Electrospun polysuccinimide scaffolds containing different salts as potential wound dressing material.

In this research, we applied electrospinning to create a two-component biodegradable polymeric scaffold containing polysuccinimide (PSI) and antibacterial salts. Antibacterial agents for therapeutical purposes mostly contain silver ions which are associated with high environmental impact and, in some cases, may cause undesired immune reactions. In our work, we prepared nanofibrous systems containing antibacterial and tissue-regenerating salts of zinc acetate or strontium nitrate in different concentrations, whose structures may be suitable for developing biomedical wound dressing systems in the future. Several experiments have been conducted to optimize the physicochemical, mechanical, and biological properties of the scaffolds developed for application as wound dressings. The scaffold systems obtained by PSI synthesis, salt addition, and fiber formation were first investigated by scanning electron microscopy. In almost all cases, different salts caused a decrease in the fiber diameter of PSI polymer-based systems (<500 nm). Fourier-transform infrared spectroscopy was applied to verify the presence of salts in the scaffolds and to determine the interaction between the salt and the polymer. Another analysis, energy-dispersive X-ray spectroscopy, was carried out to determine strontium and zinc atoms in the scaffolds. Our result showed that the salts influence the mechanical properties of the polymer scaffold, both in terms of specific load capacity and relative elongation values. According to the dissolution experiments, the whole amount of strontium nitrate was dissolved from the scaffold in 8 h; however, only 50% of the zinc acetate was dissolved. In addition, antibacterial activity tests were performed with four different bacterial strains relevant to skin surface injuries, leading to the appearance of inhibition zones around the scaffold discs in most cases. We also investigated the potential cytotoxicity of the scaffolds on human tumorous and healthy cells. Except for the ones containing zinc acetate salt, the scaffolds are not cytotoxic to either tumor or healthy cells.

The Role of TRPM7 in Oncogenesis.

This review summarizes the current understanding of the role of transient receptor potential melastatin-subfamily member 7 (TRPM7) channels in the pathophysiology of neoplastic diseases. The TRPM family represents the largest and most diverse group in the TRP superfamily. Its subtypes are expressed in virtually all human organs playing a central role in (patho)physiological events. The TRPM7 protein (along with TRPM2 and TRPM6) is unique in that it has kinase activity in addition to the channel function. Numerous studies demonstrate the role of TRPM7 chanzyme in tumorigenesis and in other tumor hallmarks such as proliferation, migration, invasion and metastasis. Here we provide an up-to-date overview about the possible role of TRMP7 in a broad range of malignancies such as tumors of the nervous system, head and neck cancers, malignant neoplasms of the upper gastrointestinal tract, colorectal carcinoma, lung cancer, neoplasms of the urinary system, breast cancer, malignant tumors of the female reproductive organs, prostate cancer and other neoplastic pathologies. Experimental data show that the increased expression and/or function of TRPM7 are observed in most malignant tumor types. Thus, TRPM7 chanzyme may be a promising target in tumor therapy.

The Wisdom in Teeth: Neuronal Differentiation of Dental Pulp Cells.

Mesenchymal stem/stromal cells (MSCs) are found in almost all postnatal organs. Under appropriate environmental cues, multipotency enables MSCs to serve as progenitors for several lineage-specific, differentiated cell types. In vitro expansion and differentiation of MSCs give the opportunity to obtain hardly available somatic cells, such as neurons. The neurogenic potential of MSCs makes them a promising, autologous source to restore damaged tissue and as such, they have received much attention in the field of regenerative medicine. Several stem cell pool candidates have been studied thus far, but only a few of them showed neurogenic differentiation potential. Due to their embryonic ontology, stem cells residing in the stroma of the dental pulp chamber are an exciting source for in vitro neural cell differentiation. In this study, we review the key properties of dental pulp stem cells (DPSCs), with a particular focus on their neurogenic potential. Moreover, we summarize the various presently available methods used for neural differentiation of human DPSCs also emphasizing the difficulties in reproducibly high production of such cells. We postulate that because DPSCs are stem cells with very close ontology to neurogenic lineages, they may serve as excellent targets for neuronal differentiation in vitro and even for direct reprogramming.

Safety of chronic hypertonic bicarbonate inhalation in a cigarette smoke-induced airway irritation guinea pig model.

