Turbinate-homing IgA-secreting cells originate in the nasal lymphoid tissues.

Nasal vaccination elicits a humoral immune response that provides protection from airborne pathogens1, yet the origins and specific immune niches of antigen-specific IgA-secreting cells in the upper airways are unclear2. Here we define nasal glandular acinar structures and the turbinates as immunological niches that recruit IgA-secreting plasma cells from the nasal-associated lymphoid tissues (NALTs)3. Using intact organ imaging, we demonstrate that nasal vaccination induces B cell expansion in the subepithelial dome of the NALT, followed by invasion into commensal-bacteria-driven chronic germinal centres in a T cell-dependent manner. Initiation of the germinal centre response in the NALT requires pre-expansion of antigen-specific T cells, which interact with cognate B cells in interfollicular regions. NALT ablation and blockade of PSGL-1, which mediates interactions with endothelial cell selectins, demonstrated that NALT-derived IgA-expressing B cells home to the turbinate region through the circulation, where they are positioned primarily around glandular acinar structures. CCL28 expression was increased in the turbinates in response to vaccination and promoted homing of IgA+ B cells to this site. Thus, in response to nasal vaccination, the glandular acini and turbinates provide immunological niches that host NALT-derived IgA-secreting cells. These cellular events could be manipulated in vaccine design or in the treatment of upper airway allergic responses.

Positively Charged Amino Acids in the Pestiviral Erns Control Cell Entry, Endoribonuclease Activity and Innate Immune Evasion.

The genus Pestivirus, family Flaviviridae, includes four economically important viruses of livestock, i.e., bovine viral diarrhea virus-1 (BVDV-1) and -2 (BVDV-2), border disease virus (BDV) and classical swine fever virus (CSFV). Erns and Npro, both expressed uniquely by pestiviruses, counteract the host's innate immune defense by interfering with the induction of interferon (IFN) synthesis. The structural envelope protein Erns also exists in a soluble form and, by its endoribonuclease activity, degrades immunostimulatory RNA prior to their activation of pattern recognition receptors. Here, we show that at least three out of four positively-charged residues in the C-terminal glycosaminoglycan (GAG)-binding site of BVDV-Erns are required for efficient cell entry, and that a positively charged region more upstream is not involved in cell entry but rather in RNA-binding. Moreover, the C-terminal domain on its own determines intracellular targeting, as GFP fused to the C-terminal amino acids of Erns was found at the same compartments as wt Erns. In summary, RNase activity and uptake into cells are both required for Erns to act as an IFN antagonist, and the C-terminal amphipathic helix containing the GAG-binding site determines the efficiency of cell entry and its intracellular localization.

Isolation and development of bovine primary respiratory cells as model to study influenza D virus infection.

Influenza D virus (IDV) is a novel type of influenza virus that infects and causes respiratory illness in bovines. Lack of host-specific in vitro model that can recapitulate morphology and physiology of in vivo airway epithelial cells has impeded the study of IDV infection. Here, we established and characterized bovine primary respiratory epithelial cells from nasal turbinate, soft palate, and trachea of the same calf. All three cell types showed characteristics peculiar of epithelial cells, polarized into apical-basolateral membrane, and formed tight junctions. Furthermore, these cells expressed both α-2,3- and α-2,6-linked sialic acids with α-2,3 linkage being more abundant. IDV strains replicated to high titers in these cells, while influenza A and B viruses exhibited moderate to low titers, with influenza C virus replication not detected. These findings suggest that bovine primary airway epithelial cells can be utilized to model infection biology and pathophysiology of IDV and other respiratory pathogens.

Neural crest-derived cells in nasal conchae of adult mice contribute to bone regeneration.

