Kshama R. Mehta

Nerve growth factor modulates TRPV1 expression and function and mediates pain in chronic pancreatitis.

BACKGROUND & AIMS The pathogenesis of pain in chronic pancreatitis (CP) is poorly understood and treatment remains difficult. We hypothesized that nerve growth factor (NGF) plays a key role in this process via its effects on the transient receptor potential vanilloid 1, TRPV1. METHODS CP was induced by intraductal injection of trinitrobenzene sulfonic acid in rats. After 3 weeks, anti-NGF antibody or control serum was administered daily for 1 week. Pancreatic hyperalgesia was assessed by nocifensive behavioral response to electrical stimulation of the pancreas as well as by referred somatic pain assessed by von Frey filament testing. TRPV1 currents in pancreatic sensory neurons were examined by patch-clamp. The expression and function of TRPV1 in pancreas-specific nociceptors was examined by immunostaining and quantification of messenger RNA levels. RESULTS Blockade of NGF significantly attenuated pancreatic hyperalgesia and referred somatic pain compared with controls. It also decreased TRPV1 current density and open probability and reduced the proportion of pancreatic sensory neurons that expressed TRPV1 as well as levels of TRPV1 in these neurons. CONCLUSIONS These findings emphasize a key role for NGF in pancreatic pain and highlight the role it plays in the modulation of TRPV1 expression and activity in CP.

Treatment of Idiopathic FSGS with Adrenocorticotropic Hormone Gel

BACKGROUND AND OBJECTIVES Adrenocorticotropic hormone (ACTH) has shown efficacy as primary and secondary therapy for nephrotic syndrome due to membranous nephropathy. The data on using ACTH to treat idiopathic FSGS are limited. This report describes our experience using ACTH for nephrotic syndrome due to idiopathic FSGS in the United States. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Twenty-four patients with nephrotic syndrome from idiopathic FSGS were treated with ACTH gel at two academic medical centers between 2009 and 2012, either as part of investigator-initiated pilot studies (n=16) or by prescription for treatment-resistant FSGS (n=8). The primary outcome was remission of proteinuria. The median dose of ACTH was 80 units injected subcutaneously twice weekly. Treatment durations were not uniform. RESULTS Twenty-two patients had received immunosuppression (mean, 2.2 medications) before ACTH therapy. Six patients had steroid-dependent and 15 had steroid-resistant FSGS. At the time of ACTH initiation, the median serum creatinine (interquartile range) was 2.0 (1.1-2.7) mg/dl, estimated GFR was 36 (28-78) ml/min per 1.73 m(2), and urine protein-to-creatinine ratio was 4595 (2200-8020) mg/g. At the end of ACTH therapy, 7 of 24 patients (29%) experienced remission (n=2 complete remissions, n=5 partial remissions). All remitters had steroid-resistant (n=5) or steroid-dependent (n=2) FSGS. Two responders relapsed during the follow-up period (mean ± SD, 70±31 weeks). Adverse events occurred in 21 of 24 patients, including one episode of new-onset diabetes that resolved after stopping ACTH and two episodes of AKI. CONCLUSIONS Response to ACTH treatment among steroid-resistant or steroid-dependent patients with FSGS is low, but ACTH gel may be a viable treatment option for some patients with resistant nephrotic syndrome due to idiopathic FSGS. Further research is necessary to determine which patients will respond to therapy.

Transient Gastric Irritation in the Neonatal Rats Leads to Changes in Hypothalamic CRF Expression, Depression- and Anxiety-Like Behavior as Adults

Aims A disturbance of the brain-gut axis is a prominent feature in functional bowel disorders (such as irritable bowel syndrome and functional dyspepsia) and psychological abnormalities are often implicated in their pathogenesis. We hypothesized that psychological morbidity in these conditions may result from gastrointestinal problems, rather than causing them. Methods Functional dyspepsia was induced by neonatal gastric irritation in male rats. 10-day old male Sprague-Dawley rats received 0.1% iodoacetamide (IA) or vehicle by oral gavage for 6 days. At 8–10 weeks of age, rats were tested with sucrose preference and forced-swimming tests to examine depression-like behavior. Elevated plus maze, open field and light-dark box tests were used to test anxiety-like behaviors. ACTH and corticosterone responses to a minor stressor, saline injection, and hypothalamic CRF expression were also measured. Results Behavioral tests revealed changes of anxiety- and depression-like behaviors in IA-treated, but not control rats. As compared with controls, hypothalamic and amygdaloid CRF immunoreactivity, basal levels of plasma corticosterone and stress-induced ACTH were significantly higher in IA-treated rats. Gastric sensory ablation with resiniferatoxin had no effect on behaviors but treatment with CRF type 1 receptor antagonist, antalarmin, reversed the depression-like behavior in IA-treated rats Conclusions The present results suggest that transient gastric irritation in the neonatal period can induce a long lasting increase in depression- and anxiety-like behaviors, increased expression of CRF in the hypothalamus, and an increased sensitivity of HPA axis to stress. The depression-like behavior may be mediated by the CRF1 receptor. These findings have significant implications for the pathogenesis of psychological co-morbidity in patients with functional bowel disorders.

