Unnexin is a protein subunit of a large-pore channel expressed by unicellular organisms.

Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca2+/Mg2+ levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca2+ and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in Xenopus oocytes. Downregulation of Unx1 mRNA with morpholine contains Trypanosoma cruzi invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.

Hiding in plain sight? A review of post-convulsive leukocyte elevations.

During physiological stress responses such as vigorous exercise, emotional states of fear and rage, and asphyxia, the nervous system induces a massive release of systemic catecholamines that prepares the body for survival by increasing cardiac output and redirecting blood flow from non-essential organs into the cardiopulmonary circulation. A curious byproduct of this vital response is a sudden, transient, and redistributive leukocytosis provoked mostly by the resultant shear forces exerted by rapid blood flow on marginated leukocytes. Generalized convulsive seizures, too, result in catecholamine surges accompanied by similar leukocytoses, the magnitude of which appears to be rooted in semiological factors such as convulsive duration and intensity. This manuscript reviews the history, kinetics, physiology, and clinical significance of post-convulsive leukocyte elevations and discusses their clinical utility, including a proposed role in the scientific investigation of sudden unexpected death in epilepsy (SUDEP).

Unnexins: Homologs of innexin proteins in Trypanosomatidae parasites.

Large-pore channels, including those formed by connexin, pannexin, innexin proteins, are part of a broad family of plasma membrane channels found in vertebrates and invertebrates, which share topology features. Despite their relevance in parasitic diseases such as Chagas and malaria, it was unknown whether these large-pore channels are present in unicellular organisms. We identified 14 putative proteins in Trypanosomatidae parasites as presumptive homologs of innexin proteins. All proteins possess the canonical motif of the innexin family, a pentapeptide YYQWV, and 10 of them share a classical membrane topology of large-pore channels. A sequence similarity network analysis confirmed their closeness to innexin proteins. A bioinformatic model showed that a homolog of Trypanosoma cruzi (T. cruzi) could presumptively form a stable octamer channel with a highly positive electrostatic potential in the internal cavities and extracellular entrance due to the notable predominance of residues such as Arg or Lys. In vitro dye uptake assays showed that divalent cations-free solution increases YO-PRO-1 uptake and hyperosmotic stress increases DAPI uptake in epimastigotes of T. cruzi. Those effects were sensitive to probenecid. Furthermore, probenecid reduced the proliferation and transformation of T. cruzi. Moreover, probenecid or carbenoxolone increased the parasite sensitivity to antiparasitic drugs commonly used in therapy against Chagas. Our study suggests the existence of innexin homologs in unicellular organisms, which could be protein subunits of new large-pore channels in unicellular organisms.

Contribution of large-pore channels to inflammation induced by microorganisms.

Plasma membrane ionic channels selectively permeate potassium, sodium, calcium, and chloride ions. However, large-pore channels are permeable to ions and small molecules such as ATP and glutamate, among others. Large-pore channels are structures formed by several protein families with little or no evolutionary linkages including connexins (Cxs), pannexins (Panxs), innexin (Inxs), unnexins (Unxs), calcium homeostasis modulator (CALHMs), and Leucine-rich repeat-containing 8 (LRRC8) proteins. Large-pore channels are key players in inflammatory cell response, guiding the activation of inflammasomes, the release of pro-inflammatory cytokines such as interleukin-1 beta (IL-1ß), and the release of adenosine-5'-triphosphate (ATP), which is considered a danger signal. This review summarizes our current understanding of large-pore channels and their contribution to inflammation induced by microorganisms, virulence factors or their toxins.

A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII.

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

Anti-parasitic drugs modulate the non-selective channels formed by connexins or pannexins.

The proteins connexins, innexins, and pannexins are the subunits of non-selective channels present in the cell membrane in vertebrates (connexins and pannexins) and invertebrates (innexins). These channels allow the transfer of ions and molecules across the cell membrane or, and in many cases, between the cytoplasm of neighboring cells. These channels participate in various physiological processes, particularly under pathophysiological conditions, such as bacterial, viral, and parasitic infections. Interestingly, some anti-parasitic drugs also block connexin- or pannexin-formed channels. Their effects on host channels permeable to molecules that favor parasitic infection can further explain the anti-parasitic effects of some of these compounds. In this review, the effects of drugs with known anti-parasitic activity that modulate non-selective channels formed by connexins or pannexins are discussed. Previous studies that have reported the presence of these proteins in worms, ectoparasites, and protozoa that cause parasitic infections have also been reviewed.

The Magnitude of Postconvulsive Leukocytosis Mirrors the Severity of Periconvulsive Respiratory Compromise: A Single Center Retrospective Study.

