The Number of Interstitial Cells of Cajal Differs Among Different Subtypes of Achalasia and is Related to Patients' Prognosis.

INTRODUCTION: Achalasia is a primary esophageal motility disorder with heterogeneous manometric subtypes and prognosis, characterized by degeneration of the esophageal myenteric plexus, and reduction in interstitial cells of Cajal (ICCs). This study aimed to explore the histopathologic characteristics of lower esophageal sphincter (LES) muscle from patients with achalasia with different subtypes and different prognosis. METHODS: We examined specimens of LES muscle from 122 patients with achalasia who underwent peroral endoscopic myotomy and from 10 control patients who underwent esophagectomy for esophageal cancer. Hematoxylin-eosin staining was performed to assess inflammation infiltration, fibrosis, and atrophy. Specific immunohistochemical staining was performed to identify ICCs and neuronal nitric oxide synthase (nNOS). RESULTS: The number of ICCs in patients with type I achalasia was significantly lower than that in patients with type II achalasia, followed by that in control patients (type I vs type II vs control group= 0.4 vs 1.2 vs 9.5; P < 0.001). The number of nNOS-positive cells was significantly lower in patients with achalasia than that in control patients (type I vs type II vs control group = 0.0 vs 0.0 vs 8.0; P < 0.001). Nonrecurrent group had significantly more ICCs than recurrent group (type I: nonrecurrent vs recurrent = 1.0 vs 0.1; P = 0.010; type II: nonrecurrent vs recurrent = 2.0 vs 0.4; P = 0.004). DISCUSSION: ICCs and nNOS-positive cells reduced significantly in LES muscle of patients with achalasia. The number of ICCs differed among different achalasia subtypes and was related to patients' clinical prognosis.

Hydrogen sulfide synthesis enzymes reduced in lower esophageal sphincter of patients with achalasia.

The etiology of achalasia remains largely unknown. Considerable evidence reveals that the lower esophageal sphincter dysfunction is due to the lack of inhibitory neurotransmitter, secondary to esophageal neuronal inflammation or loss. Recent studies suggest hydrogen sulfide may act as an inhibitory transmitter in gastrointestinal tract, but study about hydrogen sulfide in human esophagus still lack. The aim of the study was to investigate if hydrogen sulfide synthesis enzymes could be detected in human esophagus and if the synthesis of the endogenous hydrogen sulfide could be affected in achalasia patients. Tissue samples in cardia, lower esophageal sphincter, 2 cm and 4 cm above lower esophageal sphincter were obtained from achalasia patients undergoing peroral endoscopic myotomy. Control tissues in lower esophageal sphincter were obtained from esophageal carcinoma patients. Expression of cystathionine-ß-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients and control were detected by immunohistochemical staining. In addition, expression of cystathionine-ß-synthase and cystathionine-γ-lyase were compared among different parts of esophagus in achalasia patients. Compared with control, the expression of cystathionine-ß-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients was significantly reduced (χ2 = 11.429, P = 0.010). The expression of cystathionine-ß-synthase and cystathionine-γ-lyase were lower in lower esophageal sphincter than that in 2 cm and 4 cm above lower esophageal sphincter, respectively (all P < 0.05). In conclusion, the expression of hydrogen sulfide synthesis enzymes, cystathionine-ß-synthase and cystathionine-γ-lyase, can be detected in human esophagus and is reduced in patients with achalasia, which implicates the involvement of the two hydrogen sulfide synthesis enzymes in the pathophysiology of achalasia.

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation.

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome.

Reduction of hydrogen sulfide synthesis enzymes in the esophagus of patients with achalasia: effect of hydrogen sulfide in achalasia.

