Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells.

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.

Regulatory T Cell Infusion Can Enhance Memory T Cell and Alloantibody Responses in Lymphodepleted Nonhuman Primate Heart Allograft Recipients.

The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4(+) CD25(+) CD127(-) Foxp3(+) (Treg) in clinically relevant large animal models. We infused ex vivo-expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin-lymphodepleted, MHC-mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti-interleukin-6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon-γ production by host CD8(+) T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed "Treg-friendly" agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.

A Consensus-Based Interpretation of the Benchmark Evidence from South American Trials: Treatment of Intracranial Pressure Trial.

Widely-varying published and presented analyses of the Benchmark Evidence From South American Trials: Treatment of Intracranial Pressure (BEST TRIP) randomized controlled trial of intracranial pressure (ICP) monitoring have suggested denying trial generalizability, questioning the need for ICP monitoring in severe traumatic brain injury (sTBI), re-assessing current clinical approaches to monitored ICP, and initiating a general ICP-monitoring moratorium. In response to this dissonance, 23 clinically-active, international opinion leaders in acute-care sTBI management met to draft a consensus statement to interpret this study. A Delphi method-based approach employed iterative pre-meeting polling to codify the group's general opinions, followed by an in-person meeting wherein individual statements were refined. Statements required an agreement threshold of more than 70% by blinded voting for approval. Seven precisely-worded statements resulted, with agreement levels of 83% to 100%. These statements, which should be read in toto to properly reflect the group's consensus positions, conclude that the BEST TRIP trial: 1) studied protocols, not ICP-monitoring per se; 2) applies only to those protocols and specific study groups and should not be generalized to other treatment approaches or patient groups; 3) strongly calls for further research on ICP interpretation and use; 4) should be applied cautiously to regions with much different treatment milieu; 5) did not investigate the utility of treating monitored ICP in the specific patient group with established intracranial hypertension; 6) should not change the practice of those currently monitoring ICP; and 7) provided a protocol, used in non-monitored study patients, that should be considered when treating without ICP monitoring. Consideration of these statements can clarify study interpretation.

Sequential monitoring and stability of ex vivo-expanded autologous and nonautologous regulatory T cells following infusion in nonhuman primates.

Ex vivo-expanded cynomolgus monkey CD4(+)CD25(+)CD127(-) regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-specific demethylation region, and potently suppressed T cell proliferation through three rounds of expansion. When carboxyfluorescein succinimidyl ester- or violet proliferation dye 450-labeled autologous (auto) and nonautologous (non-auto)-expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 postinfusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these nontransplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of nonhuman primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.

Pig-to-monkey islet xenotransplantation using multi-transgenic pigs.

The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months.

Haemophilia Experiences, Results and Opportunities (HERO) Study: influence of haemophilia on interpersonal relationships as reported by adults with haemophilia and parents of children with haemophilia.

Evidence delineating the effects of haemophilia on interpersonal relationships is sparse and largely outdated, failing to reflect the impact of current treatment strategies. HERO (Haemophilia Experiences, Results and Opportunities) was commenced to garner a more comprehensive understanding of psychosocial issues facing persons with haemophilia (PWH). This article describes the findings of the quantitative HERO survey relating to the influence of haemophilia on interpersonal relationships of adult PWH, and parents/caregivers of children with haemophilia. Separate questionnaires were completed by adult PWH and parents of minor children from 10 countries, including satisfaction with support from partners, family, friends and other social contacts; disclosure of haemophilia and carrier status and family dynamics. A total of 675 PWH and 561 parents completed the survey. Over half of PWH (57%) and parents (84%) were married. Most PWH were satisfied with support from partners (94%), family (90%) and friends (85%), with lower percentages reported among those with inhibitors. Most parents were likewise satisfied with support from partners (88%) and family (83%). Whereas PWH were reticent to disclose their diagnosis beyond family and friends, parents were more likely to share their son's diagnosis, and most were satisfied with the support from their son's peers (74%), teachers (83%) and other adults in supervisory roles (85%). PWH and parents surveyed were satisfied overall with the support they received from partners, family, friends and social contacts. Relationships are affected by haemophilia in various ways, and particularly affected in terms of disease burden, age and social life.

