Factors associated with long-term weight-loss maintenance following bariatric surgery in adolescents with severe obesity.

BACKGROUND/OBJECTIVES: Bariatric surgery produces robust weight loss, however, factors associated with long-term weight-loss maintenance among adolescents undergoing Roux-en-Y gastric bypass surgery are unknown. SUBJECTS/METHODS: Fifty adolescents (mean±s.d. age and body mass index (BMI)=17.1±1.7 years and 59±11 kg m-2) underwent Roux-en-Y gastric bypass surgery, had follow-up visits at 1 year and at a visit between 5 and 12 years following surgery (Follow-up of Adolescent Bariatric Surgery at 5 Plus years (FABS-5+) visit; mean±s.d. 8.1±1.6 years). A non-surgical comparison group (n=30; mean±s.d. age and BMI=15.3±1.7 years and BMI=52±8 kg m-2) was recruited to compare weight trajectories over time. Questionnaires (health-related and eating behaviors, health responsibility, impact of weight on quality of life (QOL), international physical activity questionnaire and dietary habits via surgery guidelines) were administered at the FABS-5+ visit. Post hoc, participants were split into two groups: long-term weight-loss maintainers (n=23; baseline BMI=58.2 kg m-2; 1-year BMI=35.8 kg m-2; FABS-5+ BMI=34.9 kg m-2) and re-gainers (n=27; baseline BMI=59.8 kg m-2; 1-year BMI=36.8 kg m-2; FABS-5+ BMI=48.0 kg m-2) to compare factors which might contribute to differences. Data were analyzed using generalized estimating equations adjusted for age, sex, baseline BMI, baseline diabetes status and length of follow-up. RESULTS: The BMI of the surgical group declined from baseline to 1 year (-38.5±6.9%), which, despite some regain, was largely maintained until FABS-5+ (-29.6±13.9% change). The BMI of the comparison group increased from baseline to the FABS-5+ visit (+10.3±20.6%). When the surgical group was split into maintainers and re-gainers, no differences in weight-related and eating behaviors, health responsibility, physical activity/inactivity, or dietary habits were observed between groups. However, at FABS-5+, maintainers had greater overall QOL scores than re-gainers (87.5±10.5 vs 65.4±20.2, P<0.001) and in each QOL sub-domain (P<0.01 all). CONCLUSIONS: Long-term weight outcomes for those who underwent weight-loss surgery were superior to those who did not undergo surgical treatment. While no behavioral factors were identified as predictors of success in long-term weight-loss maintenance, greater QOL was strongly associated with maintenance of weight loss among adolescents who underwent Roux-en-Y gastric bypass surgery surgery.

Bariatric Surgery for Adolescents with Type 2 Diabetes: an Emerging Therapeutic Strategy.

PURPOSE OF REVIEW: Type 2 diabetes (T2D) is a growing public health problem in youth, but conventional treatments are often insufficient to treat this disease and its comorbidities. We review evidence supporting an emerging role for bariatric surgery as a treatment for adolescent T2D. RECENT FINDINGS: Paralleling what has been seen in adult patients, bariatric surgery dramatically improves glycemic control in patients with T2D. In fact, remission of T2D has been observed in as many as 95-100% of adolescents with diabetes after bariatric surgery, particularly vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass (RYGB) surgery. This striking outcome may be due to both weight-dependent- and weight-independent factors, and recent studies suggest that T2D-related comorbidities may also improve after surgery. Bariatric surgery including RYGB and VSG is a powerful therapeutic option for obese adolescents with T2D. Benefits must be weighed against risk for postoperative complications such as nutritional deficiencies, but earlier surgical intervention might lead to more complete metabolic remission in obese patients with T2D.

Lipid changes 8 years post gastric bypass in adolescents with severe obesity (FABS-5+ study).