BACKGROUND: The aim of the present study was to investigate whether the synthesis of endogenous hydrogen sulfide (H2 S) was altered in achalasia patients and to determine the effects of H2 S on esophageal motility. METHODS: (1) Tissue samples in lower esophageal sphincter (LES) were obtained from 22 achalasia patients during peroral endoscopic myotomy. LES muscle from eight esophageal carcinoma patients was obtained as control. The expression of cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) was detected by immunohistochemical staining. (2) Rabbit esophageal smooth muscle strips were used to measure isometric contractions. The effects of sodium hydrosulfide (NaHS) and L-cysteine on contractile activity and bethanechol-stimulated contractile activity were evaluated. The contraction of esophageal muscle strips was also measured after the inhibition of CBS and CSE by aminooxyacetic acid (AOA) and propargylglycine (PAG). KEY RESULTS: Both CBS and CSE could be detected in biopsies from achalasia patients and controls. Compared with controls, the expression of CBS and CSE in the LES of achalasia patients was significantly reduced (p < 0.001). Both NaHS and L-cysteine concentration-dependently inhibited esophageal contractile activity (both p < 0.05). After inhibition of CBS and CSE by PAG and AOA, esophageal contractile activity increased significantly, and this effect could be restored by NaHS but not L-cysteine (p < 0.05). CONCLUSIONS & INFERENCES: H2 S synthesis enzymes are significantly reduced in patients with achalasia compared with the controls. H2 S inhibits esophageal contractile activity concentration-dependently, and the inhibition of H2 S synthesis enzymes increases esophageal contractile activity. H2 S might be involved in the development of achalasia.

Neuroimmunopathology of Trypanosoma cruzi-induced megaoesophagus: Is there a role for mast cell proteases?

Tryptase and chymase are mast cell (MC)-specific proteases, which influence in the activation of inflammatory cells. In this study, we quantified tryptase- or chymase-expressing MCs in the oesophaguses of Chagas patients, and searched for a correlation between those data with area of nerve fibres that expressed either PGP9.5 (pan-marker) or vasoactive intestinal polypeptide (VIP), which is a neuromediator that has anti-inflammatory activity. Samples from the oesophaguses of 14 individuals Trypanosoma cruzi-infected and from six uninfected individuals were analysed by immunohistochemistry. It was demonstrated that the number of tryptase-IR MCs in infected individuals increased when compared with controls, regardless of whether the individuals had megaoesophagus, whereas the number of chymase-IR MCs increased only in infected individuals without megaoesophagus. Negative correlations were observed between tryptase-IR MCs and the density of nerve fibres that expressed VIP or PGP 9.5-IR. The participation of chymase and tryptase in this type of immunopathology is discussed.

Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency.

Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.

Intracellular ROS level is increased in fibroblasts of triple A syndrome patients.

Triple A syndrome is named after the main symptoms of alacrima, achalasia, and adrenal insufficiency but also presents with a variety of neurological impairments. To investigate the causes of progressive neurodegeneration, we examined the oxidative status of fibroblast cultures derived from triple A syndrome patients in comparison to control cells. Patient cells showed a 2.1-fold increased basal level of reactive oxygen species (ROS) and a massive boost after induction of artificial oxidative stress by paraquat. We examined the expression of the ROS-detoxifying enzymes superoxide dismutase 1 and 2 (SOD1, SOD2), catalase, and glutathione reductase. The basal expression of SOD1 was significantly (1.3-fold) increased, and the expression of catalase was 0.7-fold decreased in patient cells after induction of artificial oxidative stress. We show that the mitochondrial network is 1.8-fold more extensive in patient cells compared to control fibroblasts although the maximal ATP synthesis was unchanged. Despite having the same energy potential as the controls, the patient cells showed a 1.4-fold increase in doubling time. We conclude that fibroblasts of triple A patients have a higher basal ROS level and an increased response to artificially induced oxidative stress and undergo "stress-induced premature senescence". The increased sensitivity to oxidative stress may be a major mechanism for the neurodegeneration in triple A syndrome.

Expression of ki-67 antigen and caspase-3 protein in benign lesions and esophageal carcinoma.

