platelet response to agonists ex vivo. The ingestion of water had no effect on platelet activation, whereas a caffeine-containing beverage augmented the response of platelets to epinephrine. In summary, select cocoa procyanidins and DRW added to whole blood in vitro increased expression of platelet activation markers in unstimulated platelets but suppressed the platelet activation response to epinephrine. In contrast, cocoa consumption suppressed unstimulated and stimulated platelet activation in whole blood. This suppressive effect observed on platelet reactivity may explain in part the reported cardioprotective effects of dietary polyphenols.

Patterns of behavioral adjustment and maladjustment in mental retardation: comparison of children with and without ADHD.

A variety of behavioral and emotional problems have been associated with attention deficit disorder with hyperactivity (ADHD) in children of average intellect. In contrast, little is known about concomitant behavioral and emotional problems in children with ADHD and mental retardation. In this study, we used the Personality Inventory for Children-Revised to assess the behavioral adjustment of 48 children with mental retardation and ADHD compared to that of 47 children with mental retardation without ADHD. The ADHD group had significantly more symptoms of depression, family conflict, noncompliance, anxiety, hyperactivity, inadequate social skills, and academic problems. Results are strongly suggestive of significant behavioral and emotional problems in children with ADHD and mental retardation, thus mirroring the pattern associated with ADHD in the general school-age population.

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.

Predicting HIV disease progression in children using measures of neuropsychological and neurological functioning. Pediatric AIDS clinical trials 152 study team.

BACKGROUND: Neuropsychological testing and 2 measures of neurological status, cortical atrophy, and motor dysfunction were assessed for their usefulness in predicting human immunodeficiency virus (HIV) disease progression in infants, children, and adolescents who participated in Pediatric AIDS Clinical Trials Group Protocol 152 (PACTG 152). METHODS: A cohort of 722 antiretroviral therapy-naive children with symptomatic HIV infection were assessed at study entry and at later intervals. Assessments included neurodevelopmental testing, neuroradiologic imaging, and neurological examination of motor function. CD4 cell count and plasma RNA viral load also were measured. RESULTS: Children with the lowest neuropsychological functioning (IQ < 70) at baseline had the highest risk for later HIV disease progression (56%), compared with those with borderline/low (IQ = 70-89) functioning (26%), or with average or above (IQ > 90) functioning (18%). This was also true of week 48 neuropsychological functioning. Motor dysfunction (especially reduced muscle mass) at entry also predicted disease progression. Furthermore, motor dysfunction and week 48 neuropsychological functioning provided predictive information beyond that obtainable from surrogate markers of HIV disease status (eg, CD4 count, HIV RNA level). Children with cortical atrophy also were at higher risk for later disease progression, but when CD4 count and RNA viral load were known, cortical atrophy information provided no additional predictive information. CONCLUSIONS: Measures of neuropsychological and motor function status provide unique information regarding pediatric HIV disease progression. As such, these findings have important implications for predicting long-term outcomes (eg, longevity) in pediatric patients.

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.

Dose and timing of interleukin (IL)-12 and timing and type of total-body irradiation: effects on graft-vs.-host disease inhibition and toxicity of exogenous IL-12 in murine bone marrow transplant recipients.

Paradoxically, a single injection of recombinant murine interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits graft-vs.-host disease (GVHD) while preserving graft-vs.-leukemia (GVL) effects in lethally irradiated mice receiving fully MHC-mismatched bone marrow and spleen cells. These protective effects are mediated by interferon (IFN)-gamma, whose early secretion is induced by IL-12 treatment. We investigated the relationship of IL-12 dose and timing of administration, as well as timing and type of total-body irradiation (TBI), with the ability of IL-12 to inhibit GVHD or mediate toxicity. The results show that a relatively low dose of IL-12 (as little as 50 U in a single injection) can mediate significant GVHD protection. The timing of IL-12 administration, however, is a critical factor. IL-12 administered 1 hour before BMT was most protective, but protection was still observed when it was administered 1-12 hours after BMT. Delaying IL-12 administration to 36 hours post-BMT completely obviated its protective effect. Administration of a second IL-12 injection 6 days after BMT negated the protective effect of an initial injection at the time of BMT. While IL-12 protection was evident when TBI was administered by 137Cs-irradiator in one or two fractions on day -1 or day 0, the use of an X-irradiator to deliver TBI on day -1 was associated with marked IL-12 toxicity. Whereas the protective effect of IL-12 against GVHD depended on donor-derived IFN-gamma, toxicity depended on the ability of host cells to produce IFN-gamma. Careful studies are warranted to test the effects of IL-12 in the context of BMT with various conditioning regimens in large animal preclinical models before this novel approach to GVHD protection can be applied clinically.

