Ambient air pollutants are associated with morning serum cortisol in overweight and obese Latino youth in Los Angeles.

BACKGROUND: Hypothalamic-pituitary-adrenal (HPA)-axis dysfunction has been associated with a variety of mental health and cardio-metabolic disorders. While causal models of HPA-axis dysregulation have been largely focused on either pre-existing health conditions or psychosocial stress factors, recent evidence suggests a possible role for central nervous system activation via air pollutants, such as nitrogen dioxide (NO2), ozone (O3) and particulate matter (PM). Therefore, in an observational study of Latino youth, we investigated if monthly ambient NO2, O3, and PM with aerodynamic diameter ≤ 2.5 (PM2.5) exposure were associated with morning serum cortisol levels. METHODS: In this cross-sectional study, morning serum cortisol level was assessed after a supervised overnight fast in 203 overweight and obese Latino children and adolescents (female/male: 88/115; mean age: 11.1 ± 1.7 years; pre-pubertal/pubertal/post-pubertal: 85/101/17; BMI z-score: 2.1 ± 0.4). Cumulative concentrations of NO2, O3 and PM2.5 were spatially interpolated at the residential addresses based on measurements from community monitors up to 12 months prior to testing. Single and multi-pollutant linear effects models were used to test the cumulative monthly lag effects of NO2, O3, and PM2.5 on morning serum cortisol levels after adjusting for age, sex, seasonality, social position, pubertal status, and body fat percent by DEXA. RESULTS: Single and multi-pollutant models showed that higher O3 exposure (derived from maximum 8-h exposure windows) in the prior 1-7 months was associated with higher serum morning cortisol (p < 0.05) and longer term PM2.5 exposure (4-10 months) was associated with lower serum morning cortisol levels (p < 0.05). Stratification by pubertal status showed associations in pre-pubertal children compared to pubertal and post-pubertal children. Single, but not multi-pollutant, models showed that higher NO2 over the 4-10 month exposure period associated with lower morning serum cortisol (p < 0.05). CONCLUSIONS: Chronic ambient NO2, O3 and PM2.5 differentially associate with HPA-axis dysfunction, a mechanism that may serve as an explanatory pathway in the relationship between ambient air pollution and metabolic health of youth living in polluted urban environments. Further research that uncovers how ambient air pollutants may differentially contribute to HPA-axis dysfunction are warranted.

Effects of air pollution exposure on glucose metabolism in Los Angeles minority children.

OBJECTIVES: Growing evidence indicates that ambient (AAP: NO2 , PM2.5 and O3 ) and traffic-related air pollutants (TRAP) contribute to metabolic disease risk in adults; however, few studies have examined these relationships in children. METHODS: Metabolic profiling was performed in 429 overweight and obese African-American and Latino youth living in urban Los Angeles, California. This cross-sectional study estimated individual residential air pollution exposure and used linear regression to examine relationships between air pollution and metabolic outcomes. RESULTS: AAP and TRAP exposure were associated with adverse effects on glucose metabolism independent of body fat percent. PM2.5 was associated with 25.0% higher fasting insulin (p < 0.001), 8.3% lower insulin sensitivity (p < 0.001), 14.7% higher acute insulin response to glucose (p = 0.001) and 1.7% higher fasting glucose (p < 0.001). Similar associations were observed for increased NO2 exposure. TRAP from non-freeway roads was associated with 12.1% higher insulin (p < 0.001), 6.9% lower insulin sensitivity (p = 0.02), 10.8% higher acute insulin response to glucose (p = 0.003) and 0.7% higher fasting glucose (p = 0.047). CONCLUSIONS: Elevated air pollution exposure was associated with a metabolic profile that is characteristic of increased risk for type 2 diabetes. These results indicate that increased prior year exposure to air pollution may adversely affect type 2 diabetes-related pathophysiology in overweight and obese minority children.

Associations among sugar sweetened beverage intake, visceral fat, and cortisol awakening response in minority youth.

