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.

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.

Ectopic FOXP3 Expression Preserves Primitive Features Of Human Hematopoietic Stem Cells While Impairing Functional T Cell Differentiation.

FOXP3 is the transcription factor ruling regulatory T cell function and maintenance of peripheral immune tolerance, and mutations in its coding gene causes IPEX autoimmune syndrome. FOXP3 is also a cell-cycle inhibitor and onco-suppressor in different cell types. In this work, we investigate the effect of ectopic FOXP3 expression on HSC differentiation and we challenged this approach as a possible HSC-based gene therapy for IPEX. FOXP3-expressing HSC showed reduced proliferation ability and increased maintenance of primitive markers in vitro in both liquid and OP9-ΔL1 co-cultures. When transplanted into immunodeficient mice, FOXP3-expressing HSC showed significantly enhanced engraftment ability. This was due to a pronounced increase in the frequency of repopulating cells, as assessed by extreme limiting dilution assay. Likely underlying the increased repopulating ability, FOXP3 expressing HSC showed significantly enhanced expression of genes controlling stemness features. However, peripheral T cells developed in the FOXP3-humanized mice were quantitatively reduced and hyporesponsive to cytokine and polyclonal stimulation. Our findings reveal unpredicted effects of FOXP3 in the biology of HSC and may provide new tools to manipulate primitive features in HSC for clinical applications. Moreover, they formally prove the need of preserving endogenous FOXP3 regulation for an HSC-based gene therapy approach for IPEX syndrome.

Similar binding to glutamate receptors by Rasmussen and partial epilepsy patients' sera.

Immunoreactivity of sera from patients with Rasmussen encephalitis (RE) and patients with partial epilepsy (PE) was analyzed by immunohistoblot on rat brain sections and the staining pattern compared with that obtained with antibodies to a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and NMDA receptors. Staining for anti-glutamate receptor 3 (GluR3) was found in 82% of patients with RE and 64% of patients with PE. Histoblot analysis showed a positive staining in GluR3- and NMDA-specific regions of rat brain, providing a comprehensive CNS immunolocalization.

Biofilms on right heart flow-directed catheters.

This study was designed to detect biofilm and bacteria on right heart flow-directed catheters using scanning electron microscopy and culture following scraping and dispersion of biofilm by sonication. We examined 20 consecutive catheters removed from 18 critically ill patients, an average of 2.6 days after insertion. On scanning electron microscopy, all catheters were found to be covered by a biofilm, with bacteria visible on 50 percent of them. Cultures of specimens from 40 percent of the catheters grew skin organisms (Staphylococcus warneri, Diphtheroid), anaerobes (Propionibacterium), and other potential pathogens (Proteus vulgaris, Enterobacter cloacae). Combination of the two techniques produced a bacterial detection rate of 75 percent. This study demonstrates that the presence of biofilm with bacterial adherence is common on right heart flow-directed catheters. The phenomenon could play a significant role in endogenous infection in critically ill patients.

Abdominal distension after colonic lactulose fermentation recorded by a new extensometer.

Colonic lactulose fermentation induces bloating, but whether it also causes abdominal distension is not known. The aim of this study was to assess the effect of colonic lactulose fermentation on abdominal girth using a new extensometer. We recorded abdominal girth in 24 healthy subjects by means of an extensometer that measures the phase shift of an ultrasound wave propagating in a tube encircling the abdomen. The recordings were continuously made for 3 h after the ingestion of 100 mL of tap water with (16 subjects) or without (eight subjects) 10 g of lactulose. Every 10 min, H2 in the breath was analysed and the intensity of bloating was recorded. Bloating was never reported after water ingestion, whereas it was reported by 10 subjects after lactulose ingestion (P = 0.002). The mean +/- SD changes in abdominal girth in comparison with resting conditions were statistically significant after lactulose ingestion (3 +/- 2.9 mm; P = 0.002) but not after water ingestion (-0.2 +/- 2.7 mm; P = 0.82). The area under the curve of the changes in abdominal girth after lactulose were significantly greater than after water ingestion (P = 0.03). In conclusion, colonic lactulose fermentation induces bloating and abdominal distension. The new extensometer is useful for continuously recording changes in abdominal girth.

QSAR models for discriminating between mutagenic and nonmutagenic aromatic and heteroaromatic amines.