OBJECTIVE: Severe obesity in adolescents is increasing and few effective treatments exist. Bariatric surgery is one option, but the extent to which surgery influences cardiovascular risk factors over time in youth is not clear. We hypothesized that Roux-en Y gastric bypass (RYGB) would be associated with sustained improvements in lipids over time (>5 years). PARTICIPANTS/METHODS: Youth who underwent RYGB from 2001 to 2007 were recruited for the Follow-up of Adolescent Bariatric Surgery-5+ (FABS-5+) in 2011-2014. Baseline body mass index (BMI) and lipids were abstracted from medical records. Follow-up data were obtained at a research visit. Analyses included paired t-tests to assess changes in BMI and lipids over time. General linear models were used to evaluate predictors of high-density lipoprotein (HDL) and non-HDL-cholesterol at follow-up. A non-operative group was recruited for comparison. RESULTS: Surgical participants (n=58) were a mean±s.d. age of 17±2 years at baseline and 25±2 years at long-term follow-up. Eighty-six percent were Caucasian and 64% were female. At long-term follow-up BMI decreased by 29% and all lipids (except total cholesterol) significantly improved (P<0.01). Female sex was a significant predictor of non-HDL-cholesterol level at 1 year, while change in BMI from 1 year to long-term follow-up was a significant predictor of non-HDL-cholesterol and HDL-cholesterol during the same interval (P<0.05). In the non-operative group, BMI increased by 8% and lipid parameters were unchanged. CONCLUSIONS: This is the longest and most complete follow-up of youth following RYGB. Weight loss maintenance over time was significantly associated with improvements in lipid profile over 5 years.

Weight loss surgery in adolescents corrects high-density lipoprotein subspecies and their function.

BACKGROUND/OBJECTIVE: Youth with obesity have an altered high-density lipoprotein (HDL) subspecies profile characterized by depletion of large apoE-rich HDL particles and an enrichment of small HDL particles. The goal of this study was to test the hypothesis that this atherogenic HDL profile is reversible and that HDL function would improve with metabolic surgery. METHODS: Serum samples from adolescent males with severe obesity mean±s.d. age of 17.4±1.6 years were studied at baseline and 1 year following vertical sleeve gastrectomy (VSG). HDL subspecies and HDL function were evaluated pre and post VSG using paired t-tests. A lean group of adolescents was included as a reference group. RESULTS: After VSG, body mass index decreased by 32% and insulin resistance as estimated by homeostatic model assessment of insulin resistance decreased by 75% (both P<0.01). Large apoE-rich HDL subspecies increased following VSG (P<0.01) and approached that of lean adolescents despite participants with considerable residual obesity. In addition, HDL function improved compared with baseline (cholesterol efflux capacity increased by 12%, HDL lipid peroxidation potential decreased by 30% and HDL anti-oxidative capacity improved by 25%, all P<0.01). CONCLUSIONS: Metabolic surgery results in a significant improvement in the quantity of large HDL subspecies and HDL function. Our data suggest metabolic surgery may improve cardiovascular risk in adolescents and young adults.

Changes in inflammation, oxidative stress and adipokines following bariatric surgery among adolescents with severe obesity.