Neural crest-derived cells (NCDCs), a class of adult stem cells not restricted to embryonic tissues, are attractive tissue regenerative therapy candidates because of their ease of isolation, self-renewing properties, and multipotency. Although adult NCDCs can undergo osteogenic differentiation in vitro, whether they induce bone formation in vivo remains unclear. Previously, our group reported findings showing high amounts of NCDCs scattered throughout nasal concha tissues of adult mice. In the present study, NCDCs in nasal conchae labeled with enhanced green fluorescent protein (EGFP) were collected from adult P0-Cre/CAG-CAT-EGFP double transgenic mice, then cultured in serum-free medium to increase the number. Subsequently, NCDCs were harvested and suspended in type I atelocollagen gel, then an atelocollagen sponge was used as a scaffold for the cell suspension. Atelocollagen scaffolds with NCDCs were placed on bone defects created in a mouse calvarial bone defect model. Over the ensuing 12 weeks, micro-CT and histological analysis findings showed that mice with scaffolds containing NCDCs had slightly greater bone formation as compared to those with a scaffold alone. Furthermore, Raman spectroscopy revealed spectral properties of bone in mice that received scaffolds with NCDCs similar to those of native calvarial bone. Bone regeneration is important not only for gaining bone mass but also chemical properties. These results are the first to show the validity of biomolecule-free adult nasal concha-derived NCDCs for bone regeneration, including the chemical properties of regenerated bone tissue.

The proteostatic network chaperome is downregulated in F508del homozygote cystic fibrosis.

BACKGROUND: CF patients demonstrate clinical heterogeneity and much remains unknown about how to risk stratify individuals for disease progression.  The most common cystic fibrosis mutation, F508del, is a protein folding mutation that has been shown in vitro to negatively affect proteostasis and CFTR transcription. Since CFTR is expressed in the nasal epithelium, we hypothesized that by using unbiased transcriptomics we could gain clinically relevant insights about differential gene expression and heterogeneity in CF patients as well as assess proteostatic dysfunction in the nasal epithelium. METHODS: Using nasal curettage and RNA-seq we assessed differential gene expression in F508del homozygotes compared to healthy volunteers. Gene set enrichment analysis was performed using a list of known chaperones. Pilot and validation cohorts were studied. RESULTS: PCA analysis and gene expression heatmaps exhibited greater heterogeneity among CF than healthy volunteers. Differentially expressed genes were enriched for the downregulation of ciliary/microtubular genes and the upregulation of inflammatory/immune response genes in F508del homozygotes compared to healthy volunteers. Gene set analysis identified negative enrichment for chaperone genes and decreased CFTR transcription in the F508del homozygotes. We also found preliminary evidence for the recently identified ionocyte in the nasal specimens. CONCLUSION: CF patients homozygous for F508del demonstrate heterogeneous gene expression profiles, proteostatic dysregulation, and reduced CFTR transcription. Larger studies are needed to determine whether nasal epithelial gene transcription profiles can be leveraged for insights into disease heterogeneity.

Specific bacteriophage of Bordetella bronchiseptica regulates B. bronchiseptica-induced microRNA expression profiles to decrease inflammation in swine nasal turbinate cells.

BACKGROUND: Respiratory diseases in pigs are the main health concerns for swine producers. Similar to the diseases in human and other animals, respiratory diseases are primary related to morbidity and are the result of infection with bacteria, viruses, or both. B. bronchiseptica causes serious respiratory diseases in the swine airway track. However, the B. bronchiseptica-specific bacteriophage has diverse advantages such as decreasing antibiotic overuse and possible therapeutic potential against bacteria. OBJECTIVE: The objects of this study were to investigate the therapeutic effect of specific B. bronchiseptica bacteriophages and to identify genes related to bacteriophage signaling utilizing RNA microarrays in swine nasal turbinate cells. METHODS: Bor-BRP-1 phages were applied 24 h prior to B.bronchiseptica infection (1 × 107 cfu/ml) at several concentrations of bacterial infection. Cells were incubated to detect cytokines and 24 h to detect mucin production. And real-time quantitative PCR was performed to examine related genes expression. To determine the change of total gene expression based on B.bronchiseptica and Bor-BRP-1 treatment, we performed RNA sequencing experiments. RESULTS: The results showed that B. bronchiseptica induced increased expression of several inflammatory genes such as IL-1ß, IL-6, and Muc1 in a dose-dependent manner. However, Bor-BRP-1 induced reduction of gene expression compared to the B. bronchiseptica induction group. In addition, microarrays detected Bor-BRP-1-altered inflammatory gene expression against B. bronchiseptica, reducing B. bronchiseptica-induced airway inflammation in swine epithelial cells. CONCLUSION: These results suggest that the specific bacteriophage has a therapeutic potential to defend against B. bronchiseptica infection by altering inflammatory gene expression profiles.