BACKGROUND: The present study aimed to evaluate apoptosis and cell proliferation alterations in esophageal benign lesions in comparison to esophageal carcinomas. MATERIALS AND METHODS: Immunohistochemistry was performed for caspase-3 protein (CPP32) and Ki-67 antigen expression in the esophageal mucosa from patients with Chagas disease (CD) with and without megaesophagus (CM), chronic esophagitis (CE), esophageal carcinoma (ESCC) and in normal mucosa (NM). RESULTS: The Ki-67 labeling index (LI) was similar in all groups (range: 30%-48%), having no statistically significant difference among the groups. Positive CPP32 immunostaining was observed with similar frequency in the CD (30.8%), CM (30.4%) and CE (34.8%) groups, but it was increased in the ESCC group (55.5%); however, it was not statistically different from the other groups. No associations among the levels of CPP32 and Ki-67 expression were observed in the various groups, neither among parameters such as age, gender or alcohol and tobacco consumption. CONCLUSION: There were no evident changes in cell proliferation and apoptosis in benign lesions studied.

Association between achalasia and nitric oxide synthase gene polymorphisms.

BACKGROUND: Our group previously reported the absence of nitric oxide synthase (NOS) in the gastroesophageal junction of patients with achalasia. NOS exists in three distinct isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible isoform (iNOS). Some studies have shown that NO production is regulated by NOS polymorphisms. AIM: To assess whether some functional polymorphisms in the nNOS, iNOS, or eNOS genes are involved in susceptibility to suffer from achalasia. METHODS: Genomic DNA from 80 unrelated Spanish Caucasian patients with sporadic achalasia and 144 healthy subjects matched for age (+/-5 yr) and gender was typed by PCR and RFLP methods for the 27-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 of the eNOS gene, a CA microsatellite repeat and a Nla III (C-->T) restriction fragment length polymorphism (RFLP) in exon 29 of the nNOS gene, and two nucleotide substitutions located in exon 16 (C-->T) and exon 22 (G-->A) of the iNOS gene. RESULTS: No significant differences in carriage, genotype, and allele frequencies of the nNOS, iNOS, or eNOS gene polymorphisms were found between patients with achalasia and controls. Individuals homozygous for genotype iNOS22*A/A tended to be more frequent in achalasia (20%vs 11%, odds ratio [OR] 1.79, 95% confidence interval [CI] 0.89-3.59, p= 0.09) as were those homozygous for the rare eNOS*4a allele (6.2%vs 1.4%, OR 4.5, 95% CI 0.89-22.67, p= 0.1) although the difference did not reach statistical significance. No differences in genotype and allele distribution were found with respect to epidemiological and clinical characteristics of patients with achalasia. CONCLUSION: Our data suggest that NOS gene polymorphisms are not involved in the susceptibility to and nature of the clinical course of sporadic achalasia. However, studies in a greater number of patients are required to analyze the tendency toward a higher prevalence of genotypes iNOS22*A/A and eNOS*4a4a.

Levels of muscle enzymes in the serum after esophageal pneumatic dilation in patients with achalasia.

The success rate of pneumatic dilation of the esophagus in patients with achalasia is variable. We aim to assess whether levels of muscle enzymes in the serum are useful for predicting the efficacy of this procedure. Consecutive adults with symptomatic achalasia treated with pneumatic dilation were included. Blood samples were taken immediately before the procedure and after 12, 24 and 32 h. Clinical efficacy of the pneumatic dilation was evaluated on the basis of a symptom score defined prior to, and 2 months after the procedure. Eleven patients underwent 13 pneumatic dilations. In nine patients this was the first dilation attempt. Ten dilations were clinically effective. The study was discontinued after enzyme levels did not show a trend of increase in any of our patients. Moreover, a statistically significant unexpected decrease in creatine phosphokinase values was found 12 h after the procedure, among the 10 successful dilations. We believe that levels of muscle enzymes in the serum cannot predict the efficacy of pneumatic dilation in patients with achalasia.

Nitric oxide synthase distribution in the enteric nervous system of children with cardiac achalasia.