A Randomized, Controlled Trial of Rituximab in IgA Nephropathy with Proteinuria and Renal Dysfunction

IgA nephropathy frequently leads to progressive CKD. Although interest surrounds use of immunosuppressive agents added to standard therapy, several recent studies have questioned efficacy of these agents. Depleting antibody-producing B cells potentially offers a new therapy. In this open label, multicenter study conducted over 1-year follow-up, we randomized 34 adult patients with biopsy-proven IgA nephropathy and proteinuria >1 g/d, maintained on angiotensin-converting enzyme inhibitors or angiotensin receptor blockers with well controlled BP and eGFR<90 ml/min per 1.73 m2, to receive standard therapy or rituximab with standard therapy. Primary outcome measures included change in proteinuria and change in eGFR. Median baseline serum creatinine level (range) was 1.4 (0.8-2.4) mg/dl, and proteinuria was 2.1 (0.6-5.3) g/d. Treatment with rituximab depleted B cells and was well tolerated. eGFR did not change in either group. Rituximab did not alter the level of proteinuria compared with that at baseline or in the control group; three patients in each group had ≥50% reduction in level of proteinuria. Serum levels of galactose-deficient IgA1 or antibodies against galactose-deficient IgA1 did not change. In this trial, rituximab therapy did not significantly improve renal function or proteinuria assessed over 1 year. Although rituximab effectively depleted B cells, it failed to reduce serum levels of galactose-deficient IgA1 and antigalactose-deficient IgA1 antibodies. Lack of efficacy of rituximab, at least at this stage and severity of IgA nephropathy, may reflect a failure of rituximab to reduce levels of specific antibodies assigned salient pathogenetic roles in IgA nephropathy.

Systemic administration of anti-NGF increases A-type potassium currents and decreases pancreatic nociceptor excitability in a rat model of chronic pancreatitis.

We have previously shown that pancreatic sensory neurons in rats with chronic pancreatitis (CP) display increased excitability associated with a decrease in transient inactivating potassium currents (I(A)), thus accounting in part for the hyperalgesia associated with this condition. Because of its well known role in somatic hyperalgesia, we hypothesized a role for the nerve growth factor (NGF) in driving these changes. CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. After 3 wk, anti-NGF antibody or control serum was injected intra-peritoneally daily for 1 wk. This protocol was repeated in another set of experiments in control rats (receiving intraductal PBS instead of TNBS). Pancreatic nociceptors labeled with the dye Dil were identified, and patch-clamp recordings were made from acutely dissociated DRG neurons. Sensory neurons from anti-NGF-treated rats displayed a lower resting membrane potential, increased rheobase, decreased burst discharges in response to stimulatory current, and decreased input resistance compared with those treated with control serum. Under voltage-clamp condition, neuronal I(A) density was increased in anti-NGF-treated rats compared with rats treated with control serum. However, anti-NGF treatment had no effect on electrophysiological parameters in neurons from control rats. The expression of Kv-associated channel or ancillary genes Kv1.4, 4.1, 4.2, 4.3, and DPP6, DPP10, and KCHIPs 1-4 in pancreas-specific nociceptors was examined by laser-capture microdissection and real-time PCR quantification of mRNA levels. No significant differences were seen among those. These findings emphasize a key role for NGF in maintaining neuronal excitability in CP specifically via downregulation of I(A) by as yet unknown mechanisms.

AKT-Induced Tamoxifen Resistance Is Overturned by RRM2 Inhibition

Acquired tamoxifen resistance develops in the majority of hormone-responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biologic outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNA interference (RNAi)–mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated DNA damage and proapoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM inhibitor didox significantly reduced in vitro and in vivo growth. Thus, AKT-expressing breast cancer cells upregulate RRM2 expression, leading to increased DNA repair and protection from tamoxifen-induced apoptosis. Implications: These findings identify RRM2 as an AKT-regulated gene, which plays a role in tamoxifen resistance and may prove to be a novel target for effective diagnostic and preventative strategies. Mol Cancer Res; 12(3); 394–407. ©2013 AACR.

Brain-derived neurotrophic factor is upregulated in rats with chronic pancreatitis and mediates pain behavior.

