Comparative Effectiveness of Anti-TNF in Combination With Low-Dose Methotrexate vs Anti-TNF Monotherapy in Pediatric Crohn's Disease: A Pragmatic Randomized Trial.

BACKGROUND & AIMS: Tumor necrosis factor inhibitors, including infliximab and adalimumab, are a mainstay of pediatric Crohn's disease therapy; however, nonresponse and loss of response are common. As combination therapy with methotrexate may improve response, we performed a multicenter, randomized, double-blind, placebo-controlled pragmatic trial to compare tumor necrosis factor inhibitors with oral methotrexate to tumor necrosis factor inhibitor monotherapy. METHODS: Patients with pediatric Crohn's disease initiating infliximab or adalimumab were randomized in 1:1 allocation to methotrexate or placebo and followed for 12-36 months. The primary outcome was a composite indicator of treatment failure. Secondary outcomes included anti-drug antibodies and patient-reported outcomes of pain interference and fatigue. Adverse events (AEs) and serious AEs (SAEs) were collected. RESULTS: Of 297 participants (mean age, 13.9 years, 35% were female), 156 were assigned to methotrexate (110 infliximab initiators and 46 adalimumab initiators) and 141 to placebo (102 infliximab initiators and 39 adalimumab initiators). In the overall population, time to treatment failure did not differ by study arm (hazard ratio, 0.69; 95% CI, 0.45-1.05). Among infliximab initiators, there were no differences between combination and monotherapy (hazard ratio, 0.93; 95% CI, 0.55-1.56). Among adalimumab initiators, combination therapy was associated with longer time to treatment failure (hazard ratio, 0.40; 95% CI, 0.19-0.81). A trend toward lower anti-drug antibody development in the combination therapy arm was not significant (infliximab: odds ratio, 0.72; 95% CI, 0.49-1.07; adalimumab: odds ratio, 0.71; 95% CI, 0.24-2.07). No differences in patient-reported outcomes were observed. Combination therapy resulted in more AEs but fewer SAEs. CONCLUSIONS: Among adalimumab but not infliximab initiators, patients with pediatric Crohn's disease treated with methotrexate combination therapy experienced a 2-fold reduction in treatment failure with a tolerable safety profile. CLINICALTRIALS: gov, Number: NCT02772965.

Very-Early Onset Chronic Active Colitis with Heterozygous Variants in LRBA1 and CARD11, a Case of "Immune TOR-Opathies".

BACKGROUND: A small subset of cases of inflammatory bowel disease (IBD) occurs as a result of single gene defects, and typically occurs in young or very young pediatric patients, referred to as "monogenic very-early onset IBD (VEO-IBD)". The gene variants leading to monogenic VEO-IBD are often associated with primary immunodeficiency syndromes. CASE REPORT: A six year-old girl presented to our gastroenterology clinic with LRBA deficiency with a heterozygous mutation at c.1399 A > G, p Met467Val, histopathologic chronic active colitis without granulomas and clinical chronic colitis. Her gastrointestinal symptoms began at age 5 with bloody diarrhea, abdominal pain and weight loss. Whole exome sequencing revealed a CARD11 heterozygous de novo mutation (c.220 + 1G > A). She was in clinical remission on only abatacept. DISCUSSION: We present a case of monogenic VEO-IBD associated with two heterozygous variants in LRBA1 and CARD11, both considered as key players in the newly proposed "immune TOR-opathies".

Immunosuppressant-associated eruptions in pediatric inflammatory bowel disease: A case-control study.

Paradoxically, immunosuppressive therapy for inflammatory bowel disease (IBD) can induce psoriasiform or eczematous eruptions. This case-control study identified infliximab exposure, Crohn's disease, and history of inflammatory skin conditions as significant risk factors for these eruptions in children with IBD. Our results also showed possible trends in age and race.

Reduction of Computed Tomography Use for Pediatric Closed Head Injury Evaluation at a Nonpediatric Community Emergency Department.

