Alteration in faecal bile acids, gut microbial composition and diversity after laparoscopic sleeve gastrectomy.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is a well established treatment for severe obesity and type 2 diabetes. Although the gut microbiota is linked to the efficacy of LSG, the underlying mechanisms remain elusive. The effect of LSG for morbid obesity on the gut microbiota and bile acids was assessed here. METHODS: Severely obese subjects who were candidates for LSG were included and followed until 6 months after surgery. The composition and abundance of the microbiota and bile acids in faeces were assessed by 16S ribosomal RNA sequencing, quantitative PCR and liquid chromatography-mass spectrometry. RESULTS: In total, 28 patients with a mean(s.d.) BMI of 44·2(6·6) kg/m2 were enrolled. These patients had achieved excess weight loss of 53·2(19·0) per cent and showed improvement in metabolic diseases by 6 months after LSG, accompanied by an alteration in the faecal microbial community. The increase in α-diversity and abundance of specific taxa, such as Rikenellaceae and Christensenellaceae, was strongly associated with reduced faecal bile acid levels. These changes had a significant positive association with excess weight loss and metabolic alterations. However, the total number of faecal bacteria was lower in patients before (mean(s.d.) 10·26(0·36) log10 cells per g faeces) and after (10·39(0·29) log10 cells per g faeces) operation than in healthy subjects (10·83(0·27) log10 cells per g faeces). CONCLUSION: LSG is associated with a reduction in faecal bile acids and greater abundance of specific bacterial taxa and α-diversity that may contribute to the metabolic changes.

ANTECEDENTES: La gastrectomía vertical laparoscópica (laparoscopic sleeve gastrectomy, LSG) es un tratamiento bien establecido para la obesidad grave y la diabetes tipo 2. Aunque la microbiota intestinal se ha vinculado con la eficacia de LSG, los mecanismos subyacentes siguen siendo poco conocidos. En este estudio se evaluó el efecto de LSG en la obesidad mórbida sobre la microbiota del intestino y de los ácidos biliares (bile acids, BA). MÉTODOS: Tras la aprobación del Comité ético y la obtención del consentimiento informado, los sujetos con obesidad grave que eran candidatos para LSG fueron incluidos en el estudio y seguidos durante 6 meses después de la operación. Se evaluaron la composición y abundancia de la microbiota y BA en las heces mediante secuenciación del gen 16S rRNA, PCR cuantitativa y cromatografía líquida-espectrometría de masas. RESULTADOS: En total, 28 pacientes con una mediana (rango) del IMC de 43,9 kg/m2 (35,0-61,9) fueron reclutados y a los 6 meses tras una LSG, consiguieron una pérdida del exceso de peso de 47,3% (20,7-95,1) y mejoría de las enfermedades metabólicas acompañada de una alteración en la comunidad microbiana fecal. El aumento en la diversidad α y abundancia de especies taxonómicas específicas como Rikenellaceae y Christensenellaceae, se asociaba fuertemente con niveles fecales reducidos de BA. Estos cambios se asociaban de manera positiva y significativa con la pérdida del exceso de peso y las alteraciones metabólicas. Sin embargo, el número total de bacterias fecales en los pacientes fue inferior al de los sujetos sanos (10,84 log10 células/g heces (9,46-11,35)) antes de la operación (10,26 log10 células/g heces (9,44-10,91)) y después de la misma (10,42 log10 células/g heces (9,57-10,96)). CONCLUSIÓN: LSG se asoció con menos BA fecal y mayor abundancia de especies bacterianas específicas y diversidad α lo que puede contribuir a los cambios metabólicos.

Thymus histology and concomitant autoimmune diseases in Japanese patients with muscle-specific receptor tyrosine kinase-antibody-positive myasthenia gravis.

BACKGROUND AND PURPOSE: The differences in the characteristics of thymus histology, coexisting autoimmune diseases and related autoantibodies between anti-muscle-specific receptor tyrosine kinase (MuSK)-antibody (Ab)-positive myasthenia gravis (MG) patients, and anti-acetylcholine receptor (AChR)-Ab-positive MG patients are not clearly defined. METHODS: The types of thymus histology, coexisting autoimmune diseases and associated Abs in 83 MuSK-Ab-positive patients nationwide were investigated and were compared with those in AChR-Ab-positive patients followed at our institute (n = 83). As for the autoantibodies associated with thymoma, titin Abs were measured. RESULTS: Thymoma was not present in any of the MuSK-Ab-positive patients but presented in 21 patients (25.3%) amongst the AChR-Ab-positive patients. Titin Abs were absent in MuSK-Ab-positive patients but positive in 25 (30.1%) of the AChR-Ab-positive patients. Concomitant autoimmune diseases were present in eight MuSK-Ab-positive patients (9.6%) amongst whom Hashimoto's thyroiditis and rheumatoid arthritis predominated, whereas 22 AChR-Ab-positive patients (26.5%) had one or more concomitant autoimmune diseases of which Graves' disease predominated. CONCLUSIONS: Differences in frequency of thymoma and thymic hyperplasia, coexisting autoimmune diseases and autoantibody positivity between MuSK-Ab-positive and AChR-Ab-positive MG were indicated, suggesting that, in contrast with AChR-Ab-positive MG, thymus does not seem to be involved in the pathogenic mechanisms of MuSK-Ab-positive MG.

