Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

New insights in the pathogenesis of high-altitude pulmonary edema.

High-altitude pulmonary edema is a life-threatening condition occurring in predisposed but otherwise healthy individuals. It therefore permits the study of underlying mechanisms of pulmonary edema in the absence of confounding factors such as coexisting cardiovascular or pulmonary disease, and/or drug therapy. There is evidence that some degree of asymptomatic alveolar fluid accumulation may represent a normal phenomenon in healthy humans shortly after arrival at high altitude. Two fundamental mechanisms then determine whether this fluid accumulation is cleared or whether it progresses to HAPE: the quantity of liquid escaping from the pulmonary vasculature and the rate of its clearance by the alveolar respiratory epithelium. The former is directly related to the degree of hypoxia-induced pulmonary hypertension, whereas the latter is determined by the alveolar epithelial sodium transport. Here, we will review evidence that, in HAPE-prone subjects, impaired pulmonary endothelial and epithelial NO synthesis and/or bioavailability may represent a central underlying defect predisposing to exaggerated hypoxic pulmonary vasoconstriction and, in turn, capillary stress failure and alveolar fluid flooding. We will then demonstrate that exaggerated pulmonary hypertension, although possibly a conditio sine qua non, may not always be sufficient to induce HAPE and how defective alveolar fluid clearance may represent a second important pathogenic mechanism.

The adjustment of energy expenditure and oxidation to energy intake: the role of carbohydrate and fat balance.

Evidence is accumulating that total body mass and its relative composition influence the rate of fat utilization in man. This effect can be explained by two factors operating in concert: (i) the effect of the size of the tissue mass and (ii) the nature of the fuel mix oxidized, i.e. the proportion of energy derived from fat vs. carbohydrate. In a cross-sectional study of 307 women with increasing degrees of obesity, we observed that the respiratory quotient (RQ) in post-absorptive conditions became progressively lower with increased body fatness, indicating a shift in substrate utilization. However, the RQ is known to be also influenced by the diet commonly ingested by the subjects. A short-term mixed diet overfeeding in lean and obese women has also demonstrated the high sensitivity of RQ to changes in energy balance. Following a one-day overfeeding (2500 kcal/day in excess of the previous 24 h energy expenditure), the magnitude of increase in RQ was identical in lean and obese subjects and the net efficiency of substrate utilization and storage was not influenced by the state of obesity.

Respiratory nitric oxide and pulmonary artery pressure in children of aymara and European ancestry at high altitude.

Invasive studies suggest that healthy children living at high altitude display pulmonary hypertension, but the data to support this assumption are sparse. Nitric oxide (NO) synthesized by the respiratory epithelium regulates pulmonary artery pressure, and its synthesis was reported to be increased in Aymara high-altitude dwellers. We hypothesized that pulmonary artery pressure will be lower in Aymara children than in children of European ancestry at high altitude, and that this will be related to increased respiratory NO. We therefore compared pulmonary artery pressure and exhaled NO (a marker of respiratory epithelial NO synthesis) between large groups of healthy children of Aymara (n = 200; mean +/- SD age, 9.5 +/- 3.6 years) and European ancestry (n = 77) living at high altitude (3,600 to 4,000 m). We also studied a group of European children (n = 29) living at low altitude. The systolic right ventricular to right atrial pressure gradient in the Aymara children was normal, even though significantly higher than the gradient measured in European children at low altitude (22.5 +/- 6.1 mm Hg vs 17.7 +/- 3.1 mm Hg, p < 0.001). In children of European ancestry studied at high altitude, the pressure gradient was 33% higher than in the Aymara children (30.0 +/- 5.3 mm Hg vs 22.5 +/- 6.1 mm Hg, p < 0.0001). In contrast to what was expected, exhaled NO tended to be lower in Aymara children than in European children living at the same altitude (12.4 +/- 8.8 parts per billion [ppb] vs 16.1 +/- 11.1 ppb, p = 0.06) and was not related to pulmonary artery pressure in either group. Aymara children are protected from hypoxic pulmonary hypertension at high altitude. This protection does not appear to be related to increased respiratory NO synthesis.

Safety of combined heat and moisture exchanger filters in long-term mechanical ventilation.

