Circulating miRNAs as Biomarkers for Mitochondrial Neuro-Gastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disease for which there are currently no validated outcome measures for assessing therapeutic intervention efficacy. The aim of this study was to identify a plasma and/or serum microRNA (miRNA) biomarker panel for MNGIE. Sixty-five patients and 65 age and sex matched healthy controls were recruited and assigned to one of four study phases: (i) discovery for sample size determination; (ii) candidate screening; (iii) candidate validation; and (iv) verifying the performance of the validated miRNA panel in four patients treated with erythrocyte-encapsulated thymidine phosphorylase (EE-TP), an enzyme replacement under development for MNGIE. Quantitative PCR (qPCR) was used to profile miRNAs in serum and/or plasma samples collected for the discovery, validation and performance phases, and next generation sequencing (NGS) analysis was applied to serum samples assigned to the candidate screening phase. Forty-one differentially expressed candidate miRNAs were identified in the sera of patients (p < 0.05, log2 fold change > 1). The validation cohort revealed that of those, 27 miRNAs were upregulated in plasma and three miRNAs were upregulated in sera (p < 0.05). Through binary logistic regression analyses, five plasma miRNAs (miR-192-5p, miR-193a-5p, miR-194-5p, miR-215-5p and miR-34a-5p) and three serum miRNAs (miR-192-5p, miR-194-5p and miR-34a-5p) were shown to robustly distinguish MNGIE from healthy controls. Reduced longitudinal miRNA expression of miR-34a-5p was observed in all four patients treated with EE-TP and coincided with biochemical and clinical improvements. We recommend the inclusion of the plasma exploratory miRNA biomarker panel in future clinical trials of investigational therapies for MNGIE; it may have prognostic value for assessing clinical status.

Long-Term Sustained Effect of Liver-Targeted Adeno-Associated Virus Gene Therapy for Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in TYMP, the gene encoding the enzyme thymidine phosphorylase (TP). TP dysfunction results in systemic accumulation of the noxious TP substrates thymidine and deoxyuridine. Gene therapy using either a lentiviral vector or adeno-associated vector (AAV) has proven to be a feasible strategy, as both vectors restore biochemical homeostasis in a murine model of the disease. This study shows that the effect of an AAV containing the TYMP coding sequence transcriptionally targeted to the liver persists long term in mice. Although the vector copy number was diluted and AAV-mediated liver TP activity eventually reduced or lost after 21 months at the lowest vector doses, the effect was sustained (with a negligible decrease in TP activity) and fully effective on nucleoside homeostasis for at least 21 months at a dose of 2 × 1012 vg/kg. Macroscopic visual inspection of the animals' organs at completion of the study showed no adverse effects associated with the treatment. These results further support the feasibility of gene therapy for MNGIE.

Gain-of-Function Mutation in Filamin A Potentiates Platelet Integrin αIIbß3 Activation.

OBJECTIVE: Dominant mutations of the X-linked filamin A (FLNA) gene are responsible for filaminopathies A, which are rare disorders including brain periventricular nodular heterotopia, congenital intestinal pseudo-obstruction, cardiac valves or skeleton malformations, and often macrothrombocytopenia. APPROACH AND RESULTS: We studied a male patient with periventricular nodular heterotopia and congenital intestinal pseudo-obstruction, his unique X-linked FLNA allele carrying a stop codon mutation resulting in a 100-amino acid-long FLNa C-terminal extension (NP_001447.2: p.Ter2648SerextTer101). Platelet counts were normal, with few enlarged platelets. FLNa was detectable in all platelets but at 30% of control levels. Surprisingly, all platelet functions were significantly upregulated, including platelet aggregation and secretion, as induced by ADP, collagen, or von Willebrand factor in the presence of ristocetin, as well as thrombus formation in blood flow on a collagen or on a von Willebrand factor matrix. Most importantly, patient platelets stimulated with ADP exhibited a marked increase in αIIbß3 integrin activation and a parallel increase in talin recruitment to ß3, contrasting with normal Rap1 activation. These results are consistent with the mutant FLNa affecting the last step of αIIbß3 activation. Overexpression of mutant FLNa in the HEL megakaryocytic cell line correlated with an increase (compared with wild-type FLNa) in PMA-induced fibrinogen binding to and in talin and kindlin-3 recruitment by αIIbß3. CONCLUSIONS: Altogether, our results are consistent with a less binding of mutant FLNa to ß3 and the facilitated recruitment of talin by ß3 on platelet stimulation, explaining the increased αIIbß3 activation and the ensuing gain-of-platelet functions.