OBJECTIVE: The purpose of this study was to determine if implementation of a Pediatric Emergency Care Applied Research Network (PECARN)-based Closed Head Injury Assessment Tool could safely decrease computed tomography (CT) use for pediatric head injury evaluation at a nonpediatric community emergency department (ED). METHODS: A quality improvement project was initiated at a nonpediatric community ED to implement an institution-specific, PECARN-based Pediatric Closed Head Injury Assessment Tool. Baseline head CT use at the participating ED was determined for children with closed head injury through retrospective chart review from March 2014 through November 2015. Head injury patients were identified using International Classification of Disease (ICD)-9 codes for head injury, unspecified (959.01) and concussion with and without loss of consciousness (850-850.9) until October 2015, after which ICD-9 was no longer used. To identify eligible patients after October 2015, lists of all pediatric patients evaluated at the participating ED were reviewed, and patients were included in the analysis if they had a physician-assigned discharge diagnosis of head injury or concussion. Exclusion criteria were age ≥ 18 years, penetrating head trauma, history of brain tumor, ventriculoperitoneal shunt, bleeding disorder, or presentation > 24 hours postinjury. Medical history, injury mechanism, symptoms, head CT use, and disposition were recorded. Implementation of the Pediatric Closed Head Injury Assessment Tool was achieved through provider education sessions beginning in December 2015 and ending in August 2016. Head CT use was monitored for 12 months postimplementation, from September 2016 through August 2017. Patients were classified into low, intermediate, or high risk for clinically important traumatic brain injury (ciTBI) by chart review. ED length of stay (LOS), disposition, and ED returns within 72 hours were recorded. Categorical variables were compared using chi-square test or Fisher's exact test, and continuous variables, using Kruskal-Wallis test. RESULTS: A total of 252 children with closed head injury were evaluated preimplementation (March 2014 through November 2015), 132 children were evaluated during implementation (December 2015 through August 2016), and 172 children were evaluated postimplementation (September 2016 through August 2017). Overall CT use decreased from 37.7% (95% confidence interval [CI] = 31.7-43.7) preimplementation to 16.9% (95% CI = 11.3-22.5) postimplementation (p < 0.001). Only 1% (95% CI = 0%-2.9%) of low-risk patients received a head CT postimplementation compared to 22.6% (95% CI = 16.1%-29.1%) preimplementation (p < 0.001). CT use among patients ≥ 24 months decreased from 42.9% (95% CI = 36.5%-49.6%) to 19.6% (95% CI = 13.1%-26.1%; p < 0.001) and remained low and unchanged for patients < 24 months. Transfers to a pediatric trauma center and ED returns within 72 hours were unchanged, while median ED LOS improved from 1.5 to 1.3 hours (p = 0.03). There were no missed ciTBIs after implementation of the guideline. CONCLUSION: Implementation of the PECARN-based Pediatric Closed Head Injury Assessment Tool reduced head CT use in a nonpediatric ED. The greatest impact was seen among children aged ≥ 24 months at very low risk for ciTBI.

Remission in Pediatric Inflammatory Bowel Disease Correlates With Prescription Refill Adherence Rates.

OBJECTIVES: The aim of the study was to use pharmacy benefit management (PBM) prescription claims data to assess refill adherence in pediatric inflammatory bowel disease (IBD) and correlate adherence with clinical outcomes in pediatric IBD. METHODS: We identified 362 pediatric patients with IBD seen at Washington University from 9/1/2012 to 8/31/2013 and matched them within Express Scripts' member eligibility files for clients allowing use of prescription drug data for research purposes. Maintenance IBD medication possession ratios (MPR) were determined through PBM prescription claims data and chart review. Demographic and prospectively captured physician global assessments (PGA) were retrospectively extracted from the medical record. MPR was analyzed as continuous data and also dichotomized as greater or less than 80%. RESULTS: Among our 362 patients, we matched 228 (63%) within Express Scripts' eligibility data files. Of those, 78 patients were continuously eligible for benefits and had at least one outpatient prescription IBD medication prescribed. Their mean MPR was 0.63 ±â€Š0.31 (standard deviation) and 40% had an MPR ≥80%. Patients in clinical remission had a higher mean MPR than those with an active PGA (0.72 ±â€Š0.28 vs 0.51 ±â€Š0.32, P = 0.002) and patients whose MPR were ≥80% were more likely to have a PGA of remission than those with whose MPR were <80% (84% vs 43%, P = <0.001). CONCLUSIONS: We found a significant association between refill adherence and clinical remission. Nonadherence was common and was more common in adolescents. Use of PBM databases to identify and intervene on patients with poor adherence may improve outcomes in pediatric IBD.

Budesonide use in pediatric Crohn disease.