Influence of Cl- on organic anion transport in short-term cultured rat hepatocytes and isolated perfused rat liver.

Transport of 35S-labeled sulfobromophthalein [35S]BSP was studied in short-term cultured rat hepatocytes incubated in bovine serum albumin. At 37 degrees C, initial uptake of [35S]BSP was 5-10-fold that at 4 degrees C, linear for at least 15 min, saturable, inhibited by bilirubin, and reduced by greater than 70% after ATP depletion or isosmotic substitution of sucrose for NaCl in medium. Replacement of Na+ by K+ or Li+ did not alter uptake, whereas replacement of Cl- by HCO-3 or gluconate- reduced uptake by approximately 40%. Substitution of Cl- by the more permeant NO-3 enhanced initial BSP uptake by 30%. Efflux of [35S]BSP from cells to media was inhibited by 40% after ATP depletion or sucrose substitution. To confirm these results in a more physiologic system, transport of [3H]bilirubin was studied in isolated livers perfused with control medium or medium in which Cl- was replaced by gluconate-. Perfusion data analyzed by the model of Goresky, revealed 40-50% reductions in influx and efflux with gluconate- substitution. These results are consistent with existence of a Cl-/organic anion-exchange mechanism similar to that described by others in renal tubules.

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Purification and characterization of thymidylate synthetase from rat regenerating liver.

Thymidylate synthetase (EC 2.1.1.45) from rat regenerating liver has been purified over 5000-fold to apparent homogeneity by a procedure involving two affinity methods. Molecular weight of the native enzyme was found to be about 68,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight of 35,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The Michaelis constants for deoxyuridylate (dUMP) and (+/-)L-5,10-methylenetetrahydrofolate are 6.8 microM and 65 microM, respectively. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be ordered sequential. 5-Fluoro-dUMP, halogenated analog of the nucleotide substrate is a competitive inhibitor of the enzyme, with an apparent Ki value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent Ki value of 23 microM.

A new immunoblotting assay for thymidylate synthetase and its application to the regulation of enzyme activity in regenerating rat liver.

A highly sensitive and specific immunoblot assay has been developed to quantitate the content of rat liver thymidylate synthetase (EC 2.1.1.45). Applying the method, it is demonstrated that the increase of the activity of thymidylate synthetase in liver regeneration after partial hepatectomy is due to the de novo synthesis of the enzyme protein. Administration of cycloheximide, phenoxybenzamine, phorbol 12-myristate 13-acetate, nifedipine, dexamethasone or indomethacin to partially hepatectomized rats prevented the synthesis of thymidylate synthetase in regenerating liver. Thyroparathyroidectomy also inhibited the increase of the enzyme in liver regeneration. These observations are discussed in relation to the signal transduction concerning the alpha 1-receptor, which was shown to regulate liver regeneration in our previous papers.

Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor.

The human platelet-activating factor (PAF) receptor gene exists as a single copy on chromosome 1. We identified two 5'-noncoding exons, each of which has distinct transcriptional initiation sites. These exons are alternatively spliced to a common splice acceptor site on a third exon that contains the total open reading frame to yield two different species of functional mRNA (Transcript 1 and 2). Transcript 1 has consensus sequences for transcription factor NF-kappa B and Sp-1, and the Initiator (Inr) sequence homologous to the murine terminal deoxynucleotidyltransferase gene. Transcript 2 also contains consensus sequences for transcription factor AP-1, AP-2, and Sp-1. Transcripts 1 and 2 were both detected in heart, lung, spleen, and kidney, whereas only Transcript 1 was found in peripheral leukocytes, a differentiated human eosinophilic cell line (EoL-1 cells), and brain. Existence of distinct promoters was thus suggested to play a role in the regulatory control of PAF receptor gene expression in different human tissues and cells.

Change in caspase-3-like protease in the liver and plasma during rat liver regeneration following partial hepatectomy.

Recent studies have shown that many factors orchestrate liver regeneration after a two-thirds partial hepatectomy (PH). However, the termination mechanism in liver regeneration has not been thoroughly studied. In this paper, we report that the activity of liver caspase-3-like protease, which is specifically activated in apoptosis, increases 18, 36, and 48 hr after PH during maximal hepatocyte proliferative activity. This is the first study that shows the activation of an apoptosis-executing enzyme during physiological liver regeneration. These results suggest that apoptosis is induced in each surge of DNA synthesis as the termination mechanism. When phenoxybenzamine, an alpha-blocker that has been reported to inhibit DNA synthesis during liver regeneration, was injected 8 hr after PH, the caspase-3-like activity in the liver peaked at 15 hr after PH and the enzyme activity also increased in plasma at 18 and 24 hr after PH in sharp contrast to the case of normal regeneration. These results indicate that extensive apoptosis is caused by phenoxybenzamine and that the secondary necrosis of apoptotic cells results in the increase of caspase-3-like protease activity in the plasma.