Hyperlactataemia in HIV-infected subjects initiating antiretroviral therapy in a large randomized study (a substudy of the INITIO trial).

OBJECTIVE: The aim of the study was to evaluate the predictive value of clinical and molecular risk factors, including peripheral blood mononuclear cell (PBMC) mitochondrial DNA (mtDNA) and mitochondrial RNA (mtRNA), for the development of lactic acidosis (LA) and symptomatic hyperlactataemia (SHL). METHODS: In a substudy of a large multicentre, randomized trial of three antiretroviral regimens, all containing didanosine (ddI) and stavudine (d4T), in antiretroviralnaïve, HIV-1-infected patients, patients with LA/SHL ('cases') were compared with those without LA/SHL in a univariate analysis, with significant parameters analysed in a multivariate model. In a molecular substudy, PBMC mtDNA and mtRNA from cases and matched controls at baseline and time of event were examined. RESULTS: In 911 subjects followed for a median of 192 weeks, 24 cases were identified (14 SHL and 10 LA). In univariate analysis, cases were more likely to be female (P=0.05) and to have a high body mass index (BMI) (P=0.02). In multivariate analyses, only BMI remained an independent predictor of the development of LA/SHL (P=0.03). Between cases and controls there was no significant difference in mtDNA copy number at baseline (389 vs. 411 copies/cell, respectively; P=0.60) or at time of event (329 vs. 474 copies/cell, respectively; P=0.21), in the change in mtDNA copy number from baseline to event (-65 vs. +113 copies/cell, respectively; P=0.12), in mtRNA expression at baseline or time of event, or in the change in mtRNA expression from baseline to event. CONCLUSION: The development of LA/SHL was associated with increased BMI, but PBMC mtDNA and mtRNA did not predict LA/SHL. This demonstrates the ineffectiveness of routine measurement of PBMC mtDNA in patients on ddI and d4T as a means of predicting development of LA/SHL.

Antiviral activity of apricitabine in treatment-experienced HIV-1-infected patients with M184V who are failing combination therapy.

OBJECTIVES: Apricitabine (ATC) is a novel deoxycytidine analogue nucleoside reverse transcriptase inhibitor (NRTI) with significant antiviral activity in vitro, including activity against HIV-1 with reverse transcriptase mutations that confer resistance to other NRTIs. ATC has shown promising antiviral activity and good tolerability when given as monotherapy for 10 days in treatment-naïve HIV-1-infected patients. METHODS: In this Phase II randomized, double-blind study, 51 treatment-experienced HIV-1-infected patients with the reverse transcriptase mutation M184V who were failing therapy which included lamivudine (3TC) were randomized to receive twice-daily 600 mg ATC, 800 mg ATC or 150 mg 3TC for 21 days. Patients remained on their existing background regimen until day 21, when background therapy could be optimized according to genotype at screening. RESULTS: At day 21, the mean change in viral load was -0.71 and -0.90 log(10) HIV-1 RNA copies/mL in the 600 and 800 mg ATC groups, respectively, compared with a -0.03 log(10) change in the 3TC group. In patients with at least three thymidine analogue mutations (TAMs) at baseline, greater reductions in viral load were observed in the 800 mg ATC group at day 21 than in the 600 mg ATC group. Few genotypic changes were detected at day 21 [two patients (600 mg ATC) lost and three patients (800 mg ATC) gained a TAM] and all patients with detectable virus retained the M184V mutation. The safety profiles of the two ATC doses were similar to that of 3TC. CONCLUSIONS: Over the 21-day treatment period, ATC showed promising antiviral activity and was well tolerated in treatment-experienced patients with M184V, with or without additional TAMs.

Investigation of lymphocyte depletion and repopulation using alemtuzumab (Campath-1H) in cynomolgus monkeys.