CONTEXT: Abdominal adiposity has long been associated with excess caloric intake possibly resulting from increased psychosocial stress and associated cortisol dysfunction. However, the relationship of sugar-sweetened beverage (SSB) intake specifically with cortisol variability and visceral adipose tissue (VAT) is unknown. OBJECTIVE: To examine the relationships between SSB intake, VAT, and cortisol response in minority youth. DESIGN: A cross-sectional analysis. SETTING: The University of Southern California. PARTICIPANTS: 60 overweight/obese Non-Hispanic Black and Hispanic adolescents ages 14-18years. MAIN OUTCOME MEASURES: VAT via Magnet Resonance Imaging (MRI), cortisol awakening response (CAR) via multiple salivary samples, and SSB intake via multiple 24-hour diet recalls. SSB intake was divided into the following: low SSB consumers (<1 servings per day), medium SSB consumers (≥1-<2 servings per day), high SSB consumers (≥2 servings per day). Analysis of covariance were run with VAT and CAR as dependent variables and SSB intake categories (independent variable) with the following a priori covariates: sex, Tanner stage, ethnicity, caloric intake, and body mass index. RESULTS: The high SSB intake group exhibited a 7% higher VAT compared to the low SSB intake group (ß=0.25, CI:(0.03, 0.33), p=0.02). CAR was associated with VAT (ß=0.31, CI:(0.01,0.23), p=0.02). The high SSB intake group exhibited 22% higher CAR compared to the low SSB intake group (ß=0.30, CI:(0.02,0.48), p=0.04). CONCLUSION: This is the first study exploring the relationship between SSB, VAT, and CAR. SSB consumption appears to be independently associated greater abdominal adiposity and higher morning cortisol variability in overweight and obese minority youth. This study highlights potential targets for interventions specifically to reduce SSB intake in a minority youth population.

Saturation of subcutaneous adipose tissue expansion and accumulation of ectopic fat associated with metabolic dysfunction during late and post-pubertal growth.

BACKGROUND/OBJECTIVE: Puberty is a period defined by large changes in adipose tissue accumulation and distribution; however, longitudinal patterns of ectopic fat development have not been shown. We have previously shown significant declines in beta-cell function (BCF) across puberty and hypothesize that accumulation of ectopic fat deposition, particularly hepatic fat, will predict this fall. SUBJECT/METHODS: We conducted a longitudinal study and examined 2-year change in abdominal fat distribution and type 2 diabetes risk markers in 76 Hispanic children and young adults (16.1±0.5 years, 66% obese, 52% male, 51% post-pubertal). Subcutaneous abdominal adipose tissue (SAAT), visceral adipose tissue (VAT), hepatic fat fraction (HFF) and pancreatic fat fraction (PFF) were measured by 3-Tesla magnetic resonance imaging, and markers of type 2 diabetes risk were collected at fasting and during an oral glucose tolerance test (OGTT). RESULTS: Baseline pubertal status significantly moderated the 2-year change in ectopic fat deposition, such that VAT, HFF and PFF increased in individuals during late and post-pubertal growth, whereas children earlier in their pubertal development decreased ectopic accumulation and had less VAT accumulation (VAT: pTanner*time=0.044, 0.31±0.08 l vs 0.03±0.10 l; HFF: pTanner*time=0.007, 1.34±0.87% vs -2.61±1.11%; PFF: pTanner*time<0.001, 1.61±0.39% vs -0.96±0.50%). Independent of pubertal status, the 2-year increase in HFF and VAT significantly associated with a decline in BCF (ß=-1.04, P=0.038; ß=-1.81, P=0.020) and metabolic function, while accumulation of SAAT significantly associated with BCF (ß=1.36, P=0.012) and metabolic improvement. HFF accumulation was the only depot to significantly predict clinical markers of type 2 diabetes risk, fasting glucose and HbA1c, and circulating free fatty acid levels (ß=1.00, P=0.034; ß=1.00, P=0.015; ß=01.01, P=0.024). CONCLUSIONS: The accumulation of SAAT defends against type 2 diabetes risk and potentially ectopic fat accumulation. Intra-abdominal VAT and HFF accumulation both associate with metabolic decline and BCF, while HFF predicts an even greater number of metabolic risk features.