Background: Generalized epileptic convulsions frequently exhibit transient respiratory symptoms and non-infectious leukocytosis. While these peri-ictal effects appear to arise independently from one another, the possibility that they stem from a common ictal pathophysiological response has yet to be explored. We aimed to investigate whether peri-ictal respiratory symptoms and postictal leukocytosis coexist. Methods: We performed a single center retrospective chart review of 446 patients brought to our emergency department between January 1, 2017 and August 23, 2018 for the care of generalized epileptic convulsions with or without status epilepticus. We included 152 patients who were stratified based on the presence (PeCRC+) or absence (PeCRC-) of overt periconvulsive respiratory compromise (PeCRC). In addition, patients were stratified based on the presence or absence of postconvulsive leukocytosis (PoCL), defined as an initial postconvulsive white blood cell (WBC) count ≥ 11,000 cells/mm3. Triage vital signs, and chest x ray (CXR) abnormalities were also examined. Results: Overt PeCRC was observed in 31.6% of patients, 43% of whom required emergent endotracheal intubations. PoCL was observed in 37.5% of patients, and was more likely to occur in PeCRC+ than in PeCRC- patients (79.2 vs. 18.2%; OR = 17.0; 95% CI = 7.2-40.9; p < 0.001). Notably, the magnitude of PoCL was proportional to the severity of PeCRC, as the postconvulsive WBC count demonstrated a negative correlation with triage hemoglobin oxygen saturation (R = -0.22; p < 0.01; CI = -0.48 to -0.07). Moreover, a receiver operating characteristic analysis of the WBC count's performance as predictor of endotracheal intubation reached a significant area under the curve value of 0.81 (95% CI = 0.71-0.90; p < 0.001). Finally, PeCRC+ patients demonstrated frequent CXR abnormalities, and their postconvulsive WBC counts correlated directly with triage heart rate (R = 0.53; p < 0.001). Conclusion: Our data support the existence of an ictal pathophysiological response, which induces proportional degrees of PoCL and PeCRC. We suggest this response is at least partially propelled by systemic catecholamines.

Assessing the Committed Effective Dose From 226Ra in Thermal Spring Water From San Diego De Alcala, Chihuahua, Mexico.

The oral administration of mineral-rich spring water is known as hydropinic treatment and is used to treat certain ailments. Health benefits are attributed to thermal spring water containing radioactive elements such as radium; this has popularized use of such radioactive water in various parts of the world, causing those who ingest it to increase their internal radiation dose. The goal of this study was to assess the activity concentrations of Ra present in the thermal spring waters of San Diego de Alcala, in the state of Chihuahua, Mexico, and to estimate the health risk posed to patients by the effective dose received from ingesting this water during hydropinic treatments. Water samples were taken from different areas of the San Diego de Alcala thermal springs, and pH, temperature, electrical conductivity, and total dissolved solids were measured. The Ra activity concentrations were measured with a liquid scintillation counter. The activity concentrations of Ra in sampled water varied from 125 to 452 mBq L with an average of 276 ± 40 mBq L. The committed effective dose from each of the Ra activity concentrations found in samples ranged from 9.80 × 10 to 4.0 × 10 mSv for hydropinic treatments being carried out in San Diego de Alcala thermal spring spas. Different treatments had different intake rates (200, 600, 1,000, and 1,500 mL d) and occurred over periods of 2 or 3 wk. According to the guidelines of the US Environmental Protection Agency, the maximum permissible amount of radium in drinking water is 185 mBq L; the Ra content in most of the collected samples exceeded this limit. The committed effective doses varied with Ra concentration and intake rate; none exceeded the World Health Organization's reference dose for drinking water of 0.1 mSv y, which is the maximum amount to which the population should be exposed.

Innexins: Expression, Regulation, and Functions.

The innexin (Inx) proteins form gap junction channels and non-junctional channels (named hemichannels) in invertebrates. These channels participate in cellular communication playing a relevant role in several physiological processes. Pioneer studies conducted mainly in worms and flies have shown that innexins participate in embryo development and behavior. However, recent studies have elucidated new functions of innexins in Arthropoda, Nematoda, Annelida, and Cnidaria, such as immune response, and apoptosis. This review describes emerging data of possible new roles of innexins and summarizes the data available to date.

Ictal Mammalian Dive Response: A Likely Cause of Sudden Unexpected Death in Epilepsy.

