CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells.

The prototypical genetic autoimmune disease is immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a severe pediatric disease with limited treatment options. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which plays a critical role in immune regulation. As a monogenic disease, IPEX is an ideal candidate for a therapeutic approach in which autologous hematopoietic stem and progenitor (HSPC) cells or T cells are gene edited ex vivo and reinfused. Here, we describe a CRISPR-based gene correction permitting regulated expression of FOXP3 protein. We demonstrate that gene editing preserves HSPC differentiation potential, and that edited regulatory and effector T cells maintain their in vitro phenotype and function. Additionally, we show that this strategy is suitable for IPEX patient cells with diverse mutations. These results demonstrate the feasibility of gene correction, which will be instrumental for the development of therapeutic approaches for other genetic autoimmune diseases.

Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome.

Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.

Clinical outcome of patients examined by capsule endoscopy for suspected small bowel Crohn's disease.

BACKGROUND: Capsule endoscopy has a greater diagnostic yield than radiology for detecting subtle inflammatory changes of the small bowel mucosa, but the clinical significance of these abnormalities is still uncertain because of the lack of long-term follow-ups. AIM AND METHODS: To verify the accuracy of capsule endoscopy in a cohort of patients with suspected Crohn's disease of the small bowel, taking as 'gold standard' the final diagnosis made after a long follow-up. From April 2002 to March 2005, we enrolled and examined by capsule endoscopy 27 consecutive patients with abdominal pain and diarrhea lasting more than 3 months and at least one of the following: anaemia, weight loss, fever, extra-intestinal manifestation(s) of inflammatory bowel disease. All patients already had an unremarkable pan-endoscopy, serology for celiac disease and intestinal radiology inconclusive for small bowel abnormality. On the basis of capsule endoscopy findings, patients were distributed in three groups; Group A had severe stricturing lesions requiring surgery; Group B, moderate inflammatory lesions further investigated invasively; Group C, minimal inflammatory changes or normal findings, clinically observed every 3 months (median 21 months, range 15-29). RESULTS: Small bowel inflammatory lesions were found in 16 of the 27 patients (diagnostic yield 59%). Three had surgery (Group A) and Crohn's disease was confirmed in two; the remainder had ileal adenocarcinoma in a pathological context of chronic inflammation. Crohn's disease was histologically confirmed in four of the five patients in Group B. Group C comprised 19 patients; Crohn's disease was confirmed in seven out of eight with positive capsule endoscopy, while only one of the patients with normal findings later developed overt ileal Crohn's disease. Sensitivity, specificity, positive and negative likelihood ratio were, respectively, 93%, 84%, 5.8 and 0.08. Assuming a 50% pre-test probability of disease, capsule endoscopy gave a post-test probability of 85%. CONCLUSIONS: In our selected cohort, capsule endoscopy was highly sensitive in detecting small bowel inflammatory changes, enhancing by nearly 35% the pre-test probability of structural small bowel disease. Focal erythema and luminal debris may limit the specificity of capsule endoscopy.

Ameliorating effects of RU 47213, a novel oral and long-lasting cholinomimetic agent, on working memory impairments in rats.

The anti-amnesic effects of RU 47213 [1-(4-chlorophenoxycarbonyl)-1,2,5, 6-tetrahydropyridine-3-carboxaldehyde-O-methyloxime], a prodrug with oral and long-lasting cholinergic activity, were evaluated on working memory impairments, using tasks of unequal levels of difficulty involving the same reinforcement and motivation in rats: a spatial-based task in a radial maze and a delayed reinforced alternation task in a T-maze. Tetrahydroaminoacridine (THA; tacrine), a cholinesterase inhibitor was used as a reference. Groups of rats were trained in an automated radial maze or T-maze until they had attained an asymptotic level of performance. On test days, memory impairment was produced by administration of scopolamine (0.1 mg/kg s.c.) 15 min prior to testing. Both THA (1.3, and 5 mg/kg) and RU 47213 (0.2, 0.5, 1, and 2 mg/kg) given prior to testing markedly reduced or suppressed the scopolamine induced working memory deficits in both tasks. This activity was evidenced by either a significant decrease in the number of errors or an increase in the number of correct responses. These results show that RU 47213 possesses the capacity to reduce memory deficits induced by an impairment of cholinergic transmission in the rat.