BACKGROUND/OBJECTIVES: Inflammation, oxidative stress and dysregulation of adipokines are thought to be pathophysiological mechanisms linking obesity to the development of insulin resistance and atherosclerosis. In adults, bariatric surgery reduces inflammation and oxidative stress, and beneficially changes the levels of several adipokines, but little is known about the postsurgical changes among adolescents. SUBJECTS/METHODS: In two separate longitudinal cohorts we evaluated change from baseline of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), monocyte chemo-attractant protein-1 (MCP-1), oxidized low-density lipoprotein cholesterol (oxLDL), adiponectin, leptin and resistin up to 12 months following elective laparoscopic Roux-en-Y gastric bypass (RYGB) or vertical sleeve gastrectomy (VSG) surgery in adolescents with severe obesity. RESULTS: In cohort 1, which consisted of 39 adolescents (mean age 16.5±1.6 years; 29 females) undergoing either RYGB or VSG, IL-6 (baseline: 2.3±3.4 pg ml(-1) vs 12 months: 0.8±0.6 pg ml(-1), P<0.01), leptin (baseline: 178±224 ng ml(-1) vs 12 months: 41.4±31.9 ng ml(-1), P<0.001) and oxLDL (baseline: 41.6±11.6 U l(-1) vs 12 months: 35.5±11.1 U l(-1), P=0.001) significantly decreased and adiponectin significantly increased (baseline: 5.4±2.4 µg ml(-1) vs 12 months: 13.5±8.9 µg ml(-1), P<0.001). In cohort 2, which consisted of 13 adolescents (mean age 16.5±1.6 years; 10 females) undergoing RYGB, results were similar: IL-6 (baseline: 1.7±0.9 pg ml(-1) vs 12 months: 0.4±0.9 pg ml(-1), P<0.05) and leptin (baseline: 92.9±31.3 ng ml(-1) vs 12 months: 37.3±33.4 ng ml(-1), P<0.001) significantly decreased and adiponectin significantly increased (baseline: 6.1±2.9 µg ml(-1) vs 12 months: 15.4±8.0 µg ml(-1), P<0.001). When the cohorts were combined to evaluate changes at 12 months, oxLDL also significantly decreased (baseline: 39.8±16.7 U l(-1) vs 12 months: 32.7±11.9 U l(-1), P=0.03). CONCLUSIONS: Bariatric surgery produced robust improvements in markers of inflammation, oxidative stress and several adipokines among adolescents with severe obesity, suggesting potential reductions in risk for type 2 diabetes and cardiovascular disease.

An evolving scientific basis for the prevention and treatment of pediatric obesity.

The 2013 Pennington Biomedical Research Center's Scientific Symposium focused on the treatment and management of pediatric obesity and was designed to (i) review recent scientific advances in the prevention, clinical treatment and management of pediatric obesity, (ii) integrate the latest published and unpublished findings and (iii) explore how these advances can be integrated into clinical and public health approaches. The symposium provided an overview of important new advances in the field, which led to several recommendations for incorporating the scientific evidence into practice. The science presented covered a range of topics related to pediatric obesity, including the role of genetic differences, epigenetic events influenced by in utero development, pre-pregnancy maternal obesity status, maternal nutrition and maternal weight gain on developmental programming of adiposity in offspring. Finally, the relative merits of a range of various behavioral approaches targeted at pediatric obesity were covered, together with the specific roles of pharmacotherapy and bariatric surgery in pediatric populations. In summary, pediatric obesity is a very challenging problem that is unprecedented in evolutionary terms; one which has the capacity to negate many of the health benefits that have contributed to the increased longevity observed in the developed world.

Three-decade metabolic outcome of neonatal gastrectomy and early Roux-en-Y.

Little information is available about long-term outcomes of major gastric surgery when performed very early in life and adverse consequences in growing children might be expected. In this case, gastrectomy with Roux-en-Y esophagojejunostomy was performed in early childhood. Despite stomach loss, growth velocity paralleled the third percentile for age during development. Maintained on a daily multivitamin and monthly B12 injections, no overt nutritional deficiencies were detected in adulthood. However, dual energy X-ray absorptiometry scan at age 31 revealed that the patient had abnormally low bone mineral density. This case study demonstrates that even after gastrectomy and reconstruction early in life, linear growth can be achieved. However, bone density can be adversely affected, even in the face of normal serum calcium and vitamin D levels.

Bariatric surgery for adolescents.

Obesity is no longer just an adult disease. An increasing number of youth are overweight, defined as body mass index (BMI) at or greater than the 95th percentile for age (1). Between 2009 and 2010, 16.9% of children aged 2­19 yr were classified as overweight based on BMI (2), as compared with only 5% of children affected by obesity in 1976­1980 (3). This is a problem of enormous proportion from a public health standpoint, as without intervention these children will grow up to become overweight and obese adults. For an obese child, the risk of becoming an obese adult may be as high as 77%, compared with 7%for a child of healthy weight (4). Morbid obesity is a major risk factor for later complications such as cardiovascular disease, type 2 diabetes, obstructive sleep apnea (OSA), polycystic ovary syndrome (PCOS), and degenerative joint disease (4­10). Obesity is also an expensive problem: the US government spends $147 billion yearly on obesity-related healthcare costs (11). Thus, there is an urgent need to target obesity in the pediatric population, before the expensive and life-threatening consequences of obesity manifest. Unfortunately, the effectiveness of medical treatments for obesity is limited. Behaviorally based dietary and physical activity interventions offer little benefit for pediatric obesity, while pharmacologic therapy is also limited and carries low success rates and recidivism (12­14) (Table 1).