BACKGROUND AND AIM: Budesonide (BUD) is being used in pediatric Crohn disease (CD) because it is believed to have the potential to reduce corticosteroid-related toxicity; however, few data are available describing its use. The aim of the present study was to describe BUD use in an inception cohort of pediatric patients with CD. METHODS: Data were derived from the prospective Pediatric IBD Collaborative Research Group Registry established in 2002 in North America. Use of BUD in children with CD was examined. RESULTS: BUD was used in 119 of 932 (13%) of children with newly diagnosed CD, with 56 of 119 (47%) starting BUD ≤ 30 days of diagnosis (26/56 with ileum and/or ascending colon [IAC] disease). BUD was used as monotherapy (9%), in combination with 5-aminosalicylates (77%), or in combination with immunomodulators (43%). Forty-three percent (24/56) went on to receive conventional corticosteroid at some point following their first BUD course. For the 63 of 119 (53%) who started BUD beyond the diagnosis period, 51 of 63 (81%) also received prednisone, with BUD used as a means of weaning from prednisone in 17 of 63 (27%). Patients with IAC disease who received BUD ≤ 30 days of diagnosis were just as likely to have received conventional corticosteroids by 1 year as were those who did not receive BUD ≤ 30 days of diagnosis. Two-thirds (77/119) of patients received BUD for ≤ 6 months. CONCLUSIONS: BUD is being used among pediatric patients newly diagnosed as having CD, although the majority does not have disease limited to the IAC. BUD monotherapy was rare, and further data are required to better define the role of BUD in the treatment of pediatric CD.

Granulocyte-macrophage colony stimulating factor blockade promotes ccr9(+) lymphocyte expansion in Nod2 deficient mice.

BACKGROUND: Ileal involvement in Crohn's disease (CD) is associated with NOD2 mutations and granulocyte-macrophage colony stimulating factor autoantibodies (GM-CSF Ab), and GM-CSF blockade promotes ileitis in Nod2/Card15-deficient (C15KO) mice. RALDH2-expressing dendritic cells (DC) and IL-4 promote CCR9 imprinting and small bowel homing of T lymphocytes, in conjunction with CCL25 expression by ileal epithelial cells (IEC). We hypothesized that GM-CSF neutralization promotes ileal disease by modulating expression of CCL25 by IEC and CCR9 by T lymphocytes via Nod2-dependent and independent pathways. METHODS: CCL25 and CCR9 expression were determined in pediatric CD patients stratified by GM-CSF Ab. Ileitis was induced in C15KO mice via GM-CSF Ab administration followed by nonsteroidal antiinflammatory drug (NSAID) exposure, and expression of CCL25, CCR9, FOXP3, intracellular cytokines, and RALDH2 was determined in IEC and immune cell populations. RESULTS: The frequency of CCL25(+) IEC and CCR9(+) T lymphocytes was increased in CD patients with elevated GM-CSF Ab. In the murine model, GM-CSF blockade alone induced IEC CCL25 expression, and reduced the frequency of mesenteric lymph node (MLN) CD4(+) FOXP3(+) cells, while Card15 deficiency alone enhanced MLN DC RALDH2 expression. Both GM-CSF neutralization and Card15 deficiency were required for downregulation of MLN DC IL-10 expression; under these conditions NSAID exposure led to an expansion of IL-4(+) and IL-17(+) CCR9(+) lymphocytes in the ileum. CONCLUSIONS: GM-CSF prevents ileal expansion of CCR9(+) lymphocytes via Nod2-dependent and independent pathways. CCR9 blockade may be beneficial in CD patients with elevated GM-CSF Ab.

Presentation and outcome of histoplasmosis in pediatric inflammatory bowel disease patients treated with antitumor necrosis factor alpha therapy: a case series.

BACKGROUND: Antitumor necrosis factor alpha (aTNF) therapies are commonly used in the treatment of pediatric inflammatory bowel disease (IBD). However, inhibition of the TNF-alpha pathway predisposes to serious infections, including histoplasmosis, which is the most common invasive fungal infection in individuals on aTNF therapy and carries a high mortality rate when associated with delayed diagnosis. Few data exist on the frequency, presentation, and appropriate treatment of pediatric patients with histoplasmosis on aTNF therapy. METHODS: Following Institutional Review Board approval, cases were identified then reviewed with their primary gastroenterologist and infectious disease specialists. RESULTS: Herein we describe histoplasmosis in five pediatric patients receiving aTNF therapy for IBD in an endemic area. CONCLUSIONS: Histoplasmosis is an important complication of treatment with TNF-alpha neutralizing agents. Children with IBD treated with aTNF therapy who develop the infection may present with minimal pulmonary symptoms. While discontinuation of aTNF therapy is important initially, few data exist to determine when and how aTNF therapy can be reinstituted. Recognition of Histoplasma capsulatum is often delayed due to the overlap of symptoms with some of the extraintestinal manifestations of IBD and other more prevalent infectious complications.

Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury.

BACKGROUND: Administration of granulocyte-macrophage colony stimulating factor (GM-CSF) relieves symptoms in Crohn's disease (CD). It has been reported that reduced GM-CSF bioactivity is associated with more aggressive ileal behaviour and that GM-CSF-null mice exhibit ileal barrier dysfunction and develop a transmural ileitis following exposure to non-steroidal anti-inflammatory drugs (NSAIDs). STAT5 signalling is central to GM-CSF action. It was therefore hypothesised that GM-CSF signalling in non-haematopoietic cells is required for ileal homeostasis. METHODS: Bone marrow (BM) chimeras were generated by reconstituting irradiated GM-CSF receptor (gm-csfr) beta chain or GM-CSF (gm-csf) deficient mice with wild type BM (WTBM-->GMRKO and WTBM-->GMKO). Intestinal barrier function and the response to NSAID-induced ileal injury were examined. Expression of gm-csf, gm-csfr or stat5 in Caco-2 and HT-29 intestinal epithelial cell (IEC) lines was knocked down and the effect of GM-CSF signalling on IEC survival and proliferation was determined. RESULTS: Elevated levels of GM-CSF autoantibodies in ileal CD were found to be associated with dysregulation of IEC survival and proliferation. GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras exhibited ileal hyperpermeability. NSAID exposure induced a transmural ileitis in GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras. Transplantation of wild type BM into GM-CSF-deficient mice prevented NSAID ileal injury and restored ileal barrier function. Ileal crypt IEC proliferation was reduced in WTBM-->GMRKO chimeras, while STAT5 activation in ileal IEC following NSAID exposure was abrogated in WTBM-->GMRKO chimeras. Following knock down of gm-csf, gm-csfr alpha or beta chain or stat5a/b expression in Caco-2 cells, basal proliferation was suppressed. GM-CSF normalised proliferation of Caco-2 cells exposed to NSAID, which was blocked by stat5a/b RNA interference. CONCLUSIONS: Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury; furthermore, GM-CSF signalling in non-haematopoietic cells regulates ileal epithelial homeostasis via the STAT5 pathway. The therapeutic use of GM-CSF may therefore be beneficial in chronic ileitis associated with CD.

Granulocyte-macrophage colony-stimulating factor autoantibodies in murine ileitis and progressive ileal Crohn's disease.

BACKGROUND & AIMS: Genetic variations that affect innate immunity increase risk of ileal Crohn's disease (CD). However, the penetrance of susceptibility genes, including NOD2, is low, suggesting additional risk factors. Neutralizing autoantibodies (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF Ab) reduce neutrophil antimicrobial function in patients with primary alveolar proteinosis (PAP). We investigated whether GM-CSF Ab regulates neutrophil function in CD. METHODS: Serum samples from 354 adult and pediatric patients with inflammatory bowel disease (IBD) were analyzed for GM-CSF Ab and IBD markers. Levels of GM-CSF Ab were compared with patients' CD features and neutrophil function. Intestinal barrier function and nonsteroidal anti-inflammatory drug (NSAID)-induced injury were assessed in GM-CSF-null and NOD2-null mice. RESULTS: Median GM-CSF Ab levels increased from 0.4 microg/mL in control serum to 2.4 microg/mL in pediatric CD and 11.7 microg/mL in adult CD serum and were associated with ileal involvement (P<.001). Ileal location, duration of disease, and increased GM-CSF Ab levels were associated with stricturing/penetrating behavior (odds ratio, 2.2; P=.018). The positive and negative predictive values of GM-CSF Ab for stricturing/penetrating behavior were comparable with that of other IBD serum markers. CD patients with increased GM-CSF Ab had reduced neutrophil phagocytic capacity and increased accumulation of pSTAT3+ neutrophils in the affected ileum. GM-CSF-null mice and NOD2-null mice in which GM-CSF was neutralized had defects in mucosal barrier function and developed a transmural ileitis following NSAID exposure. CONCLUSIONS: GM-CSF regulates ileal homeostasis in CD and in mouse models. CD patients with increases in serum GM-CSF Ab might benefit from GM-CSF administration.

TNF alpha-induced hepatocyte apoptosis is associated with alterations of the cell cycle and decreased stem loop binding protein.