In vivo Oxygen Condition of Human Nasal Inferior Turbinate-Derived Stem Cells in Human Nose.

BACKGROUND AND OBJECTIVE: Human nasal inferior turbinate-derived stem cells (hNTSCs) have been considered as a potent and useful source for regenerative medicine. To most effectively mimic the native environment of inferior turbinate could be very effective to hNTSCs biology. Thus, the purpose of this study was to evaluate partial pressure of oxygen (ppO2) and temperature in inferior turbinate. METHODS: Ten patients were enrolled who underwent endoscopic endonasal transsphenoidal skull base tumor surgery between January 2014 and December 2015. The commercially available OxyLab pO2 monitor gauges the ppO2 and temperature using a fluorescence quenching technique. Also, hNTSCs were isolated from 10 patients and cultivated under hypercapnic condition (5, 10, and 15%) to mimic hypoxic intranasal conditions. RESULTS: The measured oxygen concentration in submucosa tissue was higher than that at the surface of the inferior turbinate and the temperature in submucosa tissue was higher than the value at the surface of inferior turbinate. The patterns of proliferation were significantly different according to hypercapnic cultivation conditions and there were statistically significant decreased proliferation rates after the exposure of higher CO2 over a period of 5 days. CONCLUSIONS: Intranasal turbinate tissue showed the hypoxia state in concordance with the result of the other tissues or organs. However, indirectly induced hypoxia influenced the influence on the hNTSCs proliferation negatively. Further study is needed to mimic the real hypoxic state, but our results could be used to optimize the culture environment of hNTSCs, thereby producing the stem cells for regenerative therapies.

An injectable cationic hydrogel electrostatically interacted with BMP2 to enhance in vivo osteogenic differentiation of human turbinate mesenchymal stem cells.

We have designed and characterized an injectable, electrostatically bonded, in situ-forming hydrogel system consisting of a cationic polyelectrolyte [(methoxy)polyethylene glycol-b-(poly(ε-caprolactone)-ran-poly(L-lactic acid)] (MP) copolymer derivatized with an amine group (MP-NH2) and anionic BMP2. To the best of our knowledge, there have been hardly any studies that have investigated electrostatically bonded, in situ-forming hydrogel systems consisting of MP-NH2 and BMP2, with respect to how they promote in vivo osteogenic differentiation of human turbinate mesenchymal stem cells (hTMSCs). Injectable formulations almost immediately formed an electrostatically loaded hydrogel depot containing BMP2, upon injection into mice. The hydrogel features and stability of BMP2 inside the hydrogel were significantly affected by the electrostatic attraction between BMP2 and MP-NH2. Additionally, the time BMP2 spent inside the hydrogel depot was prolonged in vivo, as evidenced by in vivo near-infrared fluorescence imaging. Biocompatibility was demonstrated by the fact that hTMSCs survived in vivo, even after 8 weeks and even though relatively few macrophages were in the hydrogel depot. The osteogenic capacity of the electrostatically loaded hydrogel implants containing BMP2 was higher than that of a hydrogel that was simply loaded with BMP2, as evidenced by Alizarin Red S, von Kossa, and hematoxylin and eosin staining as well as osteonectin, osteopontin, osteocalcin, and type 1α collagen mRNA expression. The results confirmed that our injectable, in situ-forming hydrogel system, electrostatically loaded with BMP2, can enhance in vivo osteogenic differentiation of hTMSCs.

The Ability of Conditioned Media From Stem Cells to Repair Vocal Fold Injuries.

OBJECTIVE: This study investigated the ability of hypoxia-induced 25-fold concentrated conditioned media (hCM) from human nasal inferior turbinate-derived mesenchymal stem cells (hTMSC) to repair injured vocal folds during the early phase of the wound-healing process. METHODS: The vocal fold was injured in Sprague-Dawley rats. Next, hCM from hTMSC (the hCM group) or hTMSC (the hTMSC group) were injected into the injured vocal folds. As a control, saline (the phosphate-buffered saline group) or 25-fold concentrated media (the media group) was injected in the same manner. The vocal folds were harvested for quantitative real-time polymerase chain reaction (PCR) at 1 week and 2 weeks after injury. Histologic evaluation was performed at 3 weeks postinjury. RESULTS: In the hCM group at 1 week after injury, PCR showed that the genes encoding hyaluronan synthase (HAS), HAS 1, and HAS 2 were significantly upregulated compared to the media and normal groups. The gene encoding procollagen III was significantly downregulated compared to the media group. Nearly identical results were obtained for the hTMSC group at 1 week after injury. Histological examination showed that the hCM group was similar to or better than the hTMSC group in collagen deposition and hyaluronic acid production. CONCLUSION: The injection of hCM into injured vocal folds produced antifibrotic effects in the early phase of wound healing. These effects were equivalent to those produced by the injection of hTMSC. These results provide a foundation for the future clinical use of hCM for vocal fold regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1867-1875, 2019.