As the target CD52 molecule is expressed on erythrocytes of most nonhuman primate strains, using alemtuzumab in these species would cause massive hemolysis. Six cynomolgus monkeys of Indonesian origin, screened by agglutination assay for absence of CD52 on erythrocytes, were administered alemtuzumab in a cumulative dose to a maximum of 60 mg/kg. In two monkeys, mycophenolate mofetil (MMF) was added as maintenance therapy. Complete depletion of T and B lymphocytes (>99.5%) was achieved with 20 mg/kg alemtuzumab and was more profound than in monkeys treated with antithymocyte globulin (n = 5), as quantified by flow cytometry. Repopulation was suppressed by weekly injections of 10 mg/kg. Without MMF, repopulation of CD20(+)B cells and CD8(+)T cells was complete within 2 and 3 months, respectively, and repopulation of CD4(+)T cells was 67% after 1 year. MMF significantly delayed CD4(+)T-cell repopulation. Among repopulating CD4(+) and CD8(+) T cells, a phenotypic shift was observed from CD45RA(hi)CD62L(hi) naïve cells toward CD45RA(lo)CD62L(lo) effector memory cells. In lymph nodes, the depletion of naïve cells was more profound than of memory cells, which may have initiated a proliferation of memory cells. This model offers opportunities to investigate lymphocyte depletion/repopulation phenomena, as well as the efficacy of alemtuzumab in preclinical transplantation models.

Inflammation-induced modulation of cellular galectin-1 and -3 expression in a model of rat peritonitis.

OBJECTIVE AND DESIGN: To investigate the effect of galectin-1 (Gal-1) and -3 (Gal-3) on leukocyte migration and analyze the expression of both galectins in inflammatory cells using a model of rat peritonitis. MATERIAL OR SUBJECTS: Sprague-Dawley rats (n = 4 per group). TREATMENT: Peritonitis was induced in animals through intraperitoneal injection of carrageenin (1.5 mg/kg) and rat mesenteries were analyzed at different time points (0, 4, 24 and 48h). For pharmacological treatment, rats received intravenous injection of Gal-1 or -3 (3microg/kg) followed by carrageenin. METHODS: Western blotting and immunoelectron microscopy analysis. Statistical analysis was performed using ANOVA followed by Bonferroni test. RESULTS: Pharmacological treatment with Gal-1, but not Gal-3, inhibited (approximately 50 %) leukocyte recruitment into the peritoneal cavity at 4 h time-point. In this early phase, immunogold staining of mesenteries showed a diminished Gal-3 expression in degranulated mast cells and Gal-1 in transmigrated neutrophils (approximately 20 % reduction compared to intravascular cells). In the later phases (24 and 48h), leukocyte turnover was associated with augmented Gal-1 expression in neutrophils and macrophages and Gal-3 in mast cells and macrophages. CONCLUSIONS: These results point to a balanced expression of cell-associated-Gal-1/Gal-3 and might impact on the development of new therapeutic strategies for inflammatory diseases.

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials.

cDNAs encoding IgM heavy chain constant region (Cmu) were isolated from two metatherians (marsupials)--the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cmu in both species. The domain size and structure of the marsupial Cmu sequences were compared with other Cmu sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.

Intravenous synthetic alphaGal saccharides delay hyperacute rejection following pig-to-baboon heart transplantation.

Several oligosaccharides containing the terminal structure Gal(alpha)1-3Gal (alphaGal) and different side chains were tested in vitro for their ability to block natural anti(alpha)Gal antibodies. A di-and a trisaccharide (di(alpha)Gal and tri(alpha)Gal) were selected. A blood group B baboon, having IgG and IgM natural antipig titers of 1:256 and 1:1024 and a hemolytic titer (to pig red blood cells, RBCs) of 1:8, was chosen to measure pharmacokinetic parameters of the saccharides and to assess the extent of in vivo neutralization of the antibodies. Three grams each of the di(alpha)Gal and the tri(alpha)Gal dissolved in saline were administered by bolus intravenous (i.v.) injection. Blood samples were collected at various times and urine was collected at 8 and 24 h. Plasma and urine concentrations of the alphaGal saccharides were estimated by an ELISA specially developed for this study. A fast distribution phase followed by equilibrium and excretion phases were observed, indicating a T1/2 in the order of 1 h. Fifty-eight per cent of the saccharides were recovered in the urine within 24 h. Determination of antipig antibody binding by FACS analysis and of serum cytotoxicity titers for pig endothelial cells demonstrated that a 70% reduction in binding and cytotoxicity could be achieved with plasma saccharide levels of 300-400 microg/ml. Six months later, a pig heart was transplanted heterotopically into the baboon. A 3-g bolus of the saccharide mixture (1.5 g of each saccharide) was given i.v. before allowing blood reperfusion of the transplanted heart, followed by an i.v. infusion of 1 g/hr for 1 hr and 0.5 g/hr for the 3 succeeding hours. Blood concentrations of the saccharides, CH50, hematology and cytotoxicity for PK15 cells were estimated in blood samples taken at various times. Heart function was observed to be satisfactory for 8 h, but was found to have ceased at 18 h. Myocardial biopsies taken at 3 and 5 h showed congestion only, suggestive of minimal vascular rejection, but by 18 h demonstrated severe vascular rejection. In conclusion, alphaGal saccharide therapy given for a period of 4 h delayed, but did not totally prevent, the development of vascular rejection in the pig-to-baboon heart transplant model. alphaGal saccharide therapy may be one of several useful approaches for the prevention of hyperacute rejection in pig-to-primate organ transplantation.

Effect of exercise training on energy expenditure, muscle volume, and maximal oxygen uptake in female adolescents.

OBJECTIVES: American female adolescents are at high risk of a physically inactive lifestyle that likely leads to health problems later in life. We hypothesized that a brief program of endurance exercise training in female adolescents would result in increased energy expenditure and quantifiable structural and functional adaptations. STUDY DESIGN: Forty-four high school girls (aged 15 to 17 years, none were elite athletes) enrolled in a 5-day per week anatomy class for 5 weeks and were randomly assigned to control (n = 22) and training groups. All subjects participated in a 2-hour daily teaching program. During the remaining time (2 hours), the training group members underwent endurance-type training and control group subjects participated in a computer workshop. The intervention was assessed by (1) comparison of total energy expenditure between groups with the doubly labeled water technique, (2) determination of changes in thigh muscle volume by magnetic resonance imaging, and (3) determination of changes in maximal oxygen uptake by use of respiratory gas exchange responses. RESULTS: Total energy expenditure was significantly greater (15.3%) in the training group compared with the control subjects (p < 0.003). Five weeks of training led to a 4.3% +/- 1% increase in thigh muscle volume (p < 0.0002) and a 12.1% +/- 3.7% increase in maximal oxygen uptake (p < 0.004); there were no changes in the control group. The training effect was most pronounced in the least fit subjects. CONCLUSIONS: Exercise training programs for female adolescents can be successfully integrated into a high school summer curriculum. Quantifiable, substantial structural and functional responses occur with relatively short periods of training. Approximately 60% of the training response was related to factors independent of muscle size per se. These data may serve to better design physical activity programs for female adolescents.

The thyroid status of the Yanomamö Indians of southern Venezuela: 1992 update.

Previous reports described an apparent paradox in the clinical expression of endemic iodine deficiency in Amazonian Indians, who were severely iodine deficient but had no goiter and were clinically euthyroid. To confirm and explain this unique observation, we estimated the iodine intake and evaluated the functional and structural thyroid status of an isolated population of Yanomamö Indians (n = 104) in southern Venezuela. Twenty-six children, 32 adolescents, and 46 adults were studied with serum thyroid function tests, spot urinary iodide concentration determinations, and sonographic measurement of thyroid gland volume. A subset of adolescents and adults (n = 35) had 24-h fractional thyroidal 131I uptake determinations. No individual with goiter, clinical hypothyroidism, or cretinism was observed. Children had higher mean serum T4 (114.5 vs. 102.9 nmol/L; P < 0.02) and serum T3 (2.96 vs. 2.46 nmol/L; P < 0.02) concentrations than adults, with 2% and 50%, respectively, of children having levels more than 2 SD above the mean for a North American reference population. Serum TSH concentrations were also higher in children than adults (3.3 vs. 2.3 mU/L; P < 0.02), with 25% of the subjects above the reference range. Mean urinary iodide was 61 +/- 29 micrograms/L (range, 10-178 micrograms/L), reflecting borderline iodine sufficiency. Thyroidal 131I uptake values were 28 +/- 7%, with 33% above the upper limit of normal (30%). Compared to an iodine-replete Swedish population, the thyroid gland volume was above normal (> mean + 2 SD) in 71% of the study children. We conclude that the Yanomamö have borderline iodine deficiency, associated in children with predictably higher serum T3 and TSH concentrations and a high prevalence of small goiters. The basis for the apparent change in their dietary iodide intake over the past 30 yr is unclear, as is the explanation for their previously postulated resistance to goitrogenesis with more profound iodine deficiency.

PCR-Based Full-Length cDNA Cloning Utilizing the Universal-Adaptor/Specific DOS Primer-Pair Strategy.

Biological systems are often influenced by molecules that are neither present in vast quantities or easily purified to homogeneity from other cellular constituents. The development of simple, efficient molecular cloning systems coupled with the relative ease of DNA sequence determination has made nucleic acid sequence determination the choice methodology when sequence determination of the complete biological molecule is indicated. However, one bottleneck that impedes direct access to sequence determination is the necessity to screen recombinant libraries containing a large number of clones for the low-abundance member. Advances in protein microsequencing techniques combined with application of the polymerase chain reaction (PCR) help simplify this process (1-3). Utilizing only the N-terminal protein sequence information, we have developed a protocol for the selective amplification and subsequent cloning of specific full-length cDNAs, which in some instances may circumvent the time-consuming, tedious process of library screening. This approach, as illustrated in Fig. 1, employs the following steps:

Enzyme-linked immunosorbent assay for the detection of substances that carry blood group A specificity.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate the amount of material carrying blood group A activity in biologic samples. A soluble synthetic form of the A antigenic determinant (A trisaccharide, ATS) conjugated to peroxidase competes with the blood group A substance present in a biologic sample for anti-A attached to a solid phase by a second antibody coating the plastic micro-wells. A reference curve is constructed by using known quantities of ATS to compete with a fixed amount of ATS-peroxidase conjugate. The A substance activity in a sample is obtained by extrapolating the degree of inhibition of the binding of the ATS-peroxidase conjugate to an equivalent amount of ATS in the reference curve. The assay is reproducible, specific, and sensitive. It has been used in pharmacologic studies to estimate the concentration of ATS in the blood and urine of rats, rabbits, and baboons and in a study with human samples, testing the potential clinical use of ATS to neutralize anti-A when therapeutically indicated. It is also useful for the detection of ABO natural products in secretions, thus allowing the accurate classification of secretor and nonsecretor individuals.

Are obese children truly unfit? Minimizing the confounding effect of body size on the exercise response.

To test the hypothesis that obese children are unfit (i.e., have abnormal responses to exercise testing consistent with reduced levels of habitual physical activity), we used new analytic strategies in studies of 18 obese children performing cycle ergometry. The subject's weight (mean +/- SD) was 168 +/- 24% that predicted by height, and the age range was 9 to 17 years. Size-independent measures of exercise (e.g., the ratio of oxygen uptake (VO2) to work rate during progressive exercise and the temporal response of VO2, carbon dioxide output (VCO2), and minute ventilation (VE) at the onset of exercise) were used. The ability to perform external mechanical work was corrected for VO2 at unloaded pedaling (change in maximum oxygen uptake (delta VO2max) and in anaerobic threshold (delta AT). On average, obese children's responses were in the normal range: delta VO2max, 104 +/- 41% (+/- SD) predicted (by age); delta AT, 85 +/- 51%; ratio of change in VE to change in VCO2, 111 +/- 21% and ratio of change in VO2 to change in work rate, 100 +/- 24%, but six of the obese children had values of delta VO2max or delta AT that were more than 2 SD below normal. In addition, obese children did not have increased delta VO2max or delta AT with age as observed in nonobese children. Although the response time of VO2 was normal (99 +/- 32% of predicted), those for both VCO2 and VE were prolonged. We conclude that the finding of obesity in a child is not a reliable indicator of poor fitness but that testing cardiorespiratory responses to exercise can be used to identify subjects with serious impairment and to individualize therapy.