BACKGROUND: Inhibition of nuclear factor kappa B (NF kappa B) during liver regeneration induces hepatocyte apoptosis associated with normal DNA synthesis but decreased mitosis, suggesting that inhibition of NF kappa B impairs progression from S-phase through the G(2)/M phase of the cell cycle. Our aim was to determine if inhibition of NF kappa B alters cell cycle characteristics in hepatocytes treated with tumor necrosis factor alpha (TNF alpha). METHODS: Primary hepatocytes from BALB/c mice were infected with adenoviruses expressing luciferase (control; AdLuc) or the I kappa B super-repressor (AdI kappa B) and treated with or without TNF alpha (30 ng/ml). Flow cytometry was performed (0 to 40 hours) to determine apoptosis and cell cycle progression. Reverse transcriptase-polymerase chain reaction and immunoblots assessed changes in cell cycle mediators and antiapoptotic factors. RESULTS: Primary hepatocytes treated with AdI kappa B and TNF alpha demonstrated significantly more S-phase cells (14% +/- 3% vs 6% +/- 2%, P<.05) at 14 hours compared with controls. Inhibition of NF kappa B with or without TNFalpha was associated with decreased expression of stem loop bind protein, a marker of cell cycle progression through S-phase. The NF kappa B-induced antiapoptotic proteins, iNOS and TRAF2, had decreased message at 9 and 12 hours, respectively, in TNF alpha- and AdI kappa B-treated cells. CONCLUSION: Inhibition of NF kappa B in TNF alpha-treated primary mouse hepatocytes is associated with increased S-phase cell cycle retention and decreased stem loop bind protein.

Primary cirrhotic hepatocytes resist TGFbeta-induced apoptosis through a ROS-dependent mechanism.

BACKGROUND/AIMS: The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. METHODS: Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents. RESULTS: Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment. CONCLUSIONS: These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.

Cirrhotic hepatocytes exhibit decreased TGFbeta growth inhibition associated with downregulated Smad protein expression.

TGFbeta controls hepatocyte growth through cell cycle arrest and apoptosis, and resistance to TGFbeta is a mechanism of malignant transformation. The aim of this study was to assess differences in TGFbeta-mediated growth inhibition in normal and cirrhotic hepatocytes. Cirrhosis was induced in mice and normal and cirrhotic hepatocytes were isolated by collagenase perfusion and treated with or without TGFbeta (5 ng/ml). DNA synthesis, Smad protein expression, and DNA binding activity were determined. TGFbeta reduced DNA synthesis to a greater degree in normal hepatocytes than in cirrhotic hepatocytes (87% vs. 68%; p<0.05). Smad protein expression was decreased in cirrhotic hepatocytes and Smad 2/3/4 complex formation was suppressed. Furthermore, cirrhotic hepatocytes had decreased DNA binding activity at 120 min following TGFbeta treatment. In conclusion, decreased Smad protein expression may impair TGFbeta-mediated growth inhibition in cirrhotic hepatocytes.

NFkappaB inhibition decreases hepatocyte proliferation but does not alter apoptosis in obstructive jaundice.

INTRODUCTION: Cholestasis activates nuclear factor kappa B (NFkappaB), which is involved in both hepatocyte proliferation and apoptosis, depending on the cellular microenvironment. We hypothesized that NFkappaB inhibition would decrease hepatocyte proliferation and potentiate hepatocyte apoptosis in a rat model of extrahepatic biliary obstruction. AIM: To determine if NFkappaB inhibition concomitantly decreases hepatocyte proliferation and increases apoptosis in obstructive jaundice. MATERIALS AND METHODS: Male Sprague-Dawley rats underwent either sham operation or bile-duct ligation (BDL) combined with portal vein injection of vehicle or 6 x 10(9) particles of an adenovirus carrying either the control luciferase or the IkappaB super-repressor (AdIkappaBSR) transgenes. Liver was harvested 3, 5, and 7 days after sham operation or BDL, and immunohistochemistry for proliferating cell nuclear antigen and terminal dUTP nick end-labeling was performed for detection of DNA synthesis and apoptosis, respectively. RESULTS: Increased serum total bilirubin and hematoxylin and eosin-stained liver sections confirmed cholestasis in BDL animals. Western blot analysis demonstrated IkappaBSR protein expression in AdIkappaBSR-infected animals only. At day 7, NFkappaB inhibition decreased hepatocyte DNA synthesis in BDL rats compared to both adenovirus carrying the control luciferase and vehicle-treated controls. Apoptosis was increased in BDL vehicle-treated animals compared to sham-operation animals, but NFkappaB inhibition did not alter hepatocyte apoptosis in the BDL group. CONCLUSION: In obstructive cholestasis, NFkappaB is required for hepatocyte proliferation, but does not augment apoptosis.