In a previous article, we demonstrated that the structure-activity relationship model for the mutagenic potency of aromatic amines is different from that for discriminating between mutagens and nonmutagens. In this work, we present further analyses on the molecular determinants of the mutagenicity of aromatic amines. Based on the use of various methodological approaches, our results indicate that mutagenic activity is influenced by different molecular characteristics in different subclasses of aromatic amines. Thus, the general lesson of this article is that 1) in genetic toxicology, it is necessary to separately investigate the structure-activity relationships for discrimination between positive and negative chemicals, and the structure-activity relationships for the potency of the positive chemicals; 2) in structure-activity studies, it is necessary to investigate the degree of homogeneity (congenericity) of apparently similar chemicals in order to assess and describe the various mechanisms of action that may be elicited by the chemicals.

Modelling physico-chemical properties of halogenated benzenes: QSAR optimisation through variables selection.

Abstract This paper describes the development of multivariate QSAR models for halogenated benzenes. Four physico-chemical properties have been modelled: Boiling Point (BP), Melting Point (MP), Flash Point (FP) and Density (D). For the multivariate characterisation of this class of compounds 90 topological indices derived from the chemical formula were used. The models were calibrated on training sets selected by a D-optimal design and were optimised by a statistical procedure of selection of the most informative variables. The predictive capacity of the resulting models measured in terms of the parameter SDEP (Standard Deviation of the Errors of Predictions) were as follows; MP: ± 21 ±C (with MP ranging from -48° to 181°C); BP: ±13°C (75° to 286°C); D: ±0.14 (1.02 to 2.52 g/cm(3)); FP: ±17°C (-12 to 126°C). These data were confirmed "experimentally", i.e., on external validation sets of compounds, the experimental values of the standard deviation of prediction errors were in fact as follows; MP: ±30°C; BP: ±16°C D: ±0.18 g/cm(3); FP: ±16°C. The developed models therefore provide a valuable tool for estimating physico-chemical properties relevant to environmental chemistry.

[Respiratory resuscitation in Quebec. General aspects and implications of pneumologists in intensive care units in "New France"]. / Réanimation respiratoire au Québec. Aspects généraux et implication des pneumologues aux soins intensifs en "Nouvelle France".

ICU set up is a complex framework in Québec. In this respect, quebecer and french systems are very different. Pulmonologists are one of the most committed sub-specialists in ICUs, either as consultant or as MD on duty. Amongst paramedics, respiratory therapists are essential members of the team. Invasive and noninvasive ventilations are commonly performed. Critical care teaching program and structures for developing and supporting clinical research activities are in place.

Indications for intensive care unit care after carotid endarterectomy.

OBJECTIVES: To describe the complications of carotid endarterectomy and the interventions performed in the intensive care unit (ICU) after carotid endarterectomy. To identify preoperative and recovery room (RR) risk markers for these complications and interventions. DESIGN: A retrospective case study. SETTING: The ICU of a university hospital. PATIENTS: One hundred and one patients who required carotid endarterectomy over a 15-month period. INTERVENTION: Carotid endarterectomy (bilateral procedures in 11 patients). OUTCOME MEASURES: Demographic data including Goldman's cardiac risk index and the therapeutic intervention scoring system (TISS) score to measure the risk of complications. RESULTS: Most of interventions conducted in the RR and ICU were to control high blood pressure. In the RR, three patients experienced a neurologic event, one patient was reintubated for vocal cord paralysis and one had electrocardiographic abnormalities. Overall, 5 of the 101 patients had neurologic complications and 2 suffered a myocardial infarction. Two patients died, one as a result of a massive stroke and the other of myocardial infarction with cardiogenic shock. The mean (and standard deviation) TISS. score in the ICU was 12.6 (3.8). Analysis of all events in the RR was not predictive of events in the ICU. However, the absence of major complications in the RR had a negative predictive value of 97%. CONCLUSIONS: The decision to admit patients to the ICU after carotid endarterectomy should be based on major complications occurring in the RR. A low TISS score and low incidence of complications does not warrant routine admission.

Infrared spectra as chemical descriptors for QSAR models.

Infrared spectra (IR) were used as regressors for a number of QSARs and compared with both mechanistically oriented descriptors and heuristic "chemically neutral" descriptors (modified adjacency matrices eigenvalues). IR spectra usually gave results inferior to those obtained with the mechanistically driven descriptors, with one notable exception, and comparable to those obtained by adjacency matrices eigenvalues. So the IR spectra cannot be considered as an "a-priori" optimal description of molecules for QSAR. However the relation of IR with the chemicophysical bases of drug-receptor interaction suggests the use of IR spectra for elucidating mechanistic details.