Even though sudden unexpected death in epilepsy (SUDEP) takes the lives of thousands of otherwise healthy epilepsy patients every year, the physiopathology associated with this condition remains unexplained. This article explores important parallels, which exist between the clinical observations and pathological responses associated with SUDEP, and the pathological responses that can develop when a set of autonomic reflexes known as the mammalian dive response (MDR) is deployed. Mostly unknown to physicians, this evolutionarily conserved physiological response to prolonged apnea economizes oxygen for preferential use by the brain. However, the drastic cardiovascular adjustments required for its execution, which include severe bradycardia and the sequestration of a significant portion of the total blood volume inside the cardiopulmonary vasculature, can result in many of the same pathological responses associated with SUDEP. Thus, this article advances the hypothesis that prolonged apneic generalized tonic clonic seizures induce augmented forms of the MDR, which, in the most severe cases, cause SUDEP.

The Connection of Life with Respiration: Edmund Goodwyn's unexplored treasure of cardiopulmonary physiology.

The advent of artificial ventilation was largely sparked by the popular belief that drowning and other causes of asphyxia could induce death-like states known as suspended animation. While the mystical nature of such states befuddled some physicians into the early 1900s, an English medical student by the name of Edmund Goodwyn (1756-1829) published a thesis in 1786, which demonstrated that suspended animation was simply the physical manifestation of extreme hypoxia. Goodwyn's work advanced one of the earliest arguments in favor of artificial ventilation for the treatment of asphyxia over alternative resuscitation measures like heat and exsanguination. In addition, Goodwyn's remarkable dissertation contains the first account of a reflex known as diving bradycardia, and possibly the first vehement refutation of claims by his contemporaries that pulmonary circulation stopped during exhalation. While miscellaneous aspects of his thesis have occasionally been mentioned by a few medical historians, the overall visionary nature of his work has yet to be recognized. This article attempts to accomplish this goal and to provide a first biographical glimpse of a man whose scientific career appears to have ended prematurely, perhaps because of his profound aversion to controversy.

Trypanosoma cruzi Infection Induces Pannexin-1 Channel Opening in Cardiac Myocytes.

Trypanosoma cruzi, the etiological agent of Chagas diseases, invades the cardiac tissue causing acute myocarditis and heart electrical disturbances. In T. cruzi invasion, the parasite induces [Ca2+]i transients in the host cells, an essential phenomenon for invasion. To date, knowledge on the mechanism that elicits transients of [Ca2+]i during the infection of cardiac myocytes has not been fully characterized. Pannexin1 (Panx1) channel are poorly selective channels found in all vertebrates that serve as a pathway for ATP release. In this article, we demonstrate that T. cruzi infection results in the opening of Panx1 channels in cardiac myocytes. We show that pharmacological blockade of Panx1 channels inhibits T. cruzi-induced [Ca2+]i transients and invasion in cardiac myocytes. Our results indicate that opening of Panx1 channels are required for T. cruzi invasion in cardiac myocytes, and we propose that targeting Panx1 channel could provide new potential therapeutic approaches to treat Chagas disease.

Edmund Goodwyn and the first description of diving bradycardia.

Diving bradycardia is a primordial oxygen-conserving reflex by which the heart rate of air-breathing vertebrates, including humans, slows down in response to water immersion. Its discovery is attributed to Paul Bert, whose seminal observation was published in 1870 as part of a series of experiments that examined physiological adaptations to asphyxia in ducks and other animals. However, Edmund Goodwyn, a British physician who studied medicine at the University of Edinburgh, had already described this reflex in his doctoral thesis, which was originally published in Latin in 1786 and again in English in 1788. Ironically, even though Goodwyn's work has yet to be recognized in the diving physiology literature, it was referenced in the very publication that contains Bert's original observation. Thus this article brings Goodwyn's work and its historical context to light and argues that he should be credited with the first description of diving bradycardia.

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision.

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S.

Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation.

The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca(2+) concentration ([Ca(2+)]i). Putative cell membrane pathways involved in these [Ca(2+)]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca(2+). Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca(2+)]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs.

Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare autosomal dominant disease that results in visible developmental anomalies of the limbs, face, eyes and teeth. Recently analysis of human connexin43 (Cx43) DNA sequences has revealed a number of different missense, duplication and frame shift mutations resulting in this phenotype. A mouse model of this disorder has been created with a missense point mutation of the glycine amino acid at position 60 to serine (G60S). Heterozygote +/G60S mice exhibit a similar ODDD phenotype as observed in humans. In addition to the malformations listed above, ODDD patients often have neurological findings. In the brain, Cx43 is highly expressed in astrocytes and has been shown to play a role in neuroprotection. We were interested in determining the effect of the +/G60S mutation following stroke. Four days after middle cerebral artery occlusion the volume of infarct was larger in mice with the +/G60S mutation. In astrocyte-neuron co-cultures, exposure to glutamate also resulted in greater cellular death in the +/G60S mutants. Protein levels of Cx43 in the mutant mouse were found to be reduced when compared to the normal tissue. Cx43 protein was observed as a continual line of small punctate aggregates in the plasma membrane with increased intracellular localization, which is distinct from the larger plaques seen in the normal mouse astrocytes. Functionally, primary +/G60S astrocytes exhibited reduced gap junctional coupling and increased hemichannel activity, which may underlie the mechanism of increased damage during stroke. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

The ATP required for potentiation of skeletal muscle contraction is released via pannexin hemichannels.

