Increased BCR responsiveness in B cells from patients with chronic GVHD.

Although B cells have emerged as important contributors to chronic graft-versus-host-disease (cGVHD) pathogenesis, the mechanisms responsible for their sustained activation remain unknown. We previously showed that patients with cGVHD have significantly increased B cell-activating factor (BAFF) levels and that their B cells are activated and resistant to apoptosis. Exogenous BAFF confers a state of immediate responsiveness to antigen stimulation in normal murine B cells. To address this in cGVHD, we studied B-cell receptor (BCR) responsiveness in 48 patients who were >1 year out from allogeneic hematopoietic stem cell transplantation (HSCT). We found that B cells from cGVHD patients had significantly increased proliferative responses to BCR stimulation along with elevated basal levels of the proximal BCR signaling components B cell linker protein (BLNK) and Syk. After initiation of BCR signaling, cGVHD B cells exhibited increased BLNK and Syk phosphorylation compared with B cells from patients without cGVHD. Blocking Syk kinase activity prevented relative post-HSCT BCR hyper-responsiveness of cGVHD B cells. These data suggest that a lowered BCR signaling threshold in cGVHD associates with increased B-cell proliferation and activation in response to antigen. We reveal a mechanism underpinning aberrant B-cell activation in cGVHD and suggest that therapeutic inhibition of the involved kinases may benefit these patients.

B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways.

Recent data reveal an important role for B cells in the pathogenesis of chronic GVHD (cGVHD). Patients with cGVHD have delayed B-cell reconstitution and elevated BAFF to B-cell ratios compared to patients without cGVHD. The mechanisms promoting and sustaining B-cell activation in this disease, however, remain unknown. As BAFF increases murine B-cell metabolism and survival and maintains autoreactive B-cell clones, we performed ex vivo analyses of peripheral B cells from 51 patients who either had or did not have active cGVHD and were greater than 1 year from the time of allogeneic hematopoietic stem cell transplantation. We found that B cells from patients with active cGVHD were in a heightened metabolic state and were resistant to apoptosis. Exogenous BAFF treatment amplified cell size and survival in B cells from these patients. We found significantly increased signaling through ERK and AKT that associated with decreased levels of proapoptotic Bim, suggesting a mechanistic link between elevated BAFF levels and aberrant B-cell survival. Thus, we identify a role for BAFF in the pathogenesis of cGVHD and define B-cell activation and survival pathways suitable for novel therapeutic development in cGVHD.

Evaluation of the relationship between lesions in the gastroduodenal region and cyclooxygenase expression in clinically normal dogs.

OBJECTIVE: To determine whether clinically normal dogs have lesions in the pylorus and duodenum and to examine the expression of cyclooxygenase (COX) isoforms in the pylorus and duodenum of these dogs. ANIMALS: 27 clinically normal dogs. PROCEDURES: Physical examination was performed on clinically normal dogs from animal shelters and research projects; the dogs were then euthanized. After the dogs were euthanized, the pylorus and duodenum were photographed and scored for gross appearance of lesions. Samples were obtained for histologic evaluation and determination of COX expression via western blot analyses. Tissues from the pylorus and duodenum were categorized as normal, inflamed, or eroded on the basis of histologic analysis. Each histologic category of tissue was then evaluated to determine the correlation with gross appearance and COX expression. RESULTS: Of the 27 dogs, 5 had unremarkable histologic findings in the pylorus and duodenum. Inflammation was found in the pylorus of 10 dogs and in the duodenum of 5 dogs. Epithelial erosion was detected in the pylorus of 1 dog and in the duodenum of 3 dogs. Gross appearance was not significantly correlated with histologic appearance. Expression of COX-1 was not upregulated by inflammation, whereas COX-2 expression was increased by inflammation or erosion. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs that appear to be clinically normal may have underlying gastroduodenal lesions associated with upregulation of COX-2. Because of the inability to determine this during routine physical examination, practitioners should be aware of this potential situation when prescribing COX inhibitors.

Effect of nonsteroidal anti-inflammatory drugs with varied cyclooxygenase-2 selectivity on cyclooxygenase protein and prostanoid concentrations in pyloric and duodenal mucosa of dogs.

OBJECTIVE: To assess in vivo effects of short-term administration of NSAIDs with varied cyclooxygenase (COX)-2 selectivity on pyloric and duodenal mucosa. ANIMALS: 8 healthy dogs. PROCEDURES: Each dog received deracoxib (2 mg/kg, PO, q 24 h for 3 days), firocoxib (5 mg/kg, PO, q 24 h for 3 days), meloxicam (0.2 mg/kg, PO, q 24 h for 1 day followed by 0.1 mg/kg, PO, q 24 h for 2 days), or placebo orally for 3 days; there was a 4-week interval between successive treatments. Prior to and on day 3 of drug administration, pyloric and duodenal mucosae were assessed endoscopically and biopsy specimens obtained for histologic examination. Cyclooxygenase-1 and -2 protein expressions were assessed (western blotting) and prostanoid concentrations measured (ELISAs). Data were analyzed by use of an ANOVA. RESULTS: Drug administration did not significantly affect endoscopic mucosal scores, histologic scores, or COX-1 or -2 protein expression. The COX-1 protein expression was significantly higher in the pylorus than in the duodenum. Total prostaglandin and thromboxane B(2) (TXB(2)) concentrations were significantly greater in pyloric than in duodenal mucosa. Drug administration had no effect on prostaglandin or TXB(2) concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Prostanoid concentrations in gastric and duodenal tissues, and gross and histologic appearances, were not significantly affected by drugs with varied COX-2 selectivity. These findings suggested that, for these experimental conditions, there were no differences among the preferential and selective COX-2 inhibitors with regard to adverse effects on the gastric and duodenal portions of the gastrointestinal tract of dogs.