Challenges contributing to disrupted transition from paediatric to adult diabetes care in young adults with type 1 diabetes.

AIM: To examine challenges contributing to disruptions in care during the transition from paediatric to adult care among young adults with Type 1 diabetes who are primarily in ethnic minority groups and have low socio-economic status. METHODS: Participants (n = 20) were newly enrolled patients in a transition clinic for young adults with Type 1 diabetes with a history of loss to medical follow-up. Participants completed qualitative semi-structured interviews detailing their transition experiences in addition to demographic, HbA1c and psychosocial measures. Descriptive statistics were completed for quantitative data, and narrative thematic analysis of interviews was used to identify common themes. A mixed-method analysis was used to identify the associations between stressors identified in interviews and clinical and psychosocial variables. RESULTS: Three categories of challenges contributing to loss to follow-up were identified: psychosocial challenges, health provider and health system challenges and developmental challenges. Participants experienced a high degree of stressful life circumstances which were associated with higher HbA1c (r = 0.60, P = 0.005), longer duration of loss to follow-up (r = 0.51, P = 0.02), greater emergency department utilization (r = 0.45, P = 0.05), and lower life satisfaction (r = -0.62, P = 0.003). CONCLUSIONS: A confluence of challenges, including stressful life circumstances, healthcare system barriers and the developmental trajectory of young adulthood, contributes to a high risk of loss to follow-up and poor health in this population of young adults with Type 1 diabetes. An integrated approach to transition addressing medical and psychosocial needs may facilitate improved follow-up and health outcomes in clinical settings.

Disclosure of psychosocial stressors affecting diabetes care among uninsured young adults with Type 1 diabetes.

AIMS: To determine the disclosure rates of psychosocial issues affecting routine diabetes care. METHODS: A total of 20 young adults were interviewed regarding the impact of psychosocial stressors on their diabetes care. The interviewer, endocrinologist and case manager reported the prevalence rates of psychosocial stressors. Disclosure rates were compared to determine the prevalence of psychosocial issues and the different patterns of disclosure. RESULTS: Participants reported a high number of psychosocial stressors, which were associated with poorer glycaemic control (r = 0.60, P = 0.005). Approximately half of all disclosed stressors (50.9%) were identified in routine care; other stressors were identified only through intensive case management and/or in-depth interviews. CONCLUSIONS: Identifying psychosocial stressors in routine care, and providing referrals to psychological or social services, is a significant unmet need and may improve glycaemic control among certain populations with diabetes. Systematic mechanisms of capturing this information, such as by screening surveys, should be considered.

Leptin predicts a decline in moderate to vigorous physical activity in minority female children at risk for obesity.

UNLABELLED: What is already known about this subject Physical activity declines as children enter puberty. Leptin is cross-sectionally associated with physical activity, but there are conflicting findings on the magnitude and direction of this association. Leptin concentrations fluctuate during puberty, and may impact energy balance. What this study adds Leptin predicts the decline in physical activity during the start of puberty independent of central adiposity. Based on a median split of leptin, girls with low leptin levels have higher levels of physical activity than girls with high leptin levels at the start of puberty. Leptin levels at the start of puberty may provide a biological basis for the age-related physical activity decline in girls. BACKGROUND: Leptin may influence moderate to vigorous physical activity (MVPA) at the start of puberty. The direction and magnitude of this association are unclear. OBJECTIVES: To determine the effect of baseline leptin on MVPA over 1 year in minority girls at high risk for obesity. METHODS: Data came from TRANSITIONS, a longitudinal observational study on the age-related MVPA decline. Fifty peripubertal girls aged 8-11 years at baseline participated. Baseline leptin (ng mL(-1) ) was collected via a duplicated assay using a double antibody radio immune assay. MVPA (min d(-1) ) was measured using accelerometers for at least four 10-h days on a quarterly basis for up to 1 year. RESULTS: Continuous leptin was negatively related to MVPA (P = 0.001) independent of central adiposity at baseline and predicted the MVPA decline over 1 year (P = 0.002). For descriptive purposes, baseline leptin was dichotomized at the sample median into 'high leptin' and 'low leptin' categories to determine whether MVPA trajectories differed between these groups. Girls with 'low leptin' at baseline had significantly higher levels of MPVA at baseline, visit 1 and visit 2 compared to girls with 'high leptin'. CONCLUSIONS: High leptin levels predicted nearly a 12.6% decline in MVPA over 1 year. These findings provide support for the biological basis of declining MVPA as girls enter puberty.