A new instrument for the measurement of rib cage and abdomen circumference variation in respiration at rest and during exercise.

A simple and inexpensive new extensometer for measuring changes in chest wall circumference during human respiratory movements is presented. The instrument detects the delay between ultrasound emission and reception at opposite ends of two rubber tubes encircling the rib cage and abdomen. Assuming a two degree of freedom model of the chest wall and employing an isovolume procedure for determination of volume-motion coefficients, extensometer estimation of tidal volume (VT) from changes of rib cage and abdomen circumference was compared with spirometer measurements at rest and during exercise on a cycle ergometer (55-155 W) in six subjects and, in four of them, on a treadmill (4-12 km.h-1). In three subjects hypercapnic hyperpnoea at rest was also studied. The slopes of the linear relationship between extensometer and spirometer VT (litres) averaged 0.9967 (SD 0.0117) (r2 = 0.995-0.998; n = 90-143) for cycle ergometer exercise, 1.0072 (SD 0.0078) (r2 = 0.991-0.998; n = 75-93) for treadmill exercise and 0.9942 (SD 0.0188) (r2 = 0.997-0.998; n = 18-25) for hypercapnic hyperpnoea. In all instances the slope of the regression line was consistent with the model of the identity line (slope = 1). The changes in end-expiratory lung volume between respiration at rest and during exercise were determined by the extensometers, and were nearly identical (98.4% on average) to those measured with the spirometer (r2 = 0.945; n = 24). It is concluded that determination of chest wall circumference with this new instrument is suitable for quantitative measurement of ventilation and lung volume variations in humans under most physiological conditions.

Organic mercurial diuresis: inhibition of glutamine utilization in the acidotic rat.

Organic mercurials inhibit mitochondrial glutamine metabolism in vitro while metabolic acidosis, a condition in which the predominant renal fuel is glutamine, potentiates mercurial diuresis. The following studies were undertaken to determine whether potentiation of diuresis reflects mercurial inhibition of glutamine utilization. (1) All three mercurials employed (mersalyl, chlormerodrin, and p-chloromercuribenzoate) are diuretics in the rat and this effect was potentiated by NH4Cl. (2) Despite reabsorbing less sodium, mercurial-treated rats had lower kidney ATP content (4.35 +/- 0.26 and 3.84 +/- 0.43 mumol/g dry weight (mercurial plus NH4Cl) than did controls (4.95 +/- 0.31 and 4.87 +/- 0.39 mumol/g dry weight (NH4Cl). (3) Isolated kidneys from NH4Cl and NH4Cl plus mercurial treated rats were perfused with 1 mM L-[U-14C]glutamine to determine rates of extraction and oxidation. Mercurial-treated acidotic rat kidneys had a reduced rate of glutamine uptake (40.8 +/- 7.4 vs. 64.8 +/- 5.8 mumol/h per kidney), a diminished rate of glutamine conversion to CO2 (14.8 +/- 3.6 vs. 26.4 +/- 5.2 mumol/h per kidney), and a reduction in glucose production (16 +/- 5 vs. 27 +/- 4 mumol/h per kidney). These results are consistent with an effect of organic mercurials upon glutamine utilization, limiting ATP availability, and thereby reducing tubular active sodium reabsorption.

Herbicidal triazines: acute toxicity on Daphnia, fish, and plants and analysis of its relationships with structural factors.

The 24- and 48-hr EC50 immobilization test of Daphnia magna has been performed on 14 herbicidal triazines characterized by structural differences with respect to the substituent groups at the three carbon atoms in the s-triazinic ring. EC50 values of the tested compounds were found to vary within a factor of 6 to 7; such variations of toxicity have been analyzed in terms of structural variations concerning (i) the type of substituents, i.e., Cl, SCH3, or OCH3, at the carbon atom C-2, and (ii) the number and size of the alkyl groups at the aminic nitrogen atoms bound to the ring carbons C-4 and C-6. Literature data relative to the acute toxicity of the considered herbicides on fish and plants were also analyzed with reference to the above structural features. The possibility that separate contributions to toxic effects of triazines are provided by specific groups bound to the common structural moiety, constituted by the 1,3,5-triazinic ring, is examined and discussed.