Neurologic, neurocognitive, and brain growth outcomes in human immunodeficiency virus-infected children receiving different nucleoside antiretroviral regimens. Pediatric AIDS Clinical Trials Group 152 Study Team.

OBJECTIVES: To compare the impact of three different nucleoside reverse transcriptase inhibitor regimens, zidovudine (ZDV) monotherapy, didanosine (ddI) monotherapy, and ZDV plus ddI combination therapy, on central nervous system (CNS) outcomes in symptomatic human immunodeficiency virus (HIV)-infected children. METHODS: Serial neurologic examinations, neurocognitive tests, and brain growth assessments (head circumference measurements and head computed tomography or magnetic resonance imaging studies) were performed in 831 infants and children who participated in a randomized double-blind clinical trial of nucleoside reverse transcriptase inhibitors. The Pediatric AIDS Clinical Trials Group study 152 conducted between 1991 and 1995 enrolled antiretroviral therapy-naive children. Subjects were stratified by age (3 to <30 months of age or 30 months to 18 years of age) and randomized in equal proportions to the three treatment groups. RESULTS: Combination ZDV and ddI therapy was superior to either ZDV or ddI monotherapy for most of the CNS outcomes evaluated. Treatment differences were observed within both age strata. ZDV monotherapy showed a modest statistically significant improvement in cognitive performance compared with ddI monotherapy during the initial 24 weeks, but for subsequent protection against CNS deterioration no clear difference was observed between the two monotherapy arms. CONCLUSIONS: Combination therapy with ZDV and ddI was more effective than either of the two monotherapies against CNS manifestations of human immunodeficiency virus disease. The results of this study did not indicate a long-term beneficial effect for ZDV monotherapy compared with ddI monotherapy.

Developmental course of auditory processing interactions: Garner interference and Simon interference.

Previous research suggests that with increasing age children become more efficient in inhibiting conflicting responses and in resisting interference from irrelevant information. We assessed the abilities of 100 children (ages 3-16 years) and 20 adults to resist interference during the processing of 2 auditory dimensions of speech, namely the speaker's gender and spatial location. The degree of interference from irrelevant variability in either dimension did not vary with age. Apparently, young children do not have more difficulty in resisting interference when the nontarget and the target are both perceptual attributes. We also assessed the participants' abilities to inhibit conflicting task-irrelevant information from spatial location and to resist interference from spatial variability in the context of conflict. In the presence of conflicting task-irrelevant information, both interference effects declined significantly with age. Developmental change in auditory processing seems to vary as a function of (1) the nature of the target-nontarget combination and (2) the presence/absence of conflicting task-irrelevant information.

Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen.

Mixed hematopoietic chimerism can be induced in mice receiving allogeneic bone marrow transplantation (BMT) after nonmyeloablative host conditioning with depletion T cells with of anti-T cell monoclonal antibodies (mAbs), low-dose (3 Gy) total-body irradiation (TBI), and local thymic irradiation (7 Gy). These mice are specifically tolerant to donor and host antigens. When nontolerant donor T cells are given to chimeras several months after BMT, full donor-type chimerism develops, but graft-vs.-host disease (GVHD) does not occur. The induction of such lymphohematopoietic GVH reactions without GVHD could provide an approach to separating graft-vs.-leukemia (GVL) from GVHD in patients with hematologic malignancies. To make the nonmyeloablative conditioning regimen described above more cytoreductive for such malignancies, we have now modified it by replacing TBI with cyclophosphamide (CP). Treatment with anti-CD4 and anti-CD8 mAbs on day -5, 200 mg/kg CP on day -1, and 7 Gy thymic irradiation on day 0 was only slightly myelosuppressive and allowed fully major histocompatibility complex (MHC)-mismatched (with or without multiple minor antigen disparities) allogeneic bone marrow to engraft and establish long-term mixed chimerism in 40 to 82% of recipients in three different strain combinations. The administration of nontolerant donor spleen cells at 5 weeks or at 5, 8, and 11 weeks posttransplant was capable of eliminating host hematopoietic cells, leading to full or nearly full donor chimerism in six of six and two of four chimeric animals in two different strain combinations. No clinical evidence of GVHD was observed in any recipients of these donor leukocyte infusions (DLI). These studies demonstrate that induction of mixed chimerism with nonmyeloablative conditioning followed at appropriate times by DLI might allow lymphohematopoietic GVH reactions, and hence GVL effects, to eliminate chronic hematologic malignancies without causing clinically significant GVHD.

Apple juice inhibits human low density lipoprotein oxidation.