BACKGROUND: Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are often associated with airway fluid acidification. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to impaired bicarbonate secretion contributing to CF airway pathology. Chronic cigarette smoke (CS) -the major cause of COPD- is reported to induce acquired CFTR dysfunction underlying airway acidification and inflammation. We hypothesize that bicarbonate-containing aerosols could be beneficial for patients with CFTR dysfunctions. Thus, we investigated the safety of hypertonic sodium bicarbonate (NaHCO3) inhalation in CS-exposed guinea pigs. METHODS: Animals were divided into groups inhaling hypertonic NaCl (8.4%) or hypertonic NaHCO3 (8.4%) aerosol for 8 weeks. Subgroups from each treatment groups were further exposed to CS. Respiratory functions were measured at 0 and after 2, 4, 6 and 8 weeks. After 8 weeks blood tests and pulmonary histopathological assessment were performed. RESULTS: Neither smoking nor NaHCO3-inhalation affected body weight, arterial and urine pH, or histopathology significantly. NaHCO3-inhalation did not worsen respiratory parameters. Moreover, it normalized the CS-induced transient alterations in frequency, peak inspiratory flow, inspiratory and expiratory times. CONCLUSION: Long-term NaHCO3-inhalation is safe in chronic CS-exposed guinea pigs. Our data suggest that bicarbonate-containing aerosols might be carefully applied to CF patients.

User Experience and Sustainability of 3D Printing in Dentistry.

BACKGROUND: 3D printing is a rapidly developing technology in the healthcare industry and in dentistry. Its application clearly shows that this area of digital dentistry has potential for everyday usage across all fields, including prosthodontics, orthodontics, maxillofacial surgery, and oral implantology. However, despite gaining ground, there is a lack of information about how specialists (dentists and dental technicians) use additive technology. Our research group aimed to investigate the impact of social media on additive manufacturing technology among dental specialists and their everyday usage of 3D printing. METHODS: This paper investigated specialists' everyday usage of 3D printers via an online survey (Google Forms). The survey questions aimed to discover the number of 3D printers used, the accessibility of the devices, the annual cost, and the design programs. Since specialists tend to build online communities on social media, we circulated our study questionnaire using our profiles on LinkedIn, Facebook, and Instagram platforms during our research. RESULTS: A total of 120 responses were received from 20 countries, with the most significant numbers being from Hungary 23.7% (n = 27), the United States 18.4% (n = 21), and the United Kingdom 7.9% (n = 9). Most of the participants were dentists (n = 68) or dental technicians (n = 29), but some CAD/CAM specialists (n = 23) also completed our survey. The participants had an average of 3.8 years (±0.7) of experience in the 3D printing field, and owned a total of 405 printing devices (3.6 on average/person). CONCLUSIONS: The impact of social media on this research field is growing increasingly. Hence, we support specialists in joining virtual communities on professional platforms. This article intended to provide a practical overview, feedback, and direction for dentists interested in 3D printing technology. From our survey, we can conclude that additive technology is broadening dental applications and the services that we can provide for our patients.

Rheological effects of hypertonic saline and sodium bicarbonate solutions on cystic fibrosis sputum in vitro.

BACKGROUND: Cystic fibrosis (CF) is a life-threatening multiorgan genetic disease, particularly affecting the lungs, where recurrent infections are the main cause of reduced life expectancy. In CF, mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein impair transepithelial electrolyte and water transport, resulting in airway dehydration, and a thickening of the mucus associated with abnormal viscoelastic properties. Our aim was to develop a rheological method to assess the effects of hypertonic saline (NaCl) and NaHCO3 on CF sputum viscoelasticity in vitro, and to identify the critical steps in sample preparation and in the rheological measurements. METHODS: Sputum samples were mixed with hypertonic salt solutions in vitro in a ratio of either 10:4 or 10:1. Distilled water was applied as a reference treatment. The rheological properties of sputum from CF patients, and the effects of these in vitro treatments, were studied with a rheometer at constant frequency and strain, followed by frequency sweep tests, where storage modulus (G'), loss modulus (G″) and loss factor were determined. RESULTS: We identified three distinct categories of sputum: (i) highly elastic (G' > 100,000 Pa), (ii) elastic (100,000 Pa > G' > 1000 Pa), and (iii) viscoelastic (G' < 1000). At the higher additive ratio (10:4), all of the added solutions were found to significantly reduce the gel strength of the sputum, but the most pronounced changes were observed with NaHCO3 (p < 0.001). Samples with high elasticity exhibited the greatest changes while, for less elastic samples, a weakening of the gel structure was observed when they were treated with water or NaHCO3, but not with NaCl. For the viscoelastic samples, the additives did not cause significant changes in the parameters. When the lower additive ratio (10:1) was used, the mean values of the rheological parameters usually decreased, but the changes were not statistically significant. CONCLUSION: Based on the rheological properties of the initial sputum samples, we can predict with some confidence the treatment efficacy of each of the alternative additives. The marked differences between the three categories suggest that it is advisable to evaluate each sample individually using a rheological approach such as that described here.