Antagonism of scopolamine-induced memory impairments in rats by the muscarinic agonist RU 35,926 (CI-979).

The promnesic effects of RU 35,926 (CI-979), a muscarinic receptor agonist, were evaluated on memory impairments induced by the muscarinic antagonist scopolamine, using a radial arm maze task, in comparison with tetrahydroaminoacridine (THA), a cholinesterase inhibitor. Groups of rats were trained in a standard version of the radial maze until they had attained an asymptotic level of performance. The animals were then retested with one trial a day. Twenty minutes before each retest, the rats were given subcutaneous administration of 0.1 mg/kg scopolamine. Oral administration of RU 35,926 (0.02, 0.05, 0.1, 0.2, and 0.5 mg/kg) 30 min before memory retest markedly reduced or suppressed the scopolamine-induced deficit. This reduction was evidenced by a significant decrease in the different types of errors and an increase in the number of correct responses. THA (3 mg/kg, intraperitoneally or orally) given 20 min to testing also significantly reduced or suppressed the scopolamine-induced deficits. These results show that RU 35,926 possesses the capacity to reduce memory impairments induced by a deficit of cholinergic transmission in the rat.

Adjuvant therapy with essential fatty acids (EFAs) for primary liver tumors: some hypotheses.

Hepatocarcinoma is responsible for approximately 1 million deaths annually. It is usually discovered at an advanced stage and, if inoperable, has a poor prognosis. New therapies combining chemotherapy, hyperthermia, radiotherapy and immunomodulators have been recently attempted with various levels of success. Once the tumor is detected at an early stage, some possibilities of cure seem to emerge either by intratumoral percutaneous injection (PEI) of alcohol or by chemoembolization and interstitial hyperthermia. When the tumor volume is more than 5 cm, these therapies are less successful and radiotherapy can be used. All the techniques described have some limits; PEI, for instance, does not achieve a complete eradication of lesions > 3 cm and a non-homogenous alcohol distribution within the tumor leads to areas of necrosis. Radiotherapy, even if effective, is limited by dose-related radiation hepatitis. Another important limiting factor is the incomplete response to therapy and tumor recurrence. Essential fatty acids, especially gamma linolenic acid (GLA) and eicosapentaenoic acid (EPA) are discussed here for their ability to control primary tumor proliferation and increase response to chemotherapy, radiotherapy and hyperthermic treatment, thanks to their effects on cellular membranes (increased lipoperoxidation and modification of tumor stroma).

Changes in diagnostic approach and factors affecting treatment and survival of pancreatic carcinoma in a retrospective series over twelve years.

AIMS: Identify the following aspects of pancreatic carcinoma: 1) the prevalence of some risk factors, 2) the accuracy of the diagnostic techniques and the pattern of their utilization over the years, 3) the factors affecting the therapeutic choice and mortality. METHODS: Retrospective study on all patients with a final diagnosis of pancreatic carcinoma seen at the Ospedale di Busto Arsizio, from January 1978 to August 1989. RESULTS: There were 155 patients, 68 +/- 11.6 years old, with a 1.2 male to female ratio. Antemortem pathologic confirmation was obtained in 127 cases (82%); 45% were smokers and 45% drinkers. Diabetes mellitus, a history of peptic disease, a past neoplasm and gallstone disease were respectively present in 36.1%, 12.3%, 11% and 8.2% of the cases; 61.9% 23.8% and 9% of the tumors were located respectively in the head, body and tail; 1.3%, 40% and 51.5% were respectively in stage II, III and IV. Ultrasound abdominal scanning and computerized tomography sensitivity were respectively 67.5% and 72.5% (p = NS). In addition, carcinoembryonic antigen, fine needle aspiration biopsy and percutaneous transhepatic cholangiography showed respectively a 66.6%, 88.9% and 93% sensitivity. Together the non-invasive imaging procedures dramatically decreased the number of unnecessary exploratory laparotomies over the years (p = 0.005) without changing the stage at diagnosis or survival. Among the tested variables (age, sex, year of diagnosis, past diseases, co-morbidity, location and stage of the tumor), only the head location and a less advanced stage were significantly related to a surgical choice (p < 0.001). Overall one-year survival rate was 13.4%, and among the tested variables, only a less advanced stage and the aggressive treatment were associated to a longer survival (p < 0.001). CONCLUSIONS: The prevalence of diabetes mellitus in patients with pancreatic carcinoma may be higher than previously expected; the wide use of diagnostic imaging, dramatically reduced the number of unnecessary exploratory laparotomies over the years; aggressively treated patients with a less advanced stage have a slight, but significant improvement in survival.