Bariatric surgery for pediatric extreme obesity: now or later?

Obesity is a multifactorial disease of epidemic and global proportions that poses the most significant threat to the health of our younger generations. Those who are the most extremely affected bear the largest burden of health problems. In the US, extreme obesity affects approximately 9 million adults and 2 million children, and is associated with both immediate health problems and later health risk, including premature mortality. Present medical and behavioral interventions for extreme obesity in adults and children rarely result in the significant, durable weight loss necessary to improve health outcomes, prompting a search for more aggressive measures. Weight loss (bariatric) surgery has been advocated as an intervention for those with extreme obesity. In adults, bariatric surgery results in prolonged weight control and improvement in serious obesity comorbidities, namely type 2 diabetes, dyslipidemias, hypertension and obstructive sleep apnea syndrome. A surge in weight loss operations for adolescents has been observed recently, with a threefold increase in case volumes nationwide from 2000 to 2003. Current evidence suggests that after bariatric surgery, adolescents lose significant weight and serious obesity-related medical conditions and psychosocial status are improved. Thus it is reasonable to propose that bariatric surgery performed in the adolescent period may be more effective treatment for childhood-onset extreme obesity than delaying surgery for extremely obese youth until adulthood. This position has been echoed by a number of groups and an independent systematic review. Finally, it is conceivable that bariatric surgery performed in adulthood for childhood onset extreme obesity may not be as effective for comorbidity treatment as surgery performed earlier during adolescence. The purpose of this review is to examine the evidence, which supports early rather than later use of bariatric surgery in the treatment of extreme obesity, and to present this information in light of the medical and surgical risks of bariatric surgery.

Reduced hospitalization cost for patients with pectus excavatum treated using minimally invasive surgery.

BACKGROUND: Currently, few data exist regarding the relative costs associated with open and minimally invasive pectus excavatum repair. The aim of this study was to compare the surgical and hospitalization costs for these two surgical techniques and to identify factors responsible for cost differences. METHODS: A retrospective review of hospital charts, patient and parent questionnaires, and hospital accounting records was performed for 68 patients who underwent surgical correction of pectus excavatum between June 1996 and December 1999. RESULTS: In this series, 25 patients underwent open repair, whereas 43 patients underwent minimally invasive repair of pectus excavatum (MIRPE). The patient ages ranged from 4 to 19 years. The average ages for open repair (12 years) and MIRPE (11 years) did not differ significantly. As compared with open repair, MIRPE was associated with a 27% lower overall cost of hospitalization ( p < 0.05). The operating room costs were 12% higher for the patients who underwent MIRPE ( p < 0.05). The mean operative time for open repair was 3 h 15 min, whereas MIRPE required 1 h 10 min ( p < 0.001). The hospital stay for open repair averaged 4.4 days, as compared with 2.4 days for MIRPE ( p < 0.001). In contrast to other published series, the postoperative analgesia after MIRPE in this series consisted of narcotics, ketorolac, and methocarbamol. No patient received epidural analgesia, regardless of the repair technique selected. The postoperative complication rate was 4% in the open group and 14% in the MIRPE group. Most of the patients treated with either open or MIRPE reported postoperative oral narcotic usage for 2 weeks or less and returned to routine activities within 3 weeks. The patients and parents alike reported good to excellent overall outcomes in 85% or more of the open repair cases and 90% or more of the MIRPE cases. CONCLUSIONS: These data demonstrate for the first time that the use of an alternate pain management strategy including, narcotics, NSAIDs, and methocarbamol, but without epidural catheters, results in reduced hospital length of stay and decreased overall hospitalization costs for MIRPE, as compared with open pectus repair. This cost benefit was achieved without compromising pain management or patient satisfaction with surgical care.