Bicarbonate Evokes Reciprocal Changes in Intracellular Cyclic di-GMP and Cyclic AMP Levels in Pseudomonas aeruginosa.

The formation of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) is one of the most common causes of morbidity and mortality in CF patients. Cyclic di-GMP and cyclic AMP are second messengers regulating the bacterial lifestyle transition in response to environmental signals. We aimed to investigate the effects of extracellular pH and bicarbonate on intracellular c-di-GMP and cAMP levels, and on biofilm formation. P. aeruginosa was inoculated in a brain−heart infusion medium supplemented with 25 and 50 mM NaCl in ambient air (pH adjusted to 7.4 and 7.7 respectively), or with 25 and 50 mM NaHCO3 in 5% CO2 (pH 7.4 and 7.7). After 16 h incubation, c-di-GMP and cAMP were extracted and their concentrations determined. Biofilm formation was investigated using an xCelligence real-time cell analyzer and by crystal violet assay. Our results show that HCO3− exposure decreased c-di-GMP and increased cAMP levels in a dose-dependent manner. Biofilm formation was also reduced after 48 h exposure to HCO3−. The reciprocal changes in second messenger concentrations were not influenced by changes in medium pH or osmolality. These findings indicate that HCO3− per se modulates the levels of c-di-GMP and cAMP, thereby inhibiting biofilm formation and promoting the planktonic lifestyle of the bacteria.

Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation.

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization. Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation. Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF. Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis. Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts.

TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.

Antibacterial Effects of Bicarbonate in Media Modified to Mimic Cystic Fibrosis Sputum.

Cystic fibrosis (CF) is a hereditary disease caused by mutations in the gene encoding an epithelial anion channel. In CF, Cl- and HCO3- hyposecretion, together with mucin hypersecretion, leads to airway dehydration and production of viscous mucus. This habitat is ideal for colonization by pathogenic bacteria. We have recently demonstrated that HCO3- inhibits the growth and biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus when tested in laboratory culture media. Using the same bacteria our aim was to investigate the effects of HCO3- in artificial sputum medium (ASM), whose composition resembles CF mucus. Control ASM containing no NaHCO3 was incubated in ambient air (pH 7.4 or 8.0). ASM containing NaHCO3 (25 and 100 mM) was incubated in 5% CO2 (pH 7.4 and 8.0, respectively). Viable P. aeruginosa and S. aureus cells were counted by colony-forming unit assay and flow cytometry after 6 h and 17 h of incubation. Biofilm formation was assessed after 48 h. The data show that HCO3- significantly decreased viable cell counts and biofilm formation in a concentration-dependent manner. These effects were due neither to extracellular alkalinization nor to altered osmolarity. These results show that HCO3- exerts direct antibacterial and antibiofilm effects on prevalent CF bacteria.

The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel.

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.

Bicarbonate Inhibits Bacterial Growth and Biofilm Formation of Prevalent Cystic Fibrosis Pathogens.

We investigated the effects of bicarbonate on the growth of several different bacteria as well as its effects on biofilm formation and intracellular cAMP concentration in Pseudomonas aeruginosa. Biofilm formation was examined in 96-well plates, with or without bicarbonate. The cAMP production of bacteria was measured by a commercial assay kit. We found that NaHCO3 (100 mmol l-1) significantly inhibited, whereas NaCl (100 mmol l-1) did not influence the growth of planktonic bacteria. MIC and MBC measurements indicated that the effect of HCO 3 - is bacteriostatic rather than bactericidal. Moreover, NaHCO3 prevented biofilm formation as a function of concentration. Bicarbonate and alkalinization of external pH induced a significant increase in intracellular cAMP levels. In conclusion, HCO 3 - impedes the planktonic growth of different bacteria and impedes biofilm formation by P. aeruginosa that is associated with increased intracellular cAMP production. These findings suggest that aerosol inhalation therapy with HCO 3 - solutions may help improve respiratory hygiene in patients with cystic fibrosis and possibly other chronically infected lung diseases.