Cyclooxygenase expression and prostanoid production in pyloric and duodenal mucosae in dogs after administration of nonsteroidal anti-inflammatory drugs.

OBJECTIVE: To assess cyclooxygenase (COX) expression and prostanoid concentrations in pyloric and duodenal mucosae of dogs after administration of nonsteroidal anti-inflammatory drugs (NSAIDs). ANIMALS: 8 healthy dogs. PROCEDURES: Each dog received carprofen (4.4 mg/kg, q 24 h), deracoxib (2 mg/kg, q 24 h), aspirin (10 mg/kg, q 12 h), and placebo (1 dog treat, q 24 h) orally for 3 days (4-week interval between treatments). Before study commencement (baseline) and on day 3 of each treatment, pyloric and duodenal mucosal appearance was assessed endoscopically and biopsy specimens were obtained for histologic examination. Cyclooxygenase-1 and COX-2 protein expressions were assessed via western blotting, and prostanoid concentrations were measured via ELISAs. An ANOVA was used to analyze data. RESULTS: Treatments had no effect on mucosal appearance and ulceration was not evident histologically. In pyloric and duodenal mucosae, COX-1 expression was unaffected by treatments. Cyclooxygenase-2 expression remained unchanged in pyloric mucosa; in duodenal mucosa, aspirin significantly increased COX-2 expression, compared with effects of deracoxib and carprofen. At baseline, total prostaglandin and thromboxane B2 concentrations in pyloric mucosa were significantly greater than those in duodenal mucosa. Aspirin significantly decreased both prostanoid concentrations in both mucosal tissues, compared with other treatments. In pyloric mucosa, carprofen administration significantly decreased total prostaglandin and thromboxane B2 concentrations, compared with deracoxib administration. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, prostanoid synthesis was greater in pyloric mucosa than it was in duodenal mucosa. Nonselective NSAIDs significantly decreased prostanoid concentrations in these mucosae, compared with the effects of a selective COX-2 NSAID.

Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig.

Weaning in the piglet is a stressful event associated with gastrointestinal disorders and increased disease susceptibility. Although stress is thought to play a role in postweaning intestinal disease, the mechanisms by which stress influences intestinal pathophysiology in the weaned pig are not understood. The objectives of these experiments were to investigate the impact of weaning on gastrointestinal health in the pig and to assess the role of stress signaling pathways in this response. Nineteen-day-old pigs were weaned, and mucosal barrier function and ion transport were assessed in jejunal and colonic tissues mounted on Ussing chambers. Weaning caused marked disturbances in intestinal barrier function, as demonstrated by significant (P < 0.01) reductions in transepithelial electrical resistance and increases in intestinal permeability to [3H]mannitol in both the jejunum and colon compared with intestinal tissues from age-matched, unweaned control pigs. Weaned intestinal tissues exhibited increased intestinal secretory activity, as demonstrated by elevated short-circuit current that was sensitive to treatment with tetrodotoxin and indomethacin, suggesting activation of enteric neural and prostaglandin synthesis pathways in weaned intestinal tissues. Western blot analyses of mucosal homogenates showed increased expression of corticotrophin-releasing factor (CRF) receptor 1 in the jejunum and colon of weaned intestinal tissues. Pretreatment of pigs with the CRF receptor antagonist alpha-helical CRF(9-41), which was injected intraperitoneally 30 min prior to weaning, abolished the stress-induced mucosal changes. Our results indicate that weaning stress induces mucosal dysfunction mediated by intestinal CRF receptors and activated by enteric nerves and prostanoid pathways.

Cyclooxygenase expression in the early stages of equine laminitis: a cytologic study.

BACKGROUND: Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis. HYPOTHESIS: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis. ANIMALS: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n = 5), CON-3h (control group for DEV, n = 5), LAM (n = 5) and CON-10h (control group for LAM, n = 5). METHODS: Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2. RESULTS: Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.

Prostaglandin-mediated inhibition of Na+/H+ exchanger isoform 2 stimulates recovery of barrier function in ischemia-injured intestine.

Prostaglandins stimulate repair of the ischemia-injured intestinal barrier in the porcine ileum through a mechanism involving cAMP-dependent Cl- secretion and inhibition of electroneutral Na+/H+ exchanger (NHE) activity. In the present study, we focused on the role of individual NHE isoforms in the recovery of barrier function. Ischemia-injured porcine ileal mucosa was mounted on Ussing chambers. Short-circuit current (I(sc)), transepithelial electrical resistance (TER), and isotopic fluxes of 22Na were measured in response to PGE2 and selective inhibitors of epithelial NHE isoforms. Immunoassays were used to assess the expression of NHE isoforms. Forty-five minutes of intestinal ischemia resulted in a 45% reduction in TER (P < 0.01). Near-complete restitution occurred within 60 min. Inhibition of NHE2 with HOE-694 (25 microM) added to the mucosal surface of the injured ileum stimulated significant elevations in TER, independent of changes in I(sc) and histological evidence of restitution. Pharmacological inhibition of NHE3 or NHE1 with mucosal S-3226 (20 microM) or serosal cariporide (25 microM), respectively, had no effect. Ischemia-injured tissues treated with mucosal S-3226 or HOE-694 exhibited equivalent reductions in mucosal-to-serosal fluxes of 22Na+ (by approximately 35%) compared with nontreated ischemia-injured control tissues (P < 0.05). Intestinal ischemia resulted in increased expression of the cytoplasmic NHE regulatory factor EBP50 in NHE2 but not in NHE3 immunoprecipitates. Selective inhibition of NHE2, and not NHE3, induces recovery of barrier function in the ischemia-injured intestine.