Decellularization and genipin crosslinking of amniotic membrane suitable for tissue engineering applications.

Amniotic membrane has the potential to be used as scaffold in various tissue engineering applications. However, increasing its biostability at the same time maintaining its biocompatibility is important to enhance its usage as a scaffold. This studied characteristics genipin-crosslinked amniotic membrane as a bioscaffold. Redundant human amniotic membranes (HAM) divided into native (nAM), decellularized (dAM) and genipin-crosslinked (clAM) groups. The dAM and clAM group were decellularized using thermolysin (TL) and sodium hydroxide (NaOH) solution. Next, clAM group was crosslinked with 0.5% and 1.0% (w/v) genipin. The HAM was then studied for in vitro degradation, percentage of swelling, optical clarity, ultrastructure and mechanical strength. Meanwhile, fibroblasts isolated from nasal turbinates were then seeded onto nAM, dAM and clAM for biocompatibility studies. clAM had the slowest degradation rate and were still morphologically intact after 30 days of incubation in 0.01% collagenase type 1 solution. The dAM had a significantly highest percentage of swelling than other groups (p < 0.05). Besides, the dAM retained the collagen content at similar level of nAM. Although the dAM had highest mechanical strength compared to the rest of the groups, the differences were statistically insignificant. Cell attachment on dAM and 0.5% clAM was higher compared to that on nAM and 1.0% clAM. In conclusion, clAM have better biostability and biocompatibility compared to the nAM and dAM. Together with other suitable characteristics of the clAM such as percentage of swelling, structural integrity and ECM content, clAM is suitable as scaffold for various tissue engineering applications.

Effect of MeCP2 on TGF- ß1-induced Extracellular Matrix Production in Nasal Polyp-derived Fibroblasts.

Purpose Methyl-CpG-binding protein 2 (MeCP2), known as a transcriptional regulator, has been suggested to play an important role in myofibroblast differentiation in the lung. The purpose of this study was to investigate the role of MeCP2 in transforming growth factor (TGF)- ß1-induced myofibroblast differentiation and extracellular matrix (ECM) production in nasal polyp-derived fibroblasts (NPDFs). Methods To identify the expression of MeCP2 in nasal polyp tissues, immunohistochemistry staining and Western blot were performed. TGF- ß1-induced NPDFs were treated with 5-azacytidine, a DNA methylation inhibitor, and the expression levels of α-SMA and fibronectin were determined by semiquantitative reverse transcription polymerase chain reaction, immunofluorescent staining, and Western blotting. The total soluble collagen was analyzed by the Sircol collagen assay. MeCP2 silenced by MeCP2-specific small interference ( si) RNA was verified by Western blot. Results The expression levels of MeCP2 increased in nasal polyp tissues compared to normal inferior turbinate tissues. 5-Azacytidine significantly inhibited the expression of α-SMA and fibronectin mRNA in a dose-dependent manner. In addition, 5-azacytidine suppressed collagen production and the expression of MeCP2 in the same manner. The expression levels of a-SMA and collagen production were significantly blocked by MeCP2 silencing in TGF- ß1-induced NPDFs. Conclusions Our data suggest that MeCP2 plays an essential role in TGF- ß1-induced myofibroblast differentiation and ECM production in NPDFs.

The Ability of Human Nasal Inferior Turbinate-Derived Mesenchymal Stem Cells to Repair Vocal Fold Injuries.