Effects of a randomized maintenance intervention on adiposity and metabolic risk factors in overweight minority adolescents.

OBJECTIVE: The objective of this study was to assess the effects of a maintenance programme (monthly newsletters vs. monthly group classes and telephone behavioural sessions) on obesity and metabolic disease risk at 1 year in overweight minority adolescents. METHODS: After a 4-month nutrition and strength training intervention, 53 overweight Latino and African-American adolescents (15.4 ± 1.1 years) were randomized into one of two maintenance groups for 8 months: monthly newsletters (n = 23) or group classes (n = 30; monthly classes + individualized behavioural telephone sessions). The following outcomes were measured at months 4 (immediately following the intense intervention) and 12: height, weight, blood pressure, body composition via BodPod™ (Life Measurement Instruments, Concord, CA, USA), lipids and glucose/insulin indices via frequently sampled intravenous glucose tolerance test. RESULTS: There were no significant group by time interactions for any of the health outcomes. There were significant time effects in several outcomes for both groups from months 4 to 12: bench press and leg press decreased by 5% and 14%, respectively (P = 0.004 & P = 0.01), fasting insulin and acute insulin response decreased by 26% and 16%, respectively (P < 0.001 & P = 0.046); while high-density lipoprotein cholesterol and insulin sensitivity improved by 5% and 14% (P = 0.042 & P = 0.039). CONCLUSIONS: Newsletters as opposed to group classes may suffice as follow-up maintenance programmes to decrease type 2 diabetes and cardiovascular risk in overweight minority adolescents.

Adiponectin and leptin are independently associated with insulin sensitivity, but not with insulin secretion or beta-cell function in overweight Hispanic adolescents.

The aim of the study was to investigate the independent effects of leptin and adiponectin on insulin sensitivity as well as insulin secretion and beta-cell function in overweight Hispanic adolescents. Despite pubertal changes in hormone secretion, studies investigating the independent effect of both hormones on insulin sensitivity and beta-cell function in adolescents are lacking. In a cross-sectional study, 175 overweight Hispanic adolescent boys (n=101) and girls (n=74) with a family history of diabetes were recruited and insulin sensitivity (SI), acute insulin response to glucose (AIR), disposition index (DI), body composition, total serum adiponectin, and leptin were assessed. Over age, leptin significantly increased in girls but not in boys (p for age x gender interaction=0.005) while adiponectin was similar in boys and girls. Leptin was not correlated to adiponectin. Leptin (partial r=-0.180; p=0.019) and adiponectin (partial r=0.230; p=0.003) predicted SI independent of age, gender, body fat, lean body mass, and Tanner stage but together, they explained 5% of the unique variation in SI (p for R (2)-change<0.001). Leptin or adiponectin were not related to AIR or DI. With regard to SI, AIR, and DI, no significant gender, age, or Tanner stage interactions were observed suggesting similar effects of adiponectin and leptin among gender, age, and Tanner stages. Leptin and adiponectin were independently associated with SI, but not with insulin secretion or beta-cell function.