Respiratory muscle dysfunction after herpes zoster.

We report an unusual neurologic complication of herpes zoster. After thoracic herpes zoster, our patient complained of severe shortness of breath as a result of myoclonus of the abdominal muscles as documented by electromyography. The myoclonus resulted in repetitive interruption of expiratory air flow, resulting in shortness of breath and a staccato speech. This case demonstrates the need to evaluate the function of all the respiratory muscles in a patient complaining of dyspnea.

Brain hemorrhage associated with methanol poisoning.

We recently treated seven patients who were victims of accidental methanol intoxication. Five of the seven patients died within 72 h of hospital admission. The two survivors were discharged from the hospital without visual or neurologic impairment. Hemorrhagic necrosis of the brain was found on CT scan and at autopsy in two patients who died. This complication of methanol intoxication prompted us to review our experience with this poison since 1980. Of 45 methanol intoxication patients treated in our ICU, CT scan was performed in 21, and brain hemorrhage was documented in six, thereby giving an incidence of at least six of 45 or 13.5%. Although hemorrhagic and nonhemorrhagic putamenal necrosis has been described from methanol poisoning previously, we postulate that heparinization during hemodialysis may contribute to brain hemorrhage complicating methanol poisoning and recommend the use of artificial kidneys with albumin-primed biocompatible membranes so that heparin administration during hemodialysis can be minimized.

Infection with fatal outcome after endovascular metallic stent placement.

The authors describe a case in which infection developed in a patient after placement of a metallic endoprosthesis in the iliac artery. The patient died of respiratory failure secondary to sepsis. Imaging studies did not reveal an abscess, aneurysm, or stent occlusion. Stent cultures and hemocultures grew Staphylococcus aureus. As with any foreign body, infectious complications may occur after insertion of metallic stents and should be recognized as soon as possible to initiate appropriate therapy.

Biofilms on indwelling vascular catheters.

BACKGROUND AND METHODS: Some bacteria have a natural tendency to adhere to available surfaces and to form biofilms. Biofilms have been demonstrated on right heart flow-directed catheters, endocardial pacemaker leads, urinary catheters, and other medical devices. In this study, we examined arterial and central venous catheters that had been in place in ICU patients between 1 and 14 days for the presence of bacterial biofilms by scanning electron microscopy, transmission electron microscopy, and a special scraping/sonication bacterial recovery technique. The data taken from these processes were compared with skin entry site swabs and blood cultures and correlated with patient data on infection, bacterial colonization, and antibiotic use. RESULTS: Extensive biofilm formation was demonstrated by scanning electron microscopy on all 42 arterial and 26 central venous catheters. Bacteria were seen within the biofilms on 69% (29/42) of the arterial and 88% (23/26) of the central venous catheters. These two direct methods for the detection of biofilm bacteria on the catheter surfaces demonstrated the presence of adherent organisms on 81% of devices examined. Some catheters that had been in place for only 1 day were colonized by bacterial biofilms. Biofilm scraped from catheter surfaces and dispersed by mixing and sonication yielded cultures (32% of catheters) of predominantly skin bacteria. In a few instances, the recovery of bacteria from biofilms on vascular catheters coincided with positive cultures from skin entry site swabs and/or from the blood of the patient, but there was no significant correlation between bacterial recovery from the catheter surface, entry site, and/or patients' blood. CONCLUSIONS: Direct microscopic examination of 68 vascular catheters that had been in place 1 to 14 days showed that most (81%) were colonized by bacteria growing in slime-enclosed biofilms. In many cases, this colonization of catheter surfaces could be confirmed by special biofilm culture recovery methods. Although the clinical importance of bacterial biofilms on catheter surfaces is speculative, their presence and potential to serve as a nidus for infection and bacteremia in critically ill immunocompromised hosts are cause for concern.

Effect of clofibrate treatment on chronic hyperlipidemia induced by an ACTH-producing tumor.

Plasma triglyceride and cholesterol levels were significantly increased in rats bearing an MtT-F4 tumor for 31-41 days. Addition of clofibrate at a dose of 30 mg kg-1 to the diet of rats bearing the tumor resulted in the complete elimination of the tumor effect on the plasma triglycerides, and to a great extent prevented the rise in the plasma cholesterol.