No Change in Bicarbonate Transport but Tight-Junction Formation Is Delayed by Fluoride in a Novel Ameloblast Model.

We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.

Mesenchymal Stem Cells of Dental Origin-Their Potential for Antiinflammatory and Regenerative Actions in Brain and Gut Damage.

Alzheimer's disease, Parkinson's disease, traumatic brain and spinal cord injury and neuroinflammatory multiple sclerosis are diverse disorders of the central nervous system. However, they are all characterized by various levels of inappropriate inflammatory/immune response along with tissue destruction. In the gastrointestinal system, inflammatory bowel disease (IBD) is also a consequence of tissue destruction resulting from an uncontrolled inflammation. Interestingly, there are many similarities in the immunopathomechanisms of these CNS disorders and the various forms of IBD. Since it is very hard or impossible to cure them by conventional manner, novel therapeutic approaches such as the use of mesenchymal stem cells, are needed. Mesenchymal stem cells have already been isolated from various tissues including the dental pulp and periodontal ligament. Such cells possess transdifferentiating capabilities for different tissue specific cells to serve as new building blocks for regeneration. But more importantly, they are also potent immunomodulators inhibiting proinflammatory processes and stimulating anti-inflammatory mechanisms. The present review was prepared to compare the immunopathomechanisms of the above mentioned neurodegenerative, neurotraumatic and neuroinflammatory diseases with IBD. Additionally, we considered the potential use of mesenchymal stem cells, especially those from dental origin to treat such disorders. We conceive that such efforts will yield considerable advance in treatment options for central and peripheral disorders related to inflammatory degeneration.

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods.

BACKGROUND/AIMS: ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS: We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS: Our data show that ATP (< 1 µM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS: Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.

Inhibition of the human epithelial calcium channel TRPV6 by 2-aminoethoxydiphenyl borate (2-APB).

TRPV6, a highly calcium-selective member of the transient receptor potential (TRP) channel superfamily, is a major pathway for calcium absorption in the fetal and adult body. It is expressed abundantly in the duodenum, the placenta and exocrine tissues. TRVP6 was postulated to contribute to store-operated calcium channel (SOC) activity in certain cell types such as exocrine cells. In this study, we tested 2-APB, a widely used SOC inhibitor on human TRPV6 (hTRPV6) activity using fluorescence imaging, patch clamp and radioactive tracer techniques in transiently and stably transfected HEK293 cells. We found that the basal calcium and cadmium influx was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells. 2-APB inhibited hTRPV6 activity in both transient and stably transfected cells. This effect was slightly sensitive toward extracellular calcium. The extracellular sodium concentration did not affect the inhibition of hTRPV6 by 2-APB. However, N-methyl-d-glucamine significantly diminished the inhibitory effect of 2-APB presumably through direct interaction with this compound. Furthermore, 2-APB inhibited the activity of TRPV6 orthologs but not human TRPV5. 2-APB may serve as a parental compound for the development of therapeutic strategies specifically targeting the hTRPV6 calcium channel.

The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa.

BACKGROUND: Rescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT) CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer. RESULTS: Surprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ΔF508-CFTR (ΔF-CFTR), inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ΔF-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ΔF-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ΔF-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ΔF-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/ΔF heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD) recordings and in Ussing chamber recordings of short-circuit current (ISC) in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/- heterozygotes had no difference in their responses versus +/+ wild-type mice. CONCLUSIONS: Taken altogether, these data suggest that ΔF-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER). As a consequence, ΔF-CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia. Implications of these data for the relative health of CF heterozygous carriers, for CFTR protein processing in native airway epithelia, and for the relative efficacy of different CF therapeutic approaches is significant and is discussed.

Human Th1 and Th2 lymphocytes are distinguished by calcium flux regulation during the first 10 min of lymphocyte activation.