STUDY OBJECTIVE: To evaluate the safety of a combined heat and moisture exchanger filter (HMEF) for the conditioning of inspired gas in long-term mechanical ventilation (MV). DESIGN: Randomized controlled trial. SETTING: Medical ICU in a large teaching hospital. PATIENTS: One hundred fifteen consecutive patients who required > or = 48 h of MV. INTERVENTIONS: Patients were randomized at intubation time (day 1) to receive inspired gas conditioned either by a water-bath humidifier heated at 32 degrees C (HWBH) or by an HMEF (Hygroster; DAR; Mirandola, Italy). MEASUREMENTS AND MAIN RESULTS: The two study groups were comparable in terms of primary pathologic condition at the time of hospital admission, disease severity as measured by the Simplified Acute Physiology Score, and ICU mortality. They did not differ with respect to ventilator days per patient (mean +/- SD: HMEF, 7.6 +/- 6.5; HWBH, 7.8 +/- 5.8), incidence of endotracheal tube obstruction (HMEF, 0/59; HWBH, 1/56), and incidence of hypothermic episodes (HMEF, five; HWBH, two). In 41 patients receiving MV for > or = 5 days, the morphologic integrity of respiratory epithelium was evaluated on day 1 and day 5, using a cytologic examination of tracheal aspirate smears. The state of ciliated epithelium was scored on a scale from 0 (poorest integrity) to 1,200 (maximum integrity), according to a well-described method. In both patient groups, the scores slightly but significantly decreased from day 1 to day 5 (mean +/- SD: HWBH, from 787 +/- 104 to 745 +/- 88; HMEF, from 813 +/- 79 to 739 +/- 62; p < 0.01 for both groups); there were no statistically significant differences between groups. CONCLUSIONS: These data indicate acceptable safety of HMEFs of the type used in the present study for long-term mechanical ventilation.

Allergies alimentaires rares [Rare food allergies]

A limited number of foods explain the majority of food allergies. These allergies can be due to a weak allergenicity (garlic, onion, potato), or a weak (or increasing) exposure to emergent food allergens which can be imported (exotic fruits), or recently introduced (lupin, buckwheat, sesame, inulin) or modified by the industry (lysats, lecithins, traces of antibiotics, caseinates, molds, dust mite). Others are in relation with rarer cross-reactivity food allergy syndrome (Apiaceae-Compositae-mugwort syndrome, egg-bird syndrome, cat epithelium-pork meat syndrome). Others are rarely identified, because the food is masked (pepper, basilic). We illustrate rare cases of food allergy and discuss the diagnostic management which is based on a meticulous patient history. Un nombre restreint d'aliments explique la majorité des allergies alimentaires. Les allergies alimentaires rares sont dues à une faible allergénicité (ail, oignon, pomme de terre) ou à une exposition faible ou croissante à des aliments émergents, importés (fruits exotiques), introduits (lupin, sarrasin, sésame, inuline), ou modifiés par l'industrie (lysats, lécithines, traces d'antibiotiques, caséinates, moisissures, acariens). D'autres sont en relation avec des croisements d'allergènes rares (syndrome croisé ombellifères-composées-armoise, syndrome oeuf-oiseau, syndrome épithélium de chat-viande de porc). D'autres enfin sont rarement identifiées, car l'allergène est masqué (poivre, basilic). Nous décrivons des cas rares illustratifs et rappelons la démarche diagnostique qui s'appuie sur une anamnèse minutieuse.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

PPARbeta/delta regulates paneth cell differentiation via controlling the hedgehog signaling pathway.

BACKGROUND & AIMS: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. METHODS: To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. RESULTS: We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. CONCLUSIONS: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

PURPOSE: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation. METHODS: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls. RESULTS: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively. CONCLUSIONS: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

Cycle length of spermatogenesis in shrews (mammalia: soricidae) with high and low metabolic rates and different mating systems.

The aim of the present study was to establish and compare the durations of the seminiferous epithelium cycles of the common shrew Sorex araneus, which is characterized by a high metabolic rate and multiple paternity, and the greater white-toothed shrew Crocidura russula, which is characterized by a low metabolic rate and a monogamous mating system. Twelve S. araneus males and fifteen C. russula males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determinations, we applied the classical method of estimation and linear regression as a new method. With regard to variance, and even with a relatively small sample size, the new method seems to be more precise. In addition, the regression method allows the inference of information for every animal tested, enabling comparisons of different factors with cycle lengths. Our results show that not only increased testis size leads to increased sperm production, but it also reduces the duration of spermatogenesis. The calculated cycle lengths were 8.35 days for S. araneus and 12.12 days for C. russula. The data obtained in the present study provide the basis for future investigations into the effects of metabolic rate and mating systems on the speed of spermatogenesis.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice.