During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca2+ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd+) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd+ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y1R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca2+ too, which are required for potentiation of contraction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Connexin- and pannexin-based channels in normal skeletal muscles and their possible role in muscle atrophy.

Precursor cells of skeletal muscles express connexins 39, 43 and 45 and pannexin1. In these cells, most connexins form two types of membrane channels, gap junction channels and hemichannels, whereas pannexin1 forms only hemichannels. All these channels are low-resistance pathways permeable to ions and small molecules that coordinate developmental events. During late stages of skeletal muscle differentiation, myofibers become innervated and stop expressing connexins but still express pannexin1 hemichannels that are potential pathways for the ATP release required for potentiation of the contraction response. Adult injured muscles undergo regeneration, and connexins are reexpressed and form membrane channels. In vivo, connexin reexpression occurs in undifferentiated cells that form new myofibers, favoring the healing process of injured muscle. However, differentiated myofibers maintained in culture for 48 h or treated with proinflammatory cytokines for less than 3 h also reexpress connexins and only form functional hemichannels at the cell surface. We propose that opening of these hemichannels contributes to drastic changes in electrochemical gradients, including reduction of membrane potential, increases in intracellular free Ca(2+) concentration and release of diverse metabolites (e.g., NAD(+) and ATP) to the extracellular milieu, contributing to multiple metabolic and physiologic alterations that characterize muscles undergoing atrophy in several acquired and genetic human diseases. Consequently, inhibition of connexin hemichannels expressed by injured or denervated skeletal muscles might reduce or prevent deleterious changes triggered by conditions that promote muscle atrophy.

Ocular immune privilege sites.

The eye is one of the immune privilege sites of the body that is consequently protected from the detrimental and potentially blinding influences of immunologic inflammation. Within the eye, the anterior chamber has been recognized for its immune privilege property for many years now; however, a similar property detectable in the subretinal space has only recently been appreciated. These ocular sites are not only equipped with specialized mechanisms that barricade local inflammatory responses, but also induce systemic regulatory immune response. Numerous studies have characterized molecular and cellular mechanisms involved in conferring both these sites with an immune privilege status. Pigmented epithelial cells lining the anterior chamber in the iris and ciliary body area as well as those in the retina are endowed with immunomodulatory properties that contribute to ocular immune privilege. These cells, via expression of either soluble factors or membrane molecules, inhibit inflammatory T cell activation and promote the generation of regulatory T cells. In the anterior chamber resident antigen-presenting cells, influenced by the various immunosuppressive factors present in the aqueous humor, capture ocular antigens and present them in the spleen to T cells in association with NKT cells and marginal zone B cells. Immunomodulatory microenvironment created by these cells helps generate regulatory T cells, capable of interrupting the induction as well as expression of inflammatory responses. Furthermore, neural regulation of both intraocular and systemic regulatory mechanisms also contributes to ocular immune privilege.

Retinal pigment epithelial cells induce foxp3(+) regulatory T cells via membrane-bound TGF-ß.

PURPOSE: It is speculated that retinal pigment epithelial (RPE) cells convert naïve T cells into regulatory T cells (Tregs) via soluble factors such as transforming growth factor beta (TGF-ß). Yet presence or absence of similar membrane-bound mechanisms on RPE cells has yet to be addressed. Here the authors investigated the expression of surface TGF-ß by RPE cells and its participation in the conversion of naive T cells into Tregs. METHODS: They examined the phenotype of murine CD4(+) CD25(-) T cells activated in the presence of ethanol-fixed RPE cell layers as fixation preserves membrane structure while preventing the secretion of soluble factors. RESULTS: Fixed RPE cells supported the development of a de novo foxp3(+) Th3-like suppressor phenotype in activated peripheral naïve T cells through an interaction that required both RPE-derived surface TGF-ß, and T-cell derived TGF-ß1. CONCLUSIONS: Aside from soluble factors, RPE-derived surface TGF-ß can convert activated naïve T cells into Tregs.