Predictors of intestinal pseudo-obstruction in systemic lupus erythematosus complicated by digestive manifestations: data from a Southern China lupus cohort.

OBJECTIVE: To determine factors that may predict intestinal pseudo-obstruction (IpsO) in systemic lupus erythematosus (SLE) patients complicated by digestive manifestations. METHODS: SLE patients with digestive manifestations (n = 135) were followed at Southern Medical University affiliated Nanfang Hospital from 2000 until 2013. Demographic variables, clinical features, and laboratory data were compared between the two groups. Univariate and multivariate logistic regression models were used to establish factors that predispose to IpsO in these patients. RESULTS: At the end of the study period, 32 (23.7%) patients had developed IpsO. Mortality (9 patients) was infrequent and the cause of death was unrelated to IpsO. Independent predictors of IpsO in SLE were ureterectasia, anti-U1 RNP(+), peritonitis, and low C3 levels. CONCLUSIONS: Regular abdominal X-ray examinations are recommended in SLE patients with ureterectasia, anti-U1 RNP(+), peritonitis, or low C3 levels, as early diagnosis and therapy may prevent unnecessary surgical intervention and improve the disease course.

Perioperative administration of Daikenchuto (TJ-100) reduces the postoperative paralytic ileus in patients with pancreaticoduodenectomy.

BACKGROUND/AIMS: No study has reported whether perioperative administration of Daikenchuto (TJ-100) reduced paralytic ileus after pancreaticoduodenectomy (PD). METHODOLOGY: Forty-five consecutive patients that were scheduled to undergo PD at Wakayama Medical University Hospital between August 2010 and August 2011 were enrolled in this study including the first cohort (n = 15) as the control group and the subsequent cohort (n = 30) as the TJ-100 group. This trial was registered at UMIN-CTR ID# 000005056. RESULTS: Postoperative paralytic ileus occurred more frequently in the control group (73.3% of the control group and 20.0% of the TJ-100 group; p = 0.001). The first passages of flatus significantly improved earlier in the TJ-100 group than in the control group (p = 0.014). A multiple cytokine assay of the drainage and serum showed that IL-9 and IL-10 in the drainage was significantly higher on postoperative day 1 in the TJ-100 group. There were no complications associated with the preoperative administration of TJ-100 before surgery, and no significant differences were observed between the two groups in the incidence of postoperative Gradel-2 diarrhea (CTCAE4.0). CONCLUSIONS: Perioperative administration of TJ-100 was feasible and reduced the incidence of paralytic ileus in PD, and further randomized controlled trials should be conducted.

Clinical analysis of 61 systemic lupus erythematosus patients with intestinal pseudo-obstruction and/or ureterohydronephrosis: a retrospective observational study.

The objective of this article is to investigate the clinical features of intestinal pseudo-obstruction (IPO) and/or ureterohydronephrosis in systemic lupus erythematosus (SLE). Sixty-one SLE patients with IPO and/or ureterohydronephrosis were analyzed retrospectively. A total of 183 cases were randomly selected as controls from 3840 SLE inpatients without IPO and ureterohydronephrosis during the same period. Patients were assigned to 1 of the 3 groups (SLE with IPO and ureterohydronephrosis, SLE with IPO, and SLE with ureterohydronephrosis). The clinical characteristics, treatments, and prognosis were compared between the 3 groups. There were 57 females and 4 males, with a mean age of 32.0 years. IPO was the initial manifestation of SLE in 49.1% of the cases, whereas ureterohydronephrosis in 32.5%. All patients were initially treated with a high-dose steroid. Thirty-one of these patients (50.8%) also received intravenous methylprednisolone pulse therapy. Two patients died of bowel perforation and lupus encephalopathy, and the other 59 patients (96.7%) achieved remission after treatment. The incidences of fever, glomerulonephritis, nervous system involvement, serositis, erythrocyte sedimentation rate elevation, hypoalbuminemia, hypocomplementemia, and anti-SSA antibody positivity were significantly higher in patients with IPO and/or ureterohydronephrosis than in the control group (without IPO and ureterohydronephrosis). Also, patients with IPO and/or ureterohydronephrosis had higher SLE Disease Activity Index scores than control patients. Compared with SLE patients with IPO, the patients with IPO and ureterohydronephrosis had a significantly higher incidence of gallbladder wall thickening, biliary tract dilatation, and serositis, whereas the patients with ureterohydronephrosis had less mucocutaneous involvement and serositis. Eight of the 47 IPO patients who initially responded well to immunotherapy relapsed; however, all responded well to retreatment with adequate immunotherapy. Of these 8 patients, 4 relapsed following poor compliance and self-discontinuation of steroid or immunosuppressant therapy. The rate of poor compliance with immunotherapy and the number of organ systems involved in patients in the recurrent IPO group were significantly higher than those in the nonrecurrent IPO group. IPO and ureterohydronephrosis are severe complications of SLE. As patients usually respond readily to early optimal steroid treatment, early diagnosis and timely initiation of glucocorticoid are important to relieve symptoms, prevent complications, and improve prognosis.

HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency.

We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100µL) pretreatment was based on simple deproteinization by 1.2M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4µm Hydro-RP, 150×4mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7mL/min. The UV detector was set at 267nm. The chromatographic run lasted 19min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0µg/mL. The limit of quantitation was 0.5µg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma.

Serum B-cell activating factor assessment in a population of Egyptian patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a rare systemic connective tissue disease characterized by abnormal fibroblast proliferation and micro-vascular inflammatory changes. AIM: To assess serum B-cell activating factor (BAFF) levels in patients with systemic sclerosis and to correlate this with disease features and disease severity. METHODS: This is a case-control study in which patients with the established diagnosis of SSc were recruited. The diagnosis of SSc was established according to the American Rheumatology Association 1980 criteria for the classification of scleroderma. Patients' assessment included evaluation of skin involvement using the Modified Rodnan score and disease severity using the Medsger score. Twenty-five healthy matching controls were included. The sandwich enzyme-linked immunosorbent assay technique was used for direct assessment of serum BAFF in patients and controls. RESULTS: The study included 60 patients (54 female and 6 male), with a mean age of 38.18 ± 12.06 years, with mean disease duration of 7.85 ± 4.075 years. Serum BAFF in patients ranged 98.2-5015 pg/mL with mean BAFF 1100 ± 835.4 pg/mL. In controls serum BAFF levels ranged 188.5-2314 pg/mL with mean BAFF 546.1 ± 471.1 pg/mL, showing a statistically significant elevation of serum BAFF levels in SSc patients (P = 0.0001) with insignificant correlation to skin disease or total Medsgar Score of the study population (P > 0.05). Serum BAFF levels showed significant correlation with episodes of pseudo-obstruction and methotrexate (MTX) use in the patients studied (P < 0.05). CONCLUSION: Serum BAFF levels were significantly elevated in patients with SSc irrespective of disease subtype, disease duration or age of patients. This elevation in serum BAFF significantly related to gastrointestinal track involvement and MTX therapy.

Non-myeloablative bone marrow transplant and platelet infusion can transiently improve the clinical outcome of mitochondrial neurogastrointestinal encephalopathy: a case report.

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is caused by deficiency in thymidine phosphorylase (TP), that regulates thymidine (dThd) and deoxyuridine (dUrd). Toxic levels of dThd and dUrd can lead to mitochondrial dysfunction by impairing mitochondrial DNA replication, causing GI and neurologic deterioration. We studied the impact of bone marrow transplant (BMT) and platelets, as a source of TP on the clinical outcome of MNGIE. We report a case of MNGIE, who presented with severe vomiting. Over time, he was non-ambulatory and his GI symptoms got progressively worse with severe dysphagia, abdominal pain episodes, persistent vomiting and diarrhea. Being unfit for intense conditioning regimen, he received a mini BMT, with mild conditioning regimen. Bone marrow was obtained from his HLA fully matched brother. One month after transplantation, donor chimerism in peripheral blood was 33%. Excellent clinical responses were achieved 3 months after transplantation and circulating donor cell chimerism decreased to 24% with a significant increase in platelet TP activity. Ten months post transplant the patient's symptoms recurred and fresh single donor platelets were infused, with a significant increase in platelet TP activity. Mini BMT and platelet transfusion can transiently increase circulating TP activity and might prevent progress of this fatal disease.

Assessment of thymidine phosphorylase function: measurement of plasma thymidine (and deoxyuridine) and thymidine phosphorylase activity.

We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TYMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689-692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TYMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV.

Plasma procalcitonin in patients with ileus. Relations to other inflammatory parameters.