Objective This study investigated the ability of implanted human nasal inferior turbinate-derived mesenchymal stem cells (hTMSCs) to repair injured vocal folds. To this end, we used quantitative real-time polymerase chain reaction (PCR) to analyze the early phase of wound healing and histopathological analysis to explore the late phase of wound healing in xenograft animal models. Study Design Prospective animal study. Setting Research laboratory. Subjects and Methods The right-side lamina propria of the vocal fold was injured in 20 rabbits and 30 rats. Next, hTMSCs were implanted into half of the injured vocal folds (hTMSC groups). As a control, phosphate-buffered saline (PBS) was injected into the other half of the injured vocal folds (PBS groups). Rat vocal folds were harvested for polymerase chain reaction (PCR) at 1 week after injury. Rabbit vocal folds were evaluated endoscopically and the larynges harvested for histological and immunohistochemical examination at 2 and 8 weeks after injury. Results In the hTMSC group, PCR showed that hyaluronan synthase ( HAS) 1, HAS 2, and transforming growth factor ( TGF)-ß1 were significantly upregulated compared with the PBS group. Procollagen type III ( COL III) messenger RNA expression was significantly upregulated in the PBS group compared with the normal group. Histological analyses showed that hTMSC administration afforded more favorable collagen and hyaluronic acid deposition than was evident in the controls. Implanted hTMSCs were observed in injured vocal folds 2 weeks after implantation. Conclusions Our results show that hTMSCs implantation into injured vocal folds facilitated vocal fold regeneration, with presenting antifibrotic effects.

Evaluation of characteristic of human turbinate derived mesenchymal stem cells cultured in the serum free media.

We evaluated the effect of serum-free and xeno-cultivation (SFXFM) on the characterization, proliferation, and differentiation properties of human nasal stem cells (airway tissue; hTMSCs). hTMSCs were isolated from 10 patients, after which patient samples were separated into two groups, an SFXFM group and a control group. The control group was treated with bovine serum-containing medium. FACS analysis revealed that SFXFM-cultured hTMSCs maintained a characteristic mesenchymal stem cell phenotype. hTMSC proliferation was not influenced by SFXFM. In addition, upregulation of IL-8 and GM-CSF and downregulation of RANTES expression were shown in response to SFXFM. Moreover, two-lineage differentiation properties (osteocyte and adipocyte) of hTMSCs were enhanced under SFXFM. Finally, the genetic stability of SFXFM-cultured hTMSCs was demonstrated by normal karyotype results. SFXFM enables good expansion, multipotentiality, and normal genotype maintenance of MSCs. Moreover, this approach serves as a substitute to conventional media for the cultivation of capable MSCs for upcoming medical applications.

Investigating the effect of fibulin-1 on the differentiation of human nasal inferior turbinate-derived mesenchymal stem cells into osteoblasts.

Many extracellular matrix proteins have positive influences on the adhesion, proliferation, and differentiation of stem cells into specific cell linages. Fibulin-1 (FBLN1), a member of a growing family of extracellular glycoproteins, contributes to the structure of the extracellular matrix. Here, we investigated the effect of FBLN1 on the ability of human nasal inferior turbinate-derived mesenchymal stem cells (hTMSCs) to undergo osteogenic differentiation. After we generated recombinant FBLN1, the characteristics of FBLN1-treated hTMSCs were evaluated using MTT assay, ALP and mineralization activities, and quantitative real-time PCR. FBLN1 significantly enhanced the adhesion activity (p < 0.001) and proliferation of hTMSCs (p < 0.05). The ALP and mineralization activities of cells were dramatically increased (p < 0.01) after 9 and 12 days of FBLN1 treatment, respectively. This indicated the ability of FBLN1 to induce hTMSCs to differentiate into osteoblasts. Furthermore, increasing the mRNA levels of osteogenic marker genes, such as a transcriptional coactivator with a PDZ-binding motif (TAZ), alkaline phosphatase (ALP), collagen type I (Col I), and osteocalcin (OCN), improved bone repair and regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2291-2298, 2017.

Glial differentiation of human inferior turbinate-derived stem cells: a new source of cells for nerve repair.

Schwann cell (SC) transplantation as a cell-based therapy can enhance peripheral and central nerve repair experimentally, but it is limited by donor site morbidity for clinical application. We investigated whether human turbinate-derived mesenchymal stem cells (hTMSCs) isolated from discarded inferior turbinate during surgery can differentiate into functional SC-like cells. hTMSCs expressed mesenchymal cell surface markers CD29, CD44, CD90, and