New classes of antimuscarinic agents endowed with selective antispasmodic properties. 1-Arylsulfonyl pyrrolidin-2-ones and 2-thiones, 1-arylsulfonyl piperidin-2-ones and 2-thiones and 1-arylsulfonyl hexahydro-2H-azepin-2-one.

A series of 1-arylsulfonylpyrrolidin-2-ones (and 2-thiones), 1-aryl sulfonylpiperidin-2-ones (and 2-thiones) and 1-arylsulfonyl hexahydro-2H-azepin-2-one were synthesized and submitted to a battery of binding assays. The compounds showed little or no affinity for the receptors tested other than muscarinic receptors labelled either with [3H]pirenzepine or with [3H]quinuclidinyl benzilate. When tested in the isolated guinea pig ileum, they antagonized the contractions induced by acetylcholine and behaved as competitive muscarinic antagonists. After parenteral administration in mice, most compounds inhibited carbachol-induced diarrhoea but were less effective in counteracting salivation and lacrimation and showed little or no mydriatic action, thus displaying selectivity at the intestinal level. The reference drugs tested, atropine, butyl scopolamine and cimetropium bromide were far less selective. maximal in vivo activity was obtained by introducing diethylamino or 1-piperidino or 1-hexahydroazepinyl groups in the 4-position of the phenyl ring while the enlargement of a 5- to a 6-membered lactam ring or its conversion into a thiolactam had a less marked effect. The most interesting compounds were further evaluated for their ability to antagonize carbachol-induced colonic hypermotility in the rat and arecoline-induced analgesia in mice. The effect on gastric acid secretion in the rat was also investigated. The overall in vivo data showed that compounds 14, 15, 26 and 27, i.e. those bearing a 1-hexahydroazepinyl group in the 4-position of the phenyl ring, were the most potent and selective compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

[Planning of a prospective study with a biological bank]. / Progettazione di uno studio prospettico con banca biologica.

This present work aims to give a critical description of the organization and operative aspects of project ORDET (prospective study on diet and hormones in the aetiology of breast cancer). A number of problems which could arise when carrying-out a prospective study are described: they concern participant recruitment, laboratory organization, planning of a biological bank plus relative security checks in order to ensure full workability and personnel standardization in questionnaire administration and anthropometrical measurements. The methods adopted by ORDET, or at least some of them, could be useful for those engaged in the planning phase of a similarly designed project using a biological bank.

[Endobronchial lipoma (presentation of a case)]. / Il lipoma endobronchiale (presentazione di un caso).

The Authors report on a case of endobronchial lipoma in a patient operated on six years previously for a clear-cell tumour of the kidney and discuss the diagnostic and therapeutic problems posed by this disease, after stressing its extreme rarity.

A comparison of some of the pharmacological properties of the new eburnamenine derivative vindeburnol with those of vincamine, vinburnine, dihydroergotoxine mesilate and nicergoline.