Plasma procalcitonin (PCT) is a highly specific marker for the diagnosis of bacterial infections and sepsis. PCT levels are usually low in viral infections, chronic inflammation or postsurgical states. The purpose of this study was to characterize PCT plasma levels in patients with various types of ileus at preoperative stage, where the other inducing factors such as a surgical stress are excluded. The prospective study was performed on 54 patients admitted to in-patient surgical department with a proven diagnosis of ileus. Patients were divided to three groups--obstructive, vascular and paralytic ileus. Plasma levels of PCT (Kryptor analysis), TNFalpha, IL-1beta, IL-6, cortisol (ELISA) and CRP (Kryptor ultrasensitive analysis) were estimated before any invasive procedure was realized. We demonstrated significant elevation of PCT in both obstructive ileus in adhesions and vascular ileus compared with healthy subjects (p 0.01). PCT levels were not elevated in paralytic ileus. The regression coefficient was the highest for PCT and CRP (r=0.78, p 0.01), for TNFalpha and IL-8 (r=0.76, p 0.01) in vascular ileus. There was no significant correlation between PCT and other inflammatory parameters. The different types of ileus induce an elevation of plasma PCT levels and PCT shows itself as an acute phase reactant. The highest PCT concentrations were presented in patients with vascular ileus, whereas paralytic ileus revealed similar cytokine and PCT pattern as in healthy subjects. Plasma PCT estimation extended to a measurement of CRP and IL-6 may become a useful complementary examination for diagnostics of acute abdomen in patients.

Immunoreactive trypsinogen levels in pediatric patients with intestinal failure awaiting intestinal transplantation.

The aim of this study was to evaluate pancreatic function in total parenteral nutrition (TPN)-dependent children with permanent intestinal failure by measuring immunoreactive trypsinogen (IRT) levels. Between 1992 and 1996, 105 pediatric patients with permanent intestinal failure were referred to the Children's Hospital of Pittsburgh for small intestinal transplant evaluation. Serum samples were available from 55 of them. Ten suffered from intestinal pseudo-obstruction or microvillus inclusion disease, while 45 had short bowel syndrome (SBS). IRT levels were significantly higher (p < 0.001) in SBS patients (89.4 +/- 9.2 ng mL) compared to controls (43.4 +/- 5.6 ng/ nL) without liver, gastrointestinal, or kidney disease. IRT levels did not correlate with liver injury, length of bowel, or the cause of SBS. Five of 20 patients who underwent intestinal transplantation developed pancreatitis during a median post-operative follow up 15.4 months later. IRT levels failed to predict who would develop pancreatitis post-transplant. The data suggest that elevated plasma IRT levels are common among children with intestinal failure, but fail to identify patients at risk for pancreatitis post-transplant.

Plasma somatostatin levels in patients with chronic idiopathic intestinal pseudo-obstruction.

OBJECTIVES: Somatostatin participates in the control of gut motility. Recently, somatostatin analogs have been proposed as therapeutic agents for chronic intestinal pseudo-obstruction, although the endogenous somatostatin pattern has never been assessed in this syndrome. We aimed to evaluate fasting and postprandial plasma somatostatin levels in patients with chronic idiopathic intestinal pseudo-obstruction (CIIP). METHODS: We studied eight patients with CIIP and 10 healthy volunteers. Blood samples were taken at regular intervals while patients and subjects fasted and during the 3 h after a standard solid/liquid meal (550 kcal) had been eaten. Somatostatin was measured by radioimmunoassay. RESULTS: Fasting somatostatin levels were normal, whereas postprandial peptide responses were markedly impaired or even absent in patients with CIIP. CONCLUSIONS: An impaired postprandial somatostatin response in patients with CIIP seems to be characteristic of this heterogenous disorder. Whether the lack of somatostatin response to a meal identifies patients with severe gut dysmotility for whom treatment with somatostatin analogs would be useful remains to be verified.

The megacystis-microcolon-intestinal hypoperistalsis syndrome: report of a case and review of the literature.

The megacystis-microcolon-intestinal hypoperistalsis syndrome is part of a spectrum of intestinal motility disorders and is characterized by abdominal distension, lax abdominal musculature, incomplete intestinal rotation, microcolon, megacystis, bilious vomiting and decreased or absent intestinal peristalsis. In this report a newborn girl with megacystis-microcolon-intestinal hypoperistalsis syndrome is reported.

