cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media.

Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.

Characterization of a specific 20- to 25-kD interleukin-1 inhibitor from cultured human lung macrophages.

Alveolar macrophages have the ability to downregulate immune processes in vitro. We have recently suggested the presence of interleukin-1 (IL-1) inhibitors in the supernatants of human bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis or sarcoidosis. In the present study, we further analyze the cellular origin and the biologic properties of a 20- to 25-kD IL-1 inhibitor spontaneously produced by cultured human alveolar macrophages (AM). The inhibitor blocks IL-1-induced prostaglandin E2 production by human fibroblasts and the IL-1-related increase of phytohemagglutinin-induced murine thymocyte proliferation. After rigorous IL-1 alpha and IL-1 beta depletion, supernatants of lung macrophages specifically block the binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1 in a dose-dependent manner. These results indicate that AM from both normal donors and patients produce a specific IL-1 inhibitor that may be of importance in protecting the alveolar environment from the deleterious effects of excessive IL-1 production.

Cultured epithelial autografts in the management of burn injuries: a review of the literature

Introduction. The management of large burn victims has significantly improved in the last decades. Specifically autologous cultured keratinocytes (CEA) overcame the problem of limited donor sites in severely burned patients. Several studies testing CEA's in their burn centers give mixed results on the general outcomes of burn patients. Methods. A review of publications with a minimum of 15 patients per study using CEA for the management of severe burn injury from 1989 until 2011 were recruited by using an online database including Medline, Pub Med and the archives of the medical library of the CHUV in Lausanne. Results. 18 studies with a total of 977 patients were included into this review. Most of the studies did not specify if CEA's were grafted alone or in combination with split thickness skin grafts (STSG) although most of the patients seemed to have received both methodologies in reviewed studies. The mean TBSA per study ranged from 33% to 78% in patients that were grafted with CEA's. Here no common minimum TBSA making a patient eligible for CEA grafting could be found. The definition of the "take rate" is not standardized and varied largely from 26% to 73%. Mortality and hospitalization time could not be shown to correlate with CEA use in all of the studies. As late complications, some authors described the fragility of the CEA regenerated skin. Conclusion. Since the healing of large burn victims demands for a variety of different surgical and non-surgical treatment strategies and the final outcome mainly depends on the burned surface as well as the general health condition of the patient, no definitive conclusion could be drawn from the use of CEA's of reviewed studies. From our own experience, we know that selected patients significantly profit from CEA grafts although cost efficiency or the reduction of mortality cannot be demonstrated on this particular cases.

Oxygen tension controls the expression of the monocarboxylate transporter MCT4 in cultured mouse cortical astrocytes via a hypoxia-inducible factor-1α-mediated transcriptional regulation.

The monocarboxylate transporter MCT4 is a high capacity carrier important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is predominantly expressed by astrocytes. Surprisingly, MCT4 expression in cultured astrocytes is low, suggesting that a physiological characteristic, not met in culture conditions, is necessary. Here we demonstrate that reducing oxygen concentration from 21% to either 1 or 0% restored in a concentration-dependent manner the expression of MCT4 at the mRNA and protein levels in cultured astrocytes. This effect was specific for MCT4 since the expression of MCT1, the other astrocytic monocarboxylate transporter present in vitro, was not altered in such conditions. MCT4 expression was shown to be controlled by the transcription factor hypoxia-inducible factor-1α (HIF-1α) since under low oxygen levels, transfecting astrocyte cultures with a siRNA targeting HIF-1α largely prevented MCT4 induction. Moreover, the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced MCT4 expression in astrocytes cultured in presence of 21% oxygen. In parallel, glycolytic activity was enhanced by exposure to 1% oxygen as demonstrated by the increased lactate release, an effect dependent on MCT4 expression. Finally, MCT4 expression was found to be necessary for astrocyte survival when exposed for a prolonged period to 1% oxygen. These data suggest that a major determinant of astrocyte MCT4 expression in vivo is likely the oxygen tension. This could be relevant in areas of high neuronal activity and oxygen consumption, favouring astrocytic lactate supply to neurons. Moreover, it could also play an important role for neuronal recovery after an ischemic episode.