Laparoscopically assisted anorectal pull-through for high imperforate anus--a new technique.

BACKGROUND/PURPOSE: This report describes a new technique of laparoscopically assisted anorectal pull-through (LAARP) for repair of high imperforate anus. The procedure utilizes minimal perineal dissection, preservation of the distal rectum, and accurate placement of the rectum within the levator ani and external anal sphincter muscle complex. METHODS: Sharp dissection and cautery was used laparoscopically to expose the rectal pouch down to the urethral or vaginal fistula, which was clipped distally and divided. The pelvic floor musculature was then assessed and the levator sling identified. Externally, electrostimulation was used to define the center of the anal dimple. An 8-mm skin incision was made, centered at the strongest cephalad contraction. Using a hemostat, minimal blunt dissection on the perineum was guided by transillumination from the laparoscopic light source. A trocar, consisting of a radially expandable sheath over a Varess needle, was passed through this defined plane in the external sphincter muscle complex and advanced into the pelvis between the 2 bellies of the pubococcygeus muscle, guided by laparoscopic visualization. This perineal trocar therefore formed a passage through the center of the striated muscle complex and levators. The rectal fistula, which had been dissected out laparoscopically, was grasped using the perineal trocar and exteriorized to the perineum. Anorectal anastomosis was performed with absorbable interrupted suture. RESULTS: Seven patients were treated with initial colostomy in the newborn period followed by delayed LAARP 2 to 12 months later. In 4 newborn infants, the LAARP was performed as a primary procedure without prior colostomy. Laparoscopic mobilization has been possible on all cases attempted. All of the patients have a brisk and symmetric anal contraction with perineal electrostimulation. CONCLUSIONS: Lack of long-term follow-up precludes accurate assessment of the potential for fecal continence. However, short-term experience has been that this new method of pull-through for imperforate anus offers many advantages, including excellent visualization of the rectal fistula and surrounding structures, accurate placement of the bowel through the anatomic midline and levator sling, and minimally invasive abdominal and perineal wounds.

Outcome of Nissen fundoplication using intraoperative manometry in children.

BACKGROUND: Intraoperative manometry is useful in performing Nissen fundoplication (NF) in children. Long-term clinical outcome information after use of this method is lacking. METHODS: A retrospective review of the outcomes of 62 consecutive NFs using intraoperative manometry was performed. The follow-up period was 3.4 years. Approximately half of the patients were neurologically normal (NN) and half were neurologically impaired (NI). All patients with gastroesophageal reflux disease (GERD) did not respond to an adequate trial of medical treatment. RESULTS: The NF was tailored to result in a twofold increase in the lower esophageal sphincter pressure (LESP) and a 75% increase in the LES length (LESL). An accelerated growth rate in 40% of "failure to thrive" (FTT) patients was demonstrated. Eighty-four percent of caregivers reported improved quality of life after NF. There was a twofold reduction in the number of hospital admissions and a sixfold reduction in total inpatient days for both NI and NN children. The early and late mortality rate was 13%, and the complication rate was similar to other series reported in the literature, with more complications occurring in NI patients. There was a 2% incidence of wrap herniation. An improvement in long-term outcomes after NF was seen in 89% of NN children and over half of NI patients. CONCLUSIONS: Intraoperative manometry is useful in standardizing the tightness of the wrap in NF. There was a low incidence of complications, dysphagia, recurrent emesis, and GERD in this series. Long-term outcomes using this technique were deemed very good based on caregivers' responses.

Ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression.