Paralytic ileus as a complication of atropine therapy following severe organophosphate poisoning.

A 32-year-old man presented with symptoms of severe organophosphate poisoning and required an atropine infusion for 5 weeks. We believe the development of a paralytic ileus occurred as a rare feature of atropine toxicity when other signs were masked by the underlying condition. The onset of a paralytic ileus coincided with a spontaneous increase in red cell cholinesterase levels and may be an early sign of recovery from organophosphate poisoning.

[Carbamazepine poisoning: protracted course with development of intestinal atony and hepatic toxicity]. / Intoxikation mit Carbamazepin: Protrahierter Verlauf unter Entwicklung von Darmatonie und Hepatotoxizität.

A 43 year-old female patient with a history of manic-depressive illness and prophylactic carbamazepine (CBZ) medication ingested a potentially lethal overdose of 20 g of the substance. Neurotoxic symptoms reached full intensity after about 24 hours. Subsequently, the patient developed a gastrointestinal atony, which proved to be refractory to treatment for several days. Moreover, there was an increase of bilirubin. Parallel to this we observed the persistence-and even intermittent re-increase--of toxic serum CBZ concentrations for one week with corresponding protracted clinical symptomatology.

Oncotic pressure, albumin and ileus: the effect of albumin replacement on postoperative ileus.

The effect of decreased colloid oncotic pressure, as seen in hypoalbuminemia and hypoproteinemia, upon intestinal function has been well delineated in the surgical literature. Patients undergoing abdominal aortic aneurysm resection or aortoiliac or aortofemoral bypass grafts are almost uniformly hypoalbuminemic postoperatively; with these two facts in mind, a prospective, randomized clinical study was undertaken to identify the role of serum albumin concentration on the length of postoperative ileus in this population. The main hypothesis was that patients whose albumin levels dropped below 3.5 gm/dL would have a more prolonged postoperative hospital course as a result of delay in return of bowel function when compared with those patients in whom the low albumin levels were exogenously acutely replenished to > 3.5 gm/dL. Albumin was replaced to a level greater-than or equal to 3.5 g/dL in one group of 37 patients (AR), with a control group of 32 patients (NR) not receiving any albumin. Return of bowel function was measured by the postoperative day that flatus was documented, as well as the postoperative day oral intake was resumed. Mean values were determined for each group, and t tests did not reveal a significant difference in postoperative day of flatus (AR mean = 4.06 days, NR mean = 4.16 days) or postoperative day of oral intake (AR mean = 4.0, NR mean = 3.75). Additional comparisons between the groups involving the number of postoperative days until a regular diet was begun (AR mean = 6.06, NR mean = 5.48) and length of postoperative hospital stay (AR mean = 9.16, NR mean = 8.43) failed to reveal significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Cisapride in children with chronic intestinal pseudoobstruction. An acute, double-blind, crossover, placebo-controlled trial.

To assess the effect of cisapride on gastrointestinal motility and gastric emptying in children with chronic intestinal pseudoobstruction, 20 children (mean age, 4.9 years; 14 female and 6 male) who required special means of alimentation or who had severe symptoms confirmed by diary during 2 weeks before the study were studied. A motility catheter with recording sites in the antrum and duodenum was placed on the first day of the study and remained in place until the end of the 5-day study. Cisapride (0.3 mg/kg PO t.i.d.) or placebo was given in double-blind randomized crossover fashion, with a 2-day "washout" interval. Antroduodenal motility was recorded on days 2 and 5. Recording consisted of 4 hours of fasting and 2 hours after a complex liquid meal labeled with 99mTc. Gastric emptying was assessed for 1 hour after the meal. Based on manometry, 16 patients had neuropathic and 4 patients had myopathic disorders. Cisapride had no effect on the discrete, qualitative abnormalities found in individual records. Cisapride increased the postprandial duodenal motility index from 1180 +/- 256 mm Hg/30 min after placebo to 2385 +/- 430 mm Hg/30 min (P less than 0.05) but had no significant effect on the antral motility index. Cisapride did not alter the profound delay in gastric emptying; time to reach 50% of initial activity (T1/2) was 105 +/- 20 vs. 93 +/- 19 minutes and percentage of retention after 60 minutes (R60) 56% +/- 4% vs. 58% +/- 4% in control vs. cisapride, respectively. In summary, in children with chronic intestinal pseudoobstruction, cisapride increased postprandial duodenal motility but did not improve gastric emptying.