Combined association of maternal and paternal family history of diabetes with plasma leptin and adiponectin in overweight Hispanic children.

AIMS: To investigate the importance of a maternal and paternal family history of Type 2 diabetes and their combined association with plasma leptin and adiponectin levels in overweight Latino children with a family history of Type 2 diabetes (T2DM). METHODS: This cross-sectional study investigated the combined association of a maternal and paternal family history of T2DM with leptin and adiponectin in 175 overweight Latino children (age 11.1 +/- 1.7 years). All subjects had a family history of T2DM. Plasma adiponectin and leptin levels, body fat measured by dual-energy X-ray absorptiometry, Tanner stage, age and insulin sensitivity were assessed. RESULTS: After adjustment for age, gestational diabetes, insulin sensitivity and body fat, a combined maternal and paternal family history of T2DM was associated with higher leptin concentrations (P = 0.004) compared with a maternal or paternal family history alone. This association was most pronounced at Tanner stage 1 (P for interaction family history x tanner stage = 0.022). The presence of a combined maternal and paternal family history of T2DM accounted for 4% (P = 0.003) of the variation in leptin concentrations. No such combined association was observed for adiponectin levels. CONCLUSIONS: Maternal and paternal family history of T2DM may have an additive impact on leptin, but not on adiponectin levels independent of adiposity and insulin sensitivity in overweight Latino children. This may contribute to a further clinically relevant deterioration of metabolic health in this population.

Leptin-to-adiponectin ratio as independent predictor of insulin sensitivity during growth in overweight Hispanic youth.

Because leptin and adiponectin are counter-regulated in vivo and exert opposing effects on glucose metabolism, fat oxidation and insulin sensitivity, the ratio of leptin-to-adiponectin has been investigated as a potential atherogenic index, suggesting that the index is a better biomarker for atherosclerotic risk in obese Type 2 diabetic patients than either leptin or adiponectin alone. However, no information is available regarding the leptin-to-adiponectin ratio during adolescence in Hispanic adolescents. Therefore, the present study was designed to investigate the leptin-to-adiponectin ratio during growth and to establish whether the leptin-to-adiponectin ratio is a better predictor for insulin sensitivity compared to leptin and adiponectin alone in a regression model. From the age of 8 to 14, the leptin-to-adiponectin ratio increased from 2.0+/-0.8 to 5.8+/-2.2 in girls, with no significant change noted in boys (gender x age interaction p=0.007). In a multiple regression analysis, including both adiponectin and leptin as independent variables, leptin and adiponectin explained 5% of the variation in insulin sensitivity independent of gender, age, Tanner stage, total fat mass and lean body mass (p for R2-change <0.001). The leptin-to-adiponectin ratio also explained 5% of the variation in insulin sensitivity, after controlling for the same covariates (p for R2-change <0.001). These data indicate that the leptin-to-adiponectin ratio is not a better predictor of insulin sensitivity during growth than the additive effects of leptin and adiponectin levels.

Cardiovascular fitness and physical activity in children with and without impaired glucose tolerance.

OBJECTIVE: To examine differences in cardiovascular fitness (VO(2max)) and physical activity levels in overweight Hispanic children with normal glucose tolerance (NGT) vs impaired glucose tolerance (IGT). PARTICIPANTS: A total of 173 overweight (BMI percentile 97.0 +/- 3.1) Hispanic children ages 8-13 years with a family history of type 2 diabetes. METHODS: VO(2max) was measured via a maximal effort treadmill test and open circuit spirometry. Physical activity was determined by questionnaire. Glucose tolerance was established by a 2-h oral glucose challenge (1.75 g of glucose/kg body weight). IGT was defined from an oral glucose tolerance test as a 2-h plasma glucose level > or =140 and <200 mg/dl. RESULTS: IGT was detected in 46 of the 173 participants (approximately 27%); no cases of type 2 diabetes were identified. No significant differences were found between youth with NGT and those with IGT in absolute VO(2max) (2.2 +/- 0.6 vs 2.1 +/- 0.5 l/min), VO(2max) adjusted for gender, age, and body composition (2.2 +/- 0.2 vs 2.1 +/- 0.2 l/min), or recreational physical activity levels (8.7 +/- 8.2 vs 6.9 +/- 6.2 h/week). CONCLUSION: Overweight Hispanic youth with IGT exhibit similar levels of VO(2max) and physical activity compared to their NGT counterparts. Longitudinal analyses are necessary to determine whether fitness/activity measures contribute significantly to diabetes risk over time in this group.