BACKGROUND: Lymphocytes obtained from tumor-draining lymph nodes (DLN) can have potent in vivo antitumor activity after in vitro activation with bryostatin 1 and ionomycin. However, the presence of visceral metastases in the donor can inhibit the effectiveness of such lymphocytes. In the present study, we tested the ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression in a murine model. METHODS: Mice were injected with MCA-105 sarcoma cells in the footpad alone or in the footpad and the tail vein to establish lung metastases. Cyclophosphamide was given i.p. 1 day before harvesting the draining popliteal lymph nodes. For all donor groups, DLN cells were activated with 5 nM bryostatin 1 and 1 microM ionomycin and cultured for 7 days in 20 U/ml IL-2. Activated DLN cells were then adoptively transferred to syngeneic mice with 3-day lung metastases. RESULTS: The adoptive transfer of DLN cells from mice with footpad tumors only significantly reduced the number of lung metastases compared to untreated mice. However, activated DLN cells obtained from mice with both footpad and lung tumors were significantly less effective. Treatment of similar donor mice with 10 mg/kg cyclophosphamide significantly improved the anti-tumor activity of adoptively transferred cells. This dose of cyclophosphamide did not reduce the number of cells obtained from each lymph node or the expansion of cell numbers in vitro. CONCLUSIONS: These results suggest that the administration of low-dose cyclophosphamide prior to harvesting DLN cells may improve the success of adoptive immunotherapy in cancer patients.

gamma-Interferon plays a key role in T-cell-induced tumor regression.

Recent studies have demonstrated that noncytolytic T-cells can mediate regression of murine tumors. In this report, we demonstrate that MCA-105 tumor-draining lymph node cells (DLN) activated with the protein kinase C activator, bryostatin 1, plus a calcium ionophore are capable of inducing specific tumor regression in vivo when adoptively transferred to mice with established metastases. However, these activated DLN cells lack in vitro cytotoxicity against autologous tumor. Antibody against gamma-interferon (IFN-gamma) markedly inhibited the therapeutic efficacy of these activated DLN cells. Anti-tumor necrosis factor produced a statistically significant but weaker inhibition of tumor regression. IFN-gamma, but not tumor necrosis factor alpha, could be shown to be secreted by activated DLN cells in vitro in response to specific tumor. Secretion of IFN-gamma was primarily a function of CD8+ T-cells. IFN-gamma was not directly cytotoxic to sarcoma cells in vitro. Moreover, tumor cells incubated with IFN-gamma were not more susceptible to lysis by activated DLN cells. However, recombinant murine IFN-gamma had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [3H]thymidine uptake assay. Similarly, supernatants obtained from DLN/autologous tumor cocultures markedly inhibited MCA-105 proliferation; this antiproliferative effect was abrogated by the addition of anti-IFN-gamma antibody to the cultures. These results suggest that secretion of IFN-gamma by adoptively transferred DLN cells plays an essential role in tumor rejection. The dominant effect of IFN-gamma may be its demonstrated antiproliferative activity.

Bryostatin 1 activates T cells that have antitumor activity.

Several strategies have been used to stimulate the growth of tumor-specific T cells in place of tumor antigen. One approach is to use pharmacologic agents to activate the second messenger pathways of T-cell activation. In the present study, we examined the ability of the protein kinase C activator bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to stimulate the growth of lymphocytes obtained from the axillary lymph nodes (DLN) draining a progressively growing intradermal plasmacytoma tumor. Draining lymph node cells were initially cultured with autologous tumor cells and 20 U/ml of interleukin-2 (IL-2) for 7 days. The lymphocytes were then incubated with various concentrations of bryostatin 1 plus 1 microM ionomycin and cultured for an additional 14 days in IL-2. DLN cells initially cultured with autologous tumor and then restimulated with 5 nM bryostatin 1 and 1 microM ionomycin exhibited marked in vitro proliferation and 15-fold expansion of cell numbers over 2 weeks. The cells expanded with B/I were predominantly CD8+ T cells and retained specific in vitro cytotoxicity against autologous tumor. When adoptively transferred to mice with established liver metastases, DLN cells restimulated with B/I-mediated specific tumor regression.

Adoptive transfer of bryostatin 1-activated T cells provides long-term protection from tumour metastases.