The effects of a new eburnamenine derivative (3 beta,14 alpha, 16 alpha)-(+/-)-14,15-dihydro-20,21-dinoreburnamenin-14-ol (vindeburnol, RU 24722) on EEG, on brain energy metabolism and on local cerebral blood flow (LCBF) and in different experimental models of cerebral insufficiency were compared with those of vincamine, vinburnine (1-eburnamonine), dihydroergotoxine mesilate and nicergoline. Vindeburnol at 2 mg/kg i.v., increased the EEG resistance time in rats subjected to asphyxia anoxia and at 10 mg/kg s.c., significantly improved the electrocortical recovery of gerbils subjected to a 10-min cerebral ischemia. Vindeburnol (10 mg/kg i.p.) significantly retarded glucose, phosphocreatine and adenosine triphosphate utilization and lactate production in mouse brain during 10 s of decapitation ischemia. The cerebral metabolic rate was 10.34 mmol/kg/min, which was about 50% of the control value. At 10 mg/kg i.p., the product induced a slight and transient increase in LCBF. Vincamine improved the early phase of the postischemic electrocortical recovery in the gerbil, had no effect on cerebral energy substrates and slightly increased the LCBF for 15 min. Dihydroergotoxine mesilate improved the early phase of the electrocortical recovery in gerbils subjected to ischemia, did not significantly modify the energy substrates and rapidly increased the LCBF, which was normal after 30 min. Vinburnine and nicergoline were inactive in the cerebral insufficiency models used and did not significantly modify cerebral energy metabolism. These results show that vindeburnol has a different pharmacological profile from vincamine, vinburnine, dihydroergotoxine mesilate and nicergoline, and suggest that vindeburnol may be therapeutically effective in cerebral insufficiency.

[Compounds with anti-gastric secretion and antiulcer activity. V. Derivatives of 3,3,4a,6,7,11b-hexahydro-2H,5H-benzo [1,4]cyclohep[1,2-b][1,4]-oxazine and related products]. / Composti ad attività antisecretiva gastrica ed antiulcera. Nota V. Derivati di 3,4,4a,6,7,11b-esaidro-2H, 5H-benzo[1,4]cicloept-[1,2-b][1,4]ossazine e prodotti correlati.

A series of hexahydrobenzo[3,4]cyclohept[1,2-b][1,4]oxazines, with different substituents at the benzene and oxazine rings and nitrogen atom, was synthetized. The diastereomers and enantiomers of the unsubstituted compound and the related derivatives hexahydrobenzo[5,6] and exahydrobenzo[6,7]cyclohept[1,2-b][1,4]oxazine, hexahydro[1]benzoxepine and hexahydro[1]benzothiepin[5,4-b][1,4]oxazine, octahydrobenzo[3,4]cyclohept[1,2-b][1,4]diazine were also prepared. Some of these compounds inhibited at low doses gastric secretion in pylorus-ligated rats and antagonized ulcers in pylorus-ligated rats plus acetylsalicylic acid and in cold+restraint-stressed rats. When the most active compounds were tested on cats with Heidenhain pouch, they induced unwanted side effects that ruled out further development of these chemical series.

Effects of the new eburnamenine derivative RU 24722 on EEG recovery and cerebral energy metabolism after complete ischemia.

The influence of a new eburnamenine derivative RU 24722 [(3 beta, 14 alpha, 16 alpha)-(+/-)-14,15-dihydro-20,21-dinoreburnamenin -14-ol] on post-ischemic EEG recovery was studied in N2O anesthetized rats subjected to 1 min of global-compression cerebral ischemia. RU 24722 was compared with vincamine, dihydroergotoxine mesylate and nicergoline. Treatment with RU 24722 (2 mg/kg i.v.) significantly decreased the EEG recovery time and increased the electrocortical activity during the first phase of the post-ischemic recovery. Vincamine (2 mg/kg i.v.), dihydroergotoxine mesylate (0.5 mg/kg i.v.) and nicergoline (0.5 mg/kg i.v.) were devoid of activity. In an attempt to elucidate its mechanism of action, the influence of RU 24722 on changes in the cerebral metabolic energy reserves was studied in mouse brain after different periods of decapitation ischemia. The changes occurring during the first 10 s of ischemia were used to calculate the baseline cerebral metabolic rate (CMR). The activity of RU 24722 was compared with that of vincamine and pentobarbital. RU 24722 (10 mg/kg i.p.) significantly retarded glucose, phosphocreatine and adenosine triphosphate utilisation and lactate production. Vincamine (10 mg/kg i.p.) had no effect on cerebral energy substrates. Pentobarbital (100 mg/kg i.p.) markedly increased the tissue concentration of glucose and phosphocreatine and decreased lactate levels before and after ischemia. The improvement of EEG recovery suggests that RU 24722 may be therapeutically effective in cerebral insufficiency, and the decreased brain energy demand may be one of the mechanisms by which RU 24722 has a protective effect against cerebral ischemic damage.