Insulin sensitivity, insulin secretion and beta-cell function during puberty in overweight Hispanic children with a family history of type 2 diabetes.

OBJECTIVE: To examine cross-sectional differences in insulin sensitivity, insulin secretion and beta-cell function during puberty in overweight Hispanic boys and girls with a family history of type 2 diabetes. STUDY DESIGN AND PARTICIPANTS: This cross-sectional, observational study included 214 8-13-y-old Hispanic children with a BMI percentile > or = 85th percentile and family history of type 2 diabetes. METHODS AND ANALYSES: Participants underwent a physical examination, body composition measures, oral glucose tolerance test (OGTT), and frequently-sampled intravenous glucose tolerance test. Unadjusted and adjusted general linear models (GLM) tested whether insulin/glucose dynamics differed by Tanner stage and gender. RESULTS: Unadjusted group comparisons showed that fasting insulin increased whereas insulin sensitivity (SI) and the disposition index (DI) (a measure of pancreatic beta-cell function) decreased across Tanner stage groups (all P < 0.05). No differences in the acute insulin response to glucose (AIRg), fasting glucose or 2-h glucose were found. After adjusting for covariates, there was no independent effect of Tanner stage on SI (P = 0.9) or AIRg (P = 0.2), but DI was slightly lower in later Tanner stages suggesting decreased beta-cell function in the more mature groups (P = 0.10). CONCLUSIONS: Overweight Hispanic children with a family history of type 2 diabetes may represent a unique population given that pubertal insulin resistance was not evident once analyses controlled for body composition. Longitudinal analyses are required to determine whether the slightly diminished beta-cell function in later Tanner stages plays a role in the development of type 2 diabetes.

Pediatric obesity and insulin resistance: chronic disease risk and implications for treatment and prevention beyond body weight modification.

The study of childhood obesity has continued to grow exponentially in the past decade. This has been driven in part by the increasing prevalence of this problem and the widespread potential effects of increased obesity in childhood on lifelong chronic disease risk. The focus of this review is on recent findings regarding the link between obesity and disease risk during childhood and adolescence. We describe recent reports relating to type 2 diabetes in youth (2), prediabetes (69, 166), metabolic syndrome (33, 35), polycystic ovarian syndrome (77), and nonalcoholic fatty liver disease (58, 146), and the mediating role of insulin resistance in these conditions. In addition, we review the implications of this research for the design of more effective treatment and prevention strategies that focus more on the improvement of obesity-related metabolic abnormalities and chronic disease risk reduction than on the conventional energy balance approach that focuses on weight management.

Uptake of myo-inositol by early-somite rat conceptus. Transport kinetics and effects of hyperglycemia.