Odorant receptor (OR) genes constitute with 1200 members the largest gene family in the mouse genome. A mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and from one allele. The cell bodies of OSNs that express a given OR gene display a mosaic pattern within a particular region of the main olfactory epithelium. The mechanisms and cis-acting DNA elements that regulate the expression of one OR gene per OSN - OR gene choice - remain poorly understood. Here, we describe a reporter assay to identify minimal promoters for OR genes in transgenic mice, which are produced by the conventional method of pronuclear injection of DNA. The promoter transgenes are devoid of an OR coding sequence, and instead drive expression of the axonal marker tau-β-galactosidase. For four mouse OR genes (M71, M72, MOR23, and P3) and one human OR gene (hM72), a mosaic, OSN-specific pattern of reporter expression can be obtained in transgenic mice with contiguous DNA segments of only ~300 bp that are centered around the transcription start site (TSS). The ~150bp region upstream of the TSS contains three conserved sequence motifs, including homeodomain (HD) binding sites. Such HD binding sites are also present in the H and P elements, DNA sequences that are known to strongly influence OR gene expression. When a 19mer encompassing a HD binding site from the P element is multimerized nine times and added upstream of a MOR23 minigene that contains the MOR23 coding region, we observe a dramatic increase in the number of transgene-expressing founders and lines and in the number of labeled OSNs. By contrast, a nine times multimerized 19mer with a mutant HD binding site does not have these effects. We hypothesize that HD binding sites in the H and P elements and in OR promoters modulate the probability of OR gene choice.

In vivo inhibition of lens regrowth by fibroblast growth factor 2-saporin.

PURPOSE: To investigate the ability of fibroblast growth factor (FGF) 2-saporin to prevent lens regrowth in the rabbit. METHODS: Chemically conjugated and genetically fused FGF2-saporin (made in Escherichia coli) were used. Extracapsular extraction of the lens was performed on the rabbit, and the cytotoxin either was injected directly into the capsule bag or was administered by FGF2-saporin-coated, heparin surface-modified (HSM) polymethylmethacrylate intraocular lenses. The potential of the conjugate was checked by slit lamp evaluation of capsular opacification and by measuring crystallin synthesis. Toxin diffusion and sites of toxin binding were assessed by immunohistochemistry. Possible toxicity was determined by histologic analysis of ocular tissues. RESULTS: FGF2-saporin effectively inhibited lens regrowth when it was injected directly into the capsular bag. However, high concentration of the toxin induced transient corneal edema and loss of pigment in the iris. Intraocular lenses coated with FGF2-saporin reduced lens regrowth and crystallin synthesis without any detectable clinical side effect. After implantation, FGF2-saporin was shown to have bound to the capsules and, to a lesser extent, to the iris; no histologic damage was found on ocular tissues as a result of implantation of drug-loaded HSM intraocular lenses. CONCLUSIONS: Chemically conjugated (FGF2-SAP) and genetically fused FGF2-saporin (rFGF2-SAP) bound to HSM intraocular lenses can prevent lens regrowth in the rabbit.

Radiochemotherapy in locally advanced squamous cell carcinomas of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is a common disease that develops in the upper aerodigestive epithelium. The most important risk factors are tobacco and alcohol consumption. There is also increasing evidence that human papillomavirus plays an important role in the cause of SCCHN. The complex anatomy, the vital functions of the upper aerodigestive tract and the close proximity to vital structures, explain that the goal of treatment is not only to improve survival outcomes, but also to preserve organ function. Radiotherapy and surgery are the standard modalities of treatment, reflecting the locoregional predominance of SCCHN. Chemotherapy plays an important role in the treatment of patients with locoregionally advanced disease, in conjunction with radiotherapy and surgery. Indeed, standard therapy for resectable locoregionally advanced (stage III or IV) SCCHN cancers consists either of surgery and adjuvant chemoradiotherapy or definitive concomitant chemoradiotherapy, depending upon disease site, stage and resectability of the tumour, or institutional experience. Concomitant chemoradiotherapy has been shown in several randomised trials to improve disease-free and overall survival in the postoperative setting for resected disease with poor prognostic factors. Furthermore, multiple randomised studies and meta-analyses have shown that definitive chemoradiotherapy, as well anti-epidermal growth factor receptor treatment in one randomised study, improved disease-free and overall survival when compared with radiotherapy alone. This overview reviews the most relevant published studies on the multidisciplinary management of SCCHN and discusses future strategies to reduce locoregional failures.

Peroxynitrite cytotoxicity on bovine retinal pigmented epithelial cells in culture.

Peroxynitrite induced in vitro a dose dependent toxicity on retinal pigmented epithelial (RPE) cells. Cell death was partially mediated by apoptosis as demonstrated by nuclear fragmentation and TdT-mediated dUTP nick-end labeling assay. Peroxynitrite-induced tyrosine nitration was revealed by immunocytochemistry, both in the cytoplasm and in the nucleus of the cells. Nitration was not observed in RPE cells, producing nitric oxide (NO) after stimulation by lipopolysacharide and interferon-g (IFN-gamma), suggesting that peroxynitrite was not formed in vitro in such conditions. Peroxynitrite could be responsible for the retinal damages observed in pathological conditions in which NO has been demonstrated to be involved. In this context, EGb761, identified as a free radical scavenger, was showed herein to protect RPE cells against peroxynitrite injury.