Dietary phenolic compounds, ubiquitous in vegetables and fruits and their juices possess antioxidant activity that may have beneficial effects on human health. The phenolic composition of six commercial apple juices, and of the peel (RP), flesh (RF) and whole fresh Red Delicious apples (RW), was determined by high performance liquid chromatography (HPLC), and total phenols were determined by the Folin-Ciocalteau method. HPLC analysis identified and quantified several classes of phenolic compounds: cinnamates, anthocyanins, flavan-3-ols and flavonols. Phloridzin and hydroxy methyl furfural were also identified. The profile of phenolic compounds varied among the juices. The range of concentrations as a percentage of total phenolic concentration was: hydroxy methyl furfural, 4-30%; phloridzin, 22-36%; cinnamates, 25-36%; anthocyanins, n.d.; flavan-3-ols, 8-27%; flavonols, 2-10%. The phenolic profile of the Red Delicious apple extracts differed from those of the juices. The range of concentrations of phenolic classes in fresh apple extracts was: hydroxy methyl furfural, n.d.; phloridzin, 11-17%; cinnamates, 3-27%; anthocyanins, n.d.-42%; flavan-3-ols, 31-54%; flavonols, 1-10%. The ability of compounds in apple juices and extracts from fresh apple to protect LDL was assessed using an in vitro copper catalyzed human LDL oxidation system. The extent of LDL oxidation was determined as hexanal production using static headspace gas chromatography. The apple juices and extracts, tested at 5 microM gallic acid equivalents (GAE), all inhibited LDL oxidation. The inhibition by the juices ranged from 9 to 34%, and inhibition by RF, RW and RP was 21, 34 and 38%, respectively. Regression analyses revealed no significant correlation between antioxidant activity and either total phenolic concentration or any specific class of phenolics. Although the specific components in the apple juices and extracts that contributed to antioxidant activity have yet to be identified, this study found that both fresh apple and commercial apple juices inhibited copper-catalyzed LDL oxidation. The in vitro antioxidant activity of apples support the inclusion of this fruit and its juice in a healthy human diet.

Functionally and phenotypically mature mouse CD8+ T cells develop in porcine thymus grafts in mice.

Mouse CD4+ T cells efficiently develop in fetal pig thymus (FP THY) grafts and repopulate the periphery of T cell and NK cell-depleted, thymectomized (ATX) mice. However, efficient peripheral repopulation of mouse CD8+ T cells does not occur in these mice. We have therefore evaluated the maturation and function of mouse CD8 single positive (SP) thymocytes in fetal pig thymus and liver fragment (FP THY LIV) grafts. Phenotypic maturity, as measured by upregulated expression of TCR, class I MHC, and Qa-2, and downregulated expression of heat stable antigen (HSA) on CD8 SP cells in FP THY grafts, was similar to that in host thymi of euthymic control mice. Cytolytic T lymphocyte (CTL) activity of thymocytes from FP THY grafts was similar to that of thymocytes from host thymi of euthymic mice, indicating that functional maturation of CD8 SP cells had taken place in the grafts. Furthermore, similarly efficient deletion of V beta 5.1/5.2+ and V beta 11+ CD8 SP cells was observed in FP THY grafts as in host thymi of euthymic control mice. Similar percentages of V beta 6, V beta 7, and V beta 8.1/8.2 expressing cells were also detected among CD8 SP cells in FP THY grafts and host thymi of euthymic controls. Together, our results suggest that normal positive and negative selection occurs, and that mouse CD8+ cells can undergo normal functional and phenotypic maturation in FP THY grafts. Thus, other explanations must be sought for the failure of CD8+ cells to repopulate the peripheral lymphoid tissues of ATX, T cell-depleted, pig THY/LIV-grafted mice.

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.

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.

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.

Interleukin-12 inhibits graft-versus-host disease through an Fas-mediated mechanism associated with alterations in donor T-cell activation and expansion.

We have recently made the paradoxical observation that a single injection of recombinant murine interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) inhibits graft-versus-host disease (GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow and spleen cells. We have now examined the mechanism of this effect of IL-12 on acute GVHD. By day 4 post-BMT, IL-12-treated mice showed marked reductions in splenic donor CD4(+) and CD8(+) T cells compared with GVHD controls. Expression of the early activation markers IL-2R alpha chain (CD25) and CD69 on splenic donor CD4(+) cells was considerably higher at early time points (36 and 72 hours post-BMT) in IL-12-treated mice compared with GVHD controls. However, the later, GVHD-associated increase in CD25 and very late antigen-4 (VLA-4) expression on donor T cells was greatly depressed in IL-12-protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by day 4 was also completely inhibited in the IL-12-treated group. Expression of Fas was increased on donor CD4 cells of IL-12-treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least partially dependent on the ability of donor cells to express functional Fas molecules. We conclude that IL-12 treatment at the time of BMT markedly perturbs the activation of alloreactive donor CD4(+) T cells that play a critical role in the pathogenesis of acute GVHD. We hypothesize that these perturbations culminate in Fas-dependent apoptosis of donor T cells, thus impeding their expansion and their GVHD-promoting activity.