We have shown that myo-inositol in the cultured rat embryo is diminished whenever malformations are induced by hyperglycemia and that the malformations and reductions of tissue myo-inositol content are not corrected by aldose reductase inhibitors. This study was designed to evaluate the kinetics of myo-[3H]inositol uptake in vitro during 1-, 3-, and 24-h intervals in the 10.5-day rat conceptus (10-12 somites). We found that the equilibration between tissue and medium is relatively slow and that the concentration of free myo-inositol in tissue is only approximately threefold greater than in the medium even after 24 h. The integrated uptake of free myo-inositol by the intact 10.5-day conceptus is a saturable process with a Km (246 +/- 16 microM) consistent with a low-affinity system. The net rate of accumulation into the tissue pool of free myo-inositol exceeds the rate of incorporation of the accumulated myo-inositol into lipid components. Ambient glucose inhibits net myo-inositol uptake in a concentration-dependent fashion, and the inhibition is competitive in nature. The glucose-mediated inhibitions of myo-inositol transport also compromise the concurrent incorporation of myo-[3H]inositol into lipid components, although to a lesser extent. These inhibitory effects are relatively specific for D-glucose and not replicated by equimolar additions of D-mannose or D-galactose. myo-Inositol accumulation by the 10.5-day rat conceptus is also impaired by relatively specific inhibitors of D-glucose transport such as phloridzin or ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of effector cells of graft vs leukemia following allogeneic bone marrow transplantation in mice inoculated with murine B-cell leukemia.

It is now widely accepted that immunocompetent lymphocytes in allogeneic bone marrow grafts exert an antileukemic effect that contributes to the cure of leukemia. Graft vs leukemia (GVL) effects independent of graft vs host disease were investigated in allogeneic bone marrow chimeras tolerant of host and donor alloantigens. The role of Thy1.2, L3T4 and Lyt2 T lymphocytes as effector cells of GVL were investigated in (BALB/c x C57BL/6)F1 mice inoculated with murine B-cell leukemia and subsequently conditioned with total lymphoid irradiation and cyclophosphamide (200 mg/kg). Mice were reconstituted with C57BL/6 bone marrow cells depleted of well-defined T-cell subsets or enriched for stem cells by the soybean agglutination method. Detection of residual tumor cells, an indicator for efficacy of GVL, was carried out by adoptive transfer of peripheral blood or spleen cells obtained from treated chimeras into secondary naive BALB/c recipients at different time intervals following bone marrow transplantation. Treatment of the primary marrow inoculum with monoclonal anti-Thy1.2 or anti-Lyt2 abolished the GVL effects and all secondary BALB/c recipients developed leukemia within 60 days. On the other hand, the treatment with monoclonal anti-L3T4 did not influence the effect of GVL and all treated recipients remained without leukemia. The data suggest that T cells may mediate GVL effects in the absence of graft vs host disease and in circumstances where tolerance to conventional alloantigens is elicited. Effector cells of GVL across the major histocompatibility complex (MHC) in the murine B-cell leukemia tumor model system appear to be Thy1.2+ Lyt2+ L3T4-. Induction of GVL effects by allogeneic cells tolerant of host MHC suggests that these effects may be independent of graft vs host disease.

Functional clonal deletion versus suppressor cell-induced transplantation tolerance in chimeras prepared with a short course of total-lymphoid irradiation.

Allogeneic bone marrow (BM) chimeras induced by infusion of BM cells into recipients conditioned with total lymphoid irradiation (TLI) were shown to develop humoral and cell-mediated tolerance to host and donor-type alloantigens by a number of in vitro and in vivo assays. Spleen cells of tolerant chimeras exhibited suppressive activity of mixed lymphocyte reaction (MLR). MLR suppression was not abrogated by depletion of Lyt-2 cells, and neither could Lyt-2-positive cells sorted from the spleens of tolerant chimeras suppress MLR or attenuate graft-versus-host reactivity in vivo. Likewise, specifically unresponsive spleen cells obtained from chimeras could not be induced to respond in MLR against tolerizing host-type cells following depletion of Lyt-2 or passage through a nylon-wool column. Tolerance of chimera spleen cells to host alloantigens, best documented by permanent survival of donor-type skin allografts, could be adoptively transferred into syngeneic recipients treated by heavy irradiation but not into untreated or mildly irradiated recipients. Adoptive transfer of tolerance seemed to be associated with experimental conditions favoring engraftment of tolerant cells rather than suppression of host reactivity. We speculate that although host and/or donor-derived suppressor cells may be operating in reducing the pool of specific alloreactive clones by blocking cell proliferation in response to allogeneic challenge, the final outcome in tolerant chimeras is actual or functional deletion of alloreactive clones.