Gastric cytoprotective, antisecretory and antiulcer activities of (E)-4-oxo-4-(3,4,5-trimethoxyphenyl)-2-butenoic acid (RU 38086).

As a result of trials of a large series of compounds, RU 38086 (E)-4-oxo-4-(3,4,5-trimethoxyphenyl)-2-butenoic acid) was selected because of its cytoprotective, antisecretory and antiulcer properties. In pylorus-ligated rats, RU 38086 dose-dependently decreased the total acid output, at 2.5, 5 and 10 mg/kg orally and at 10 and 50 mg/kg intraduodenally. In the perfused rat stomach, 1.2 mg/kg RU 38086 in situ inhibited acid secretion stimulated by histamine or pentagastrin but was inactive against carbachol. In the same test 5 mg/kg intravenously did not antagonize pentagastrin-induced acid secretion. In the cat Heidenhain pouch, 0.6 mg/kg RU 38086 was also antisecretory, reducing the acid concentration when in contact with the mucosa of the pouch. In ulcers induced in rats by ligature of the pylorus plus acetylsalicylic acid, RU 38086 at 2.5 and 5 mg/kg demonstrated much more striking activity after oral than after intraduodenal administration. It also had antiulcer activity against stress ulcers (restraint plus cold), starting at a dose of 5 mg/kg orally. RU 38086 had marked gastric cytoprotective activity in rats against the necrotizing effects of ethanol from the low dose of 0.3 mg/kg orally. This cytoprotective activity was not significantly affected by indomethacin pre-treatment. At 4 and 20 mg/kg orally, RU 38086 strongly increased prostaglandin E2 levels in gastric juice of pylorus-ligated rats and in stomach tissue of normal rats. These data indicate that RU 38086 is an orally effective cytoprotective, antisecretory and antiulcer agent.

Cerebral energy metabolism and computerized EEG. Analysis following transient ischemia in the rat.

1. Cerebral energy metabolism and electroencephalogram (EEG) power spectra were evaluated before, during and after 1 minute of global compression ischemia induced in N2O anesthetized rats. 2. Before ischemia, the EEG of rats was characterized by theta and delta frequencies. During ischemia, the EEG flattened after 14 +/- 1 sec and the electrical activity reappeared 55 +/- 4 s after recirculation and was characterized by high amplitude slow waves. After ischemia, the total power showed a rapid rebound mainly due to a 10 fold increase of delta band. 10 minutes after recirculation, the EEG was normal. 3. The ischemia induced a decrease of phosphocreatine, ATP and glucose, and an increase of ADP, AMP and lactate, while pyruvate remained normal. During recirculation phosphocreatine, ATP, glucose and energy charge potential showed a rapid recovery. AMP normalized after 10 minutes, while ADP was slightly higher for up to 30 minutes. Lactate returned to normal levels after 30 minutes and pyruvate showed a high peak at 3 minutes and returned to normal values after 30 minutes. 4. The results suggest that there is not a correlation between EEG and the metabolic pattern of recovery after a short complete ischemia.

Influence of side chain configuration on anti-inflammatory analgesic and anti-pyretic properties of 4-biphenylyl alkanoic acids.

A study on the influence of the number of carbon atoms in the side chain as well as side chain configuration on some pharmacologic properties of 4-biphenylyl alkanoic acids is presented. Unlike the chemical structure dependent anti-inflammatory properties, mild analgesic and anti-pyretic properties were neither dependent upon number of carbon atoms nor side chain configuration.