Prolactin-releasing peptide and its homolog RFRP-1 act in hypothalamus but not in anterior pituitary gland to stimulate stress hormone secretion.

The RF-amide peptides (RFRPs), including prolactin (PRL)-releasing peptide-31 (PrRP-31) and RFRP-1, have been reported to stimulate stress hormone secretion by either direct pituitary or indirect hypothalamic actions. We examined the possible direct effects of these peptides on PRL and adrenocorticotropin (adrenocorticotropic hormone [ACTH]) release from dispersed anterior pituitary cells in culture and on PRL and ACTH secretion following intracerebroventricular (i.c.v.) administration in vivo. Neither peptide significantly altered PRL or ACTH release from cultured pituitary cells (male rat donors). Central administration of 1.0 and 3.0 nmol of PrRP-31, but only the higher dose of RFRP-1, significantly elevated serum corticosterone levels in conscious male rats. The effect of PrRP-31 was not blocked by pretreatment (i.v.) with the corticotropin-releasing hormone (CRH) antagonist, alpha-helical CRH 9-41; however, pretreatment of the animals (i.v.) with an antiserum to CRH significantly lowered the hypothalamic-pituitary- adrenal axis response to central administration of PrRP-31. On the other hand, the release of PRL was significantly elevated by 3.0 nmol of RFRP-1, but not PrRP-31, in similarly treated, conscious male rats. Pretreatment with the catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, prevented the stimulation of PRL secretion observed following central administration of RFRP-1. RFRP-1 similarly did not alter PRL secretion in rats pretreated with the dopamine, D(2) receptor blocker, domperidone. These results suggest that the RF-amide peptides are not true neuroendocrine regulators of stress hormone secretion in the rat but, instead, act centrally to alter the release of neuroendocrine factors that do act in the pituitary gland to control PRL and ACTH release. In the case of RFRP-1, stimulation of PRL secretion is potentially owing to an action of the peptide to inhibit dopamine release into the median eminence. The corticosterone secretion observed following central administration of PrRP-31 does not appear, based on our current results, to be solely owing to an action of the peptide on CRH-producing neurons but, instead, may be a result of the ability of PrRP-31 to increase as well the exposure of the corticotrophs in vivo to other ACTH secretagogues, such as oxytocin or vasopressin.

Transforming growth factor-beta1 induces hepatocyte apoptosis by a c-Jun independent mechanism.

BACKGROUND: During hepatic regeneration, transforming growth factor (TGF)-beta1 messenger RNA increases after the initial cycle of DNA synthesis, and it may control hepatocyte growth by inducing apoptosis. TGF-beta1 also induces c-Jun, a potential proapoptotic transcription factor. We hypothesized that autocrine expression of activated TGF-beta1 (Ad5aTGF-beta1) would increase c-jun expression in rat liver and limit hepatic regeneration by inducing apoptosis. METHODS: Male rats (175 to 200 g) received portal venous injections with adenoviruses expressing either luciferase (Ad5Luc), as a control, or Ad5aTGF-beta1 at a dose of 6 x 10(9) plaque-forming units. Livers were harvested 24 or 48 hours after injection and nuclear extracts and total RNA isolated. TGF-beta1 expression was confirmed by Northern blot analysis in all TGF-beta1-injected rats. RESULTS: A 2.5-fold increase in c-jun mRNA expression was detected in Ad5aTGF-beta1-infected rats compared with control rats. Transcriptional activity was assessed with an AP-1-responsive-reporter gene that increased 3-fold in rat primary hepatocytes infected with Ad5aTGF-beta1. C-Jun N-terminal kinase activity also increased 6- to 7-fold in Ad5aTGF-beta1-treated rats 24 and 48 hours after injection. Ad5aTGF-beta1-injected rats demonstrated increased AP-1 binding activity compared with Ad5Luc rats. Hepatocytes infected in vitro with Ad5aTGF-beta1 demonstrated increased apoptosis compared with Ad5Luc-infected hepatocytes (47% vs 27%) 36 hours after infection. Dual adenoviral infection with Ad5aTGF-beta1 and a dominant-negative c-Jun (Ad5TAM67) decreased AP-1-induced Ad5Luc activity but not hepatocyte apoptosis (46% with dominant-negative c-Jun and 47% without). CONCLUSIONS: These data demonstrate that TGF-beta1 induces c-Jun, but c-Jun is not proapoptotic in hepatocytes.