Deep hypothermia and rewarming alters glutamate levels and glycogen content in cultured astrocytes.

BACKGROUND: Deep hypothermia has been associated with an increased incidence of postoperative neurologic dysfunction after cardiac surgery in children. Recent studies suggest an excitotoxic mechanism involving overstimulation of glutamate receptors. Extracellular glutamate uptake occurs primarily by astrocytes. Astrocytes also store glycogen, which may be used to sustain the energy-consuming glutamate uptake. Extracellular glutamate and glycogen content were studied during temperature changes mimicking cardiopulmonary bypass in vivo. METHODS: Primary cultures of cerebral cortical astrocytes were used in a specially designed incubator allowing continuous changes of temperature and ambient gas concentrations. The sequence of events was as follows: normothermia, rapid cooling (2.8 degrees C/min) followed by 60 min of deep hypothermia (15 degrees C), followed by rewarming (3.0 degrees C/min) and subsequent 5 h of mild hyperthermia (38.5 degrees C). Two different conditions of oxygenation were studied: (1) normoxia (25% O2, 70% N2, 5% CO2); or (2) hyperoxia (95% O2, 5% CO2). The extracellular glutamate concentrations and intracellular glycogen levels were measured at nine time points. RESULTS: One hundred sixty-two cultures were studied in four independent experiments. The extracellular concentration of glutamate in the normoxic group increased significantly from 35+/-10 nM/mg protein at baseline up to 100+/-15 nM/mg protein at the end of 5 h of mild hyperthermia (P < 0.05). In contrast, extracellular glutamate levels did not vary from control in the hyperoxic group. Glycogen levels decreased significantly from 260+/-85 nM/mg protein at baseline to < 25+/-5 nM/mg protein at the end of 5 h in the normoxic group (P < 0.05) but returned to control levels after rewarming in the hyperoxic group. No morphologic changes were observed in either group. CONCLUSION: The extracellular concentration of glutamate increases, whereas the intracellular glycogen content decreases when astrocytes are exposed to a sequence of deep hypothermia and rewarming. This effect of hypothermia is prevented when astrocytes are exposed to hyperoxic conditions.

Regulation and glucocorticoid-independent induction of lipocortin I in cultured astrocytes.

Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

Detection of new cross-reacting carcinoembryonic antigen(s) on cultured tumor cells by mixed hemadsorption assay.

Antisera highly specific for carcinoembryonic antigen (CEA) from New Zealand White rabbits and a goat reacted strongly in antibody binding tests with cultured tumor cell lines, irrespective of the ability of the cell lines to produce CEA. The most reactive were colon carcinoma and melanoma cell lines, the former known to produce CEA and the latter not associated with CEA production. The reactivity was not diminished by absorption with perchloric acid extracts of normal lung or spleen, whereas absoprtion with purified CEA preparations abolished the reactivity. Quantitative absorption studies indicated that reactivity against CEA-producing cell lines could be totally removed by absorption with other CEA-producing lines but not with melanoma cell lines. Reactivity against melanoma cell lines could be completely removed by colon carcinoma cells as well as by melanoma cells. Antisera raised against purified CEA, after absorption with extracts of normal lung, still contained two populations of antibodies, one that binds a newly described antigen cross-reacting with CEA which is present on melanoma cells.

Nitric Oxide Induces the Expression of the Monocarboxylate Transporter MCT4 in Cultured Astrocytes by a cGMP-Independent Transcriptional Activation