Treatment of human cancer with tumour-specific T lymphocytes is limited by the frequent unavailability of autologous tumour to stimulate T-cell growth and by the toxicity associated with high-dose interleukin-2 (IL-2) treatment. In the present study we demonstrate that Bryostatin 1 (B) plus ionomycin (I) can substitute for tumour antigen and activate tumour-bearing hosts' T-cells which provide long-term protection against tumour challenge after adoptive transfer. Lymphocytes obtained from the popliteal lymph nodes (DLN) draining an MCA-105 footpad sarcoma were stimulated with B/I, and then cultured for 7 days with 20 U ml-1 IL-2. This in vitro stimulation protocol consistently expanded cell numbers greater than 20-fold during 7 days. Mice given B/I-stimulated draining lymph node (DLN) cells were protected from specific i.v. tumour challenge for at least 15 weeks after adoptive transfer, even in the absence of IL-2 treatment. Tumour immunity conferred by B/I-activated DLN cells was systemic and independent of host T-cells. However, resistance to tumour challenge was lost when either CD4+ or CD8+ T-cells were depleted in vivo. These studies indicate that DLN cells activated with bryostatin 1 and ionomycin persist long-term in vivo as functional memory cells after adoptive transfer.

Activation of CD8+ murine T cells from tumor-draining lymph nodes by phorbol dibutyrate plus calcium ionophore.

When lymphocytes from the lymph nodes draining the site of a progressively growing MCA-105 sarcoma are stimulated in vitro with autologous tumor and low-dose interleukin-2 (IL-2), they will grow and develop the ability to lyse autologous tumor cells in vitro; these lymphocytes can also eradicate tumor metastases in vivo. Phorbol esters and calcium ionophores activate signal transduction pathways in T cells and mimic the events triggered by antigen binding. We therefore sought to determine whether large numbers of MCA-105 tumor-specific, therapeutically active T cells could be obtained from MCA-105 draining lymph nodes (DLNs) following a brief exposure to phorbol dibutyrate (PDBu) and ionomycin (Io). DLN cells primarily stimulated with autologous tumor, followed by a secondary stimulation with PDBu-Io and cultured in 20 U/ml IL-2, demonstrated marked expansion of cell numbers during 3 weeks in culture, had moderate cytolytic activity [37% at effector:target ratio (E:T) = 80:1], and were all CD8+ T cells. In contrast, DLN cells stimulated primarily with PDBu-Io and cultured in 20 U/ml IL-2 demonstrated at least 8-10-fold greater growth than antigen-stimulated DLN cells during 3 weeks, were moderately cytolytic (31% at E:T = 80:1), and were a mixed population of CD8+ and CD4+ T lymphocytes. DLN cells that were expanded by either protocol, like cells stimulated repeatedly in vitro with tumor cells, could eliminate MCA-105 pulmonary metastases when given with IL-2 in an adoptive immunotherapy model. DLN cells stimulated primarily with PDBu-Io completely eradicated MCA-105 metastases but had no in vivo antitumor activity against the syngeneic B16 melanoma or MCA-203 sarcoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Bryostatin 1-activated T cells can traffic and mediate tumor regression.

Adoptive immunotherapy in humans may be limited by the lack of autologous tumor cells to activate and expand tumor-specific T cells. Pharmacologic manipulation of protein kinase C (PKC) and intracellular calcium may substitute for tumor antigen and stimulate T cells for adoptive immunotherapy. In the present study, we evaluated the ability of the PKC activator Bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to activate lymphocytes obtained from popliteal lymph nodes (DLN) draining an MCA-105 footpad tumor. The adoptive transfer of B/I-stimulated DLN cells eradicated MCA-105 pulmonary metastases. These lymphocytes do not require concomitant IL-2 administration to mediate regression of lung metastases. Three days after intrasplenic injection of tumor cells and splenectomy, mice were given iv injections of B/I-stimulated DLN cells. Adoptive immunotherapy with these cells induced regression of established liver metastases. In an intradermal tumor model, the adoptive transfer of B/I-stimulated MCA-105 DLN cells cured mice of MCA-105 intradermal (id) tumors, but did not induce regression of MCA-206 tumors. Mice cured of MCA-105 id tumors were protected against MCA-105, but not MCA-203, tumor challenge in the footpad 7 weeks after adoptive immunotherapy.