Functional clonal deletion versus active suppression in transplantation tolerance induced by total-lymphoid irradiation.

Transplantation tolerance and stable chimerism were established in adult mice conditioned with a short course of total-lymphoid irradiation (TLI) followed by infusion of 30 X 10(6) allogeneic bone marrow cells. Spleen cells of tolerant mice could not exert a proliferative or cytotoxic response against host-type cells in vitro and were unable to induce graft-versus-host reaction in secondary host-type recipients. The degree of suppression assessed by coculturing tolerant splenocytes in vitro in the one-way mixed lymphocyte reaction was quite variable--and, in some cases, was not at all demonstrable, although tolerance was clearly maintained. Suppression, when apparent, could not be ascribed to T lymphocytes. Suppressor cells were found to bind soybean agglutinin and could be separated from the nonsuppressive cells by means of this lectin. Dissociation of the suppressive population (SBA+ cells) from that which is normally alloreactive (SBA- cells) resulted in a suppressor cell-depleted fraction that was still unable to respond to host-type cells but regained reactivity to unrelated cells. Limiting dilution analysis of chimeric splenocytes revealed markedly reduced frequencies of cytotoxic T lymphocyte precursors (CTL-P) directed against host-type cells, as compared with normal splenocytes reacting against the same target cells. This difference was accentuated when these cells were sensitized to host-type target cells prior to plating in limiting dilution cultures. In 1:1 mixing experiments of normal and chimeric splenocytes, there was no evidence of any in vitro suppressive activity to account for hyporeactivity of chimeric cells against host-type cells. Thus, maintenance of TLI-induced tolerance seemed not to be mediated primarily through an active suppressor cell mechanism.

Lectin separation of nonlymphoid suppressor cells induced by total lymphoid irradiation.

Suppression of the mixed lymphocyte reaction (MLR) exerted by splenocytes derived from mice treated with fractionated total lymphoid irradiation (TLI, 200 rds x 8) was analyzed by various criteria in order to characterize the phenotype of the cell type(s) responsible for suppression. TLI-induced suppressor cells could not be eliminated by removal of cells bearing surface immunoglobulin, Thy-1, Lyt-2 and TL, and thus could not be ascribed to lymphocytes of the B or T cell lineage. Suppressor cells were large, and nonadherent to nylon wool, Sephadex G-10 and plastic surfaces. Suppressor activity of TLI splenocytes was predominantly located in fractions of cells bearing receptors for soybean agglutinin (SBA), peanut agglutinin (PNA) or both lectins. SBA+, PNA+, sequentially agglutinated (SBA followed by PNA) SBA+PNA+ and (PNA followed by SBA) PNA+SBA+ suppressor cells were radioresistant upon exposure to 1000 rds in vitro. Cells bearing the receptor for PNA but lacking that for SBA (PNA+SBA-) had sharply reduced suppressor activity. However, a radiosensitive PNA- suppressor cell subset was also documented in the spleen of TLI-treated mice. Thus, suppressor cells could best be physically separated from nonsuppressors by the SBA lectin. SBA+ suppressor cells were found, by scatter analysis, to include the population of large cells characteristic of TLI splenocytes, whereas SBA- cells were much smaller and almost exclusively devoid of suppressive capacity. The PNA receptor was found to further dissect the SBA+ suppressor cells into two distinct subpopulations: radioresistant SBA+PNA+ cells and radiosensitive SBA+PNA- cells. In summary, we suggest here the presence of at least two suppressive populations induced by TLI: radioresistant SBA+, PNA+, SBA+PNA+ or PNA+SBA+ cells, and radiosensitive PNA- and SBA+PNA- cells. Similar subsets of MLR suppressor cells can be isolated from normal bone marrow cells and splenocytes of nude mice, suggesting that suppression is mediated by large, immature, nonlymphoid cells which might migrate from shielded bone marrow compartments into the spleen of TLI-treated mice.