The monocarboxylate transporter MCT4 is a proton-linked carrier particularly important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is exclusively expressed by astrocytes. Surprisingly, MCT4 expression in primary cultures of mouse cortical astrocytes is conspicuously low, suggesting that an external, nonastrocytic signal is necessary to obtain the observed pattern of expression in vivo. Here, we demonstrate that nitric oxide (NO), delivered by various NO donors, time- and dose-dependently induces MCT4 expression in cultured cortical astrocytes both at the mRNA and protein levels. In contrast, NO does not enhance the expression of MCT1, the other astrocytic monocarboxylate transporter. The transcriptional effect of NO is not mediated by a cGMP-dependent mechanism as shown by the absence of effect of a cGMP analog or of a selective guanylate cyclase inhibitor. NO causes an increase in astrocytic lactate transport capacity which requires the enhancement of MCT4 expression as both are prevented by the use of a specific siRNA against MCT4. In addition, cumulated lactate release by astrocytes over a period of 24 h was also enhanced by NO treatment. Our data suggest that NO represents a putative intercellular signal to control MCT4 expression in astrocytes and in doing so, to facilitate lactate transfer to other surrounding cell types in the central nervous system. (C) 2011 Wiley-Liss, Inc.

Reactivity of antiglioma monoclonal antibodies for a large panel of cultured gliomas and other neuroectoderm derived tumors.

We produced three monoclonal antibodies, BF7, GE2 and CG12, against cultured human glioma cells. Their specificity was tested by an indirect antibody-binding radioimmunoassay on a panel of glial and non-glial tumor cell lines. BF7 and GE2 react preferentially with glioma cells and, except for one colon carcinoma line, they do not bind to the control non-neuroectodermal cells; they appear to be directed against common malignant glioma associated antigens. CG12, the third monoclonal antibody, binds to the great majority of tumor cell lines of neuroectodermal origin and does not bind to any other cell lines tested.

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.

Treatment of extended skin defects with autologous composite cultured skin grafts in a patient with epidermolysis bullosa

Rationale: The purpose of this article is to demonstrate the use of homologous culture cells in treating an advanced coccon formation of the hand and three extended squamous cell carcinomas of the lower and upper limb in a patient with recessive dystrophic epidermolysis bullosa. The preparation and application of these cells in the operation room are being described. Methods: A number of surgical approaches have been described to correct these deformities in order to improve function.We propose a new therapeutic approach of treating loss of motion and independent digital function as well as coverage of large skin defects in a patient with recessive dystrophic epidermolysis bullosa by using autologous culture cells. Surgical treatment of these patients is really difficult because of the existing skin fragility. Furthermore, surgical wounds do not easily heal because of recurrent blisters and erosions as well as due to the patients' poor nutricial status. Results: We report our experience of mutiple extended cutaneous squamous cell carcinomas arising in our patient which were successfully managed using autologous composite cultured skin grafts. The cocoon hand deformity was also treated with the limb becoming functional. Conclusion: The use of autologous keratinocytes and fibroblasts in epidermolysis bullosa is hereby outlined for the fist time.

The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.

BACKGROUND: Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-beta signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. CONCLUSIONS/SIGNIFICANCE: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-beta signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-beta signaling and connective tissue dysfunction.

Phenotyping of 60 cultured human gliomas and 34 other neuroectodermal tumors by means of monoclonal antibodies against glioma, melanoma and HLA-DR antigens.

The reactivity spectrum of three monoclonal antibodies (Mabs) to human malignant glioma, five Mabs to melanomas and one Mab anti-HLA-DR was investigated by an indirect antibody binding radioimmunoassay on a panel of cells derived from 60 glioma lines, including 47 malignant astrocytomas, 11 low-grade astrocytomas and two malignant ependymomas as well on cells from 12 melanoma, three neuroblastoma, three medulloblastoma, two schwannoma, two retinoblastoma, two choroïd plexus papilloma, ten meningioma and 12 unrelated tumor lines. The anti-glioma Mabs BF7 and GE2 reacted preferentially with gliomas, while the anti-glioma Mab CG12 reacted with gliomas, melanomas, neuroblastomas and medulloblastomas. The five anti-melanoma Mabs reacted with gliomas, neuroblastomas and medulloblastomas. The anti-HLA-DR Mab D1-12 reacted with gliomas, melanomas and some meningiomas. On the basis of the data presented, we describe three different antigenic systems; the first one is glioma-associated, the second one is related to differentiation antigens expressed on cells derived from the neuroectoderm and the third is represented by HLA-DR antigens which are expressed not only on B-lymphoblastoid cells but also on melanomas and gliomas.