Preliminary data suggest different intracellular calcium handling of Th1 and Th2 lymphocytes that may contribute to distinct cytokine production patterns. In this study we explored the contribution of the main mechanisms in charge of the elevation and decrease of cytoplasmic free calcium levels, i.e., the endoplasmic calcium release, the calcium release activated calcium (CRAC) channel, the mitochondrial calcium uniporter (MCU), the sarco/endoplasmic reticulum calcium ATPase (SERCA), and the plasma membrane calcium ATPase (PMCA) during the first 10 min of activation in human Th1 and Th2 lymphocytes applying a kinetic flow cytometry approach. We isolated peripheral blood mononuclear cells from 10 healthy individuals. Cells were stained with CD4, CXCR3 and CCR4 cell surface markers to identify Th1 and Th2 cells, respectively and loaded with Fluo-3/AM calcium sensitive dye. Cells were activated with phytohemagglutinine and alterations of cytoplasmic free calcium levels were monitored for 10 min after specific inhibition of the above mechanisms. Our results revealed delicate differences in calcium flux kinetics of Th1 and Th2 lymphocytes. The lower activity of MCU, and therefore of CRAC channels, along with the higher activity of the SERCA pump account for the notion that Th2 cells go through a lower level of lymphocyte activation compared with Th1 cells upon identical activating stimuli. The observed differences in calcium flux of Th1 and Th2 cells may contribute to different calcium handling kinetics and, hence, to distinct cytokine production patterns by these subsets.

Extracellular alkalinization stimulates calcium-activated chloride conductance in cystic fibrosis human airway epithelial cells.

BACKGROUND: The pH of the airway surface liquid (ASL) plays a pivotal role in maintaining the proper function of the respiratory epithelium. In patients with cystic fibrosis (CF) acidic ASL has been observed. Thus, alkalinization of ASL itself might be beneficial in CF. The aim of this study was to investigate the role of extracellular pH (pH(o)) on the alternative Ca(2+)-activated Cl(-) channels (CaCCs) in CF airway epithelial cells. METHODS: The [Ca(2+)](i) and viability of CF airway epithelial cells (IB3-1) were assessed using Fluo-3/AM and YO-PRO-1 fluorescent dyes, respectively. Ion currents were detected in whole-cell configuration using the patch clamp technique. RESULTS: Extracellular alkalinization (pH(o) 8.2) stimulated Ca(2+) entry and inward currents in low Na(+) containing medium. The inward currents were blocked by the removal of extracellular Ca(2+), chelating cytosolic Ca(2+), as well as by the application of niflumic acid and DIDS. While Zn(2+) promoted sustained Ca(2+) entry in pH(o)-dependent manner, it inhibited the anion conductance. The low external Na(+) concentrations and alkaline pH(o) were well tolerated by the cells. CONCLUSIONS: Stimulation of CaCCs could be achieved by alkalinization of the extracellular environment in CF airway epithelial cells. Zn(2+) directly blocked, however indirectly enhanced the activity of Cl(-) conductance.

New therapeutic targets in ulcerative colitis: the importance of ion transporters in the human colon.

BACKGROUND: The absorption of water and ions (especially Na(+) and Cl(-)) is an important function of colonic epithelial cells in both physiological and pathophysiological conditions. Despite the comprehensive animal studies, there are only scarce available data on the ion transporter activities of the normal and inflamed human colon. METHODS: In this study, 128 healthy controls and 69 patients suffering from ulcerative colitis (UC) were involved. We investigated the expressional and functional characteristics of the Na(+)/H(+) exchangers (NHE) 1-3, the epithelial sodium channel (ENaC), and the SLC26A3 Cl(-)/HCO 3- exchanger downregulated in adenoma (DRA) in primary colonic crypts isolated from human biopsy and surgical samples using microfluorometry, patch clamp, and real-time reverse-transcription polymerase chain reaction (RT-PCR) techniques. RESULTS: Data collected from colonic crypts showed that the activities of electroneutral (via NHE3) and the electrogenic Na(+) absorption (via ENaC) are in inverse ratio to each other in the proximal and distal colon. We found no significant differences in the activity of NHE2 in different segments of the colon. Surface cell Cl(-)/HCO 3- exchange is more active in the distal part of the colon. Importantly, both sodium and chloride absorptions are damaged in UC, whereas NHE1, which has been shown to promote immune response, is upregulated by 6-fold. CONCLUSIONS: These results open up new therapeutic targets in UC.