Facile degradation of apolipoprotein B by radical reactions and the presence of cleaved proteins in serum.

A facile cleavage of peptide bonds of apolipoprotein B (apoB) by radical reaction is reported. When human LDL was subjected to oxidative damage using Cu2+, extensive degradation of apoB was observed based on immunoblotting. The degradation of apoB was inhibited by radical scavengers (beta-mercaptoethanol, butylated hydroxytoluene, and probucol) and promoted by a radical initiator [2, 2'-azobis(2-amidinopropane)dihydrochloride]. When human serum was treated with Cu2+, a similar cleavage pattern of apoB was observed. The cleaved apoB proteins were also detected in normal serum on the basis of immunoblots. These results suggest that apoB is highly reactive toward radicals in vitro and in vivo, with reaction resulting in the cleavage of peptide bonds.

Alpha-adrenergic regulation of the activity of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy.

The increases in activity of hepatic thymidylate synthetase and of thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which had been given alpha-adrenoceptor antagonists (phenoxybenzamine and phentolamine) or adrenergic neuron blockers (guanethidine and reserpine). These suppressions were not observed with a beta-adrenoceptor antagonist (propranolol), or an anticholinergic agent (atropine methyl nitrate). The rise in the activity of the thymidylate-synthesizing enzymes was closely correlated with the increase in the DNA content of the liver. It is concluded that catecholamine regulates the increase in the activity of thymidylate synthetase and thymidine kinase, which are key enzymes in DNA synthesis in regenerating liver. It is also suggested that sympathetic nerves play an important role in liver regeneration.

Churg-Strauss syndrome (allergic granulomatous angiitis) with multiple perforating ulcers of the small intestine, multiple ulcers of the colon, and mononeuritis multiplex.

A case of Churg-Strauss syndrome with multiple perforations of the small intestine is described. A 31-year-old woman was admitted with a complaint of epigastric pain. She had a history of bronchial asthma. One week before admission, white blood cell count was 20,800/mm3 with 59% eosinophils. Neurological examination on admission disclosed mononeuritis multiplex with paresthesia in both the lower and upper extremities. At colonoscopy, there were scattered aphthous ulcers in the colon. Ophthalmological examination revealed allergic conjunctivitis. After admission, hypereosinophilia increased to as high as 36,000/mm3. Oral administration of prednisolone (60 mg/day) was begun. On the 3rd day of the treatment, the eosinophil count decreased dramatically, to 400/mm3, while severe abdominal pain developed. Since abdominal X-ray film revealed free air in the abdominal cavity, emergency laparotomy was performed and multiple intestinal ulcers with perforations were found. Partial ileectomy was performed. Pathological findings of the resected specimen were interpreted as a necrotizing angiitis with extravascular granuloma. Since the operation, the patient has been asymptomatic, except for neurological symptoms. Hypereosinophilia has decreased without treatment to counts averaging 270/mm3, within 3 months. On the basis of the clinical features and histopathological findings, a diagnosis of Churg-Strauss syndrome was established.

Determination of folate derivatives in rat tissues during folate deficiency.

A method for the sensitive and specific determination of folate derivatives was developed. The method involves hydrolysis by gamma-glutamyl hydrolase and high-performance liquid chromatography with electrochemical detection. The method was applied to measure the change in the level of folate derivatives in the liver, kidney, spleen and brain of rats during folate deficiency. 5,6,7,8-Tetrahydrofolic acid was the major folate derivative in the liver, kidney, spleen and brain. Total concentration of folate derivatives decreased from the second week of folate deficiency in the liver, kidney, spleen and brain followed by anemia, which appeared at the fifth week. The level of 5,6,7,8-tetrahydrofolic acid in the brain did not change during folate deficiency, but it significantly decreased in the liver, kidney and spleen.

[A case of ischemic colitis due to polyarteritis nodosa].

A 78-year-old female was admitted to our hospital with acute abdomen (abdominal pain and bloody stool). Abdominal examination revealed mild rebound tenderness on the right side. The laboratory data revealed severe inflammation (WBC: 33100/microliters, CRP:35.5 mg/dl). Panperitonitis was suspected because of diffuse and severe abdominal pain and rebound tenderness on the next day. X-ray examination by gastrografin showed mucosal irregularity and tubular narrowing of the tubular narrowing of the ascending colon which indicated ischemic colitis, and an emergency operation was performed. Histological examination of the pathologic specimens revealed fibrinoid necrosis and destruction of the internal lamina in small and medium-size arteries. We report a case diagnosed as ischemic colitis due to polyarteritis nodosa by the findings of its pathologic specimens.