OBJECTIVE: To study the distribution of nitric oxide synthase (NOS) in the enteric nervous system of children with cardiac achalasia. METHODS: Biopsy specimens of the lower esophagus, cardia, gastric fundus and pylorus from 13 patients with cardiac achalasia and 6 controls were obtained and studied histochemically with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunohistochemically with a specific polyclonal antiserum. RESULTS: NOS was abundant in the myenteric plexus and the nerve fibers of musculatures in the esophagus, cardia and gastric fundus in control group, while it was nearly absent in the patient group. The distribution of NOS in the pylorus was similar in the two groups. CONCLUSION: These findings suggest that a lack of NOS in the lower esophagus, cardia and gastric fundus is involved in the pathophysiology of cardiac achalasia in children.

Familial adrenal insufficiency, achalasia, alacrima, peripheral neuropathy, microcephaly, normal plasma very long chain fatty acids, and normal muscle mitochondrial respiratory chain enzymes.

Adrenal insufficiency has been associated with adrenoleukodystrophy and adrenomyeloneuropathy. In these diseases, plasma very long chain fatty acids are elevated. Peripheral neuropathy is frequently seen in adults with adrenomyeloneuropathy. We encountered two first cousins with adrenal insufficiency, who also developed peripheral neuropathy, achalasia, alacrima, and microcephaly. However, plasma very long chain fatty acids, pipecolic acid, phytanic acid, and cranial computed tomographic scan were normal. Muscle mitochondrial respiratory chain enzymes were also normal. This syndrome of adrenal insufficiency, achalasia, alacrima, microcephaly, and peripheral neuropathy is different from either adrenomyeloneuropathy or adrenoleukodystrophy.

Patients with achalasia lack nitric oxide synthase in the gastro-oesophageal junction.

The abnormal function of the lower oesophageal sphincter in achalasia is likely to be due to impaired nonadrenergic, noncholinergic (NANC) inhibitory input. Since recent studies in animals suggest that nitric oxide (NO) is implicated physiologically in the inhibitory responses of the lower oesophageal sphincter, we have investigated whether the synthesis of NO is altered in the gastro-oesophageal junction of patients with achalasia. NO synthase activity was investigated in samples of tissue from the gastro-oesophageal junction obtained during surgery in eight patients with typical achalasia and six non-achalasic controls who underwent oesophagectomy for reasons other than sphincter dysfunction. The NO synthase activity was determined by the transformation of 14C-L-arginine into 14C-L-citrulline in tissue homogenates. In addition, immunohistochemical staining of the tissues was performed using a polyclonal antibody raised against a peptide sequence of rat brain NO synthase. Furthermore, the relaxant response to an exogenous NO donor (sodium nitroprusside, SNP) was measured in vitro in muscle strips obtained from two patients with achalasia and in two non-achalasic controls. NO synthase activity was detected in each of the samples obtained from six control patients (0.59 +/- 0.21 pmol mg-1 min-1; mean +/- SE). By contrast, none of the samples obtained from the eight patients with achalasia had any detectable NO synthase activity. Immunohistochemical studies confirmed the presence of NO synthase in the myenteric plexus of the gastro-oesophageal junction of control patients and its absence in achalasia. SNP relaxed muscle strips precontracted with bethanechol in both control samples and those from patients with achalasia.(ABSTRACT TRUNCATED AT 250 WORDS)

[Activity of the actomyosin complex of the esophageal muscles in patients with functional cardial obstruction]. / Aktivnost' aktomiozinovogo kompleksa myshts pishchevoda u bol'nykh s funktsional'noi neprokhodimost'iu kardii.

The results of studying ATP-ase activity of the actomyosin complex of the esophageal smooth muscles in functional cardiac obstruction (FCO) are discussed. It was established that in advancement of the diseases the ATP-ase activity of the actomyosin complex of the esophageal muscular coat diminishes steadily. Analysis of surgical treatment of 63 patients with FCO showed that in 12 patients with neglected stages of the disease (stages III and IV) its results were poor due to marked reduction of the esophageal tonus, which was linked with diminished activity of the actomyosin complex of the esophageal muscles in stages III and IV of the disease. The study provides evidence of the importance of determining ATP-ase activity of actomyosin in FCO in choice of the methods of nonoperative treatment and the time of surgical management of this pathological condition.