Objectives: We examined the role of brain-derived neurotrophic factor (BDNF) in the pathogenesis of pain in an experimental model of chronic pancreatitis (CP). Methods: Pancreatitis was induced by retrograde infusion of trinitrobenzene sulfonic acid into the pancreatic duct of adult rats. Twenty-one days after injection, BDNF expression was examined in pancreas-specific dorsal root ganglia (DRGs) by immunohistochemistry, and protein levels were quantified from DRGs and spinal cord extracts. The effects of intrathecal infusion of a neutralizing antibody to BDNF on pancreatic hyperalgesia were assessed by the sensitivity of the abdominal wall to filament probing as well as the nocifensive behavior to electrical stimulation of the pancreas. Results: Levels of BDNF in DRGs and spinal cords (T9-13) were significantly higher in trinitrobenzene sulfonic acid rats compared with controls, accompanied by an increase in the number of pancreas-specific neurons expressing BDNF immunoreactivity. Brain-derived neurotrophic factor antagonism suppressed phospho-tropomyosin-related kinase B receptor levels in the spinal cord and significantly reduced behavioral responses in rats with CP. Conclusions: Brain-derived neurotrophic factor is upregulated in pancreas-specific primary afferent neurons in rats with CP, and BDNF antagonism is associated with a reduction of pain-related behavior in these animals, suggesting an important role for this neurotransmitter in the nociception of CP.

Characterization of the Inflammatory Response in Idiopathic Gastroparesis Reveals a Role for Macrophage Derived Factors

Abdominal bloating symptoms are highly prevalent in subjects with functional GI disorders and are associated with increased healthcare utilization and reduced quality of life [APT 2010]. Altered intestinal transit and abnormal intraluminal fermentation have been proposed as possible pathophysiological mechanisms for these symptoms. We have previously demonstrated slower colonic transit in subjects with abdominal bloating compared to subjects without bloating. In this study we investigated the hypothesis that abdominal bloating symptoms are associated with abnormally increased intestinal fermentation.Aim: To investigate and compare the magnitude and site of intestinal fermentation in subjects with functional GI symptoms and bloating (FGI-B) and healthy controls (HC) by measuring pH in the GI lumen. Methods: We studied a total of 47 subjects; FGI-B (n=37) and HC (n=10). Intestinal intraluminal pH was used as a surrogate marker for intestinal fermentation. The intestinal pH levels were measured at different segments of the GI tract using a wireless motility capsule (WMC). Comparisons of transit time and pH variables between the groups were done using t-test; correlation analyses were done using Spearman Correlation Coefficients. Results: Intestinal pH levels were significantly lower at the proximal colon (Q1) of subjects with FGI-B compared to HC (mean pH 6.27 vs. 6.84; p=0.008) suggesting a significantly higher intraluminal intestinal fermentation at this segment of the GI tract in these subjects. The mean intraluminal colonic pH levels in the FGI-B group were also lower than that of HC at other segments of the large intestine; however, statistical differences were noticed only in the proximal and total colon. Mean colonic pH levels at the four (proximal to distal) colonic segments and the total colon are provided in the following Table. There were no noticeable differences between the groups in mean pH levels at any of the segments of the small bowel. The average total colonic pH significantly correlated with the total colonic transit time (r=0.34; p=0.014). However, no correlation was found between the average small bowel pH and small bowel transit time.Conclusions: We provide evidence of abnormal intestinal fermentation in subjects with functional GI symptoms and bloating. We demonstrate that bloating symptoms are associated with increased fermentation in the large rather than the small bowel. WMCmay be helpful in identifying underlying physiological abnormalities and guide treatment in patients with bloating symptoms. Mean intraluminal pH levels in subjects with abdominal bloating and healthy controls

721 Immunological Correlates of Disease Progression, Exocrine and Endocrine Failure in Patients With Chronic Pancreatitis: An Analysis of Pancreatic Juice Cytokines

(33y and 36y versus 45 and 46y resp) (p=0.005 and 0.005 resp), irrespective of the amount of alcohol intake. Chronic pancreatic pain was also more frequent (p=0.05). At 20 p.y threshold, ACP and acute pancreatitis occurred earlier (p=0.0002 and <0.0001), and the pts hade more often, calcifications and ductal changes (p=0.05 and 0.005 resp) irrespective of alcohol intake. Similar results were observed at the 30 p.y threshold, but additionally pancreatic exocrine insufficiency occurred earlier (p=0.04). ConclusionTobacco intake accelerates the course of ACP in a dose-dependent fashion. ACP occurs earlier as soon as 15 p.y, and a major threshold effect is seen at 20 p.y, where the frequency of all major complications of ACP is increased.