Immunomodulatory effects of transforming growth factor-beta on T lymphocytes. Induction of CD8 expression in the CTLL-2 cell line and in normal thymocytes.

We investigated the role of transforming growth factor-beta 1 (TGF-beta) in regulation of T cell growth and differentiation. Treatment of CTLL-2 cells with TGF-beta inhibited IL-2-dependent proliferation and caused morphologic changes as well as increased adherence. A major change of phenotype in TGF-beta-treated cells was the de novo expression of CD8 alpha chain in 35% of cells, which required the continuous presence of TGF-beta. Of the CD8 alpha+ cells, 20 to 30% co-expressed CD8 beta chain. Increased CD8 expression occurred even in the total absence of cell growth, was not a consequence of growth inhibition, and was not a result of selective growth or survival of CD8+ cells. New RNA synthesis was required for TGF beta-induced CD8 alpha surface expression, inasmuch as this was prevented by treatment with actinomycin D. Northern blot analysis demonstrated that cells treated with IL-2 + TGF-beta rapidly accumulated mRNA encoding both chains of the CD8 dimer, to a level fourfold greater than control by 6 to 12 h. In contrast, the IL-2-dependent increases in IL-2R alpha, IL-2R beta, and Granzyme B mRNA levels in these cultures were profoundly inhibited by TGF-beta. When unfractionated murine thymocytes were stimulated with phorbol dibutyrate plus ionomycin and cultured with IL-2 + TGF-beta, an increase in CD8 alpha mRNA was seen and greater numbers of CD8+ cells with higher levels of CD8 alpha and CD8 beta surface expression resulted, as compared to controls treated with IL-2 alone. Furthermore, similar treatment of CD4-CD8-(double negative) thymocytes with TGF-beta induced de novo CD8 alpha expression by a substantial number of cells, and the majority of these CD8+ cells lacked TCR/CD3. These data suggest that TGF-beta has both positive and negative regulatory effects on the expression of gene products important for T lymphocyte differentiation and function.

Inhibition of tumor-specific cytotoxic T-lymphocyte responses by transforming growth factor beta 1.

Transforming growth factor beta (TGF-beta) is a potent immunosuppressive cytokine that is produced by neoplastic and normal cells. It has not been demonstrated directly, however, that TGF-beta can inhibit antigen-specific T-cell responses to tumor cells in vitro. We show here that generation of antitumor cytotoxic T-lymphocyte (CTL) activity in mixed-lymphocyte tumor cultures of splenocytes from DBA/2 mice immunized with the syngeneic P815 mastocytoma + Corynebacterium parvum was consistently and profoundly inhibited when 0.675 to 10 ng/ml of TGF-beta were added on Day 0 of culture. TGF-beta added on Day 1 or later had little or no effect. In contrast to the results with P815 immune mice, mixed-lymphocyte tumor cultures established with splenocytes from P815 tumor-bearing hosts showed variable degrees of inhibition by TGF-beta, depending on the stage of the ongoing in vivo immune response. Addition of recombinant murine tumor necrosis factor alpha (1,000 or 10,000 units/ml) partially reversed inhibition of CTL responses by TGF-beta, while recombinant interleukin 2 nearly completely reversed the suppression. These data indicate that one level at which TGF-beta may act to inhibit mixed-lymphocyte tumor cultures is that of cytokine production. To determine whether TGF-beta also has any direct effect on CTL, P815-specific CTL clones derived from tumor-bearing host mice were utilized. We found that proliferation of rested CTL clones in response to tumor cells + interleukin 2 was inhibited by 5 ng/ml of TGF-beta, while the interleukin 2-dependent reactivation of cytolytic activity was not affected by TGF-beta. In contrast to rested CTL, when TGF-beta was added to cultures of previously activated CTL, proliferation was not inhibited. These data demonstrate that TGF-beta has profound inhibitory effects on the in vitro generation of effector CTL from tumor-specific murine splenocytes, and this inhibition may be an indirect result of suppressed cytokine production as well as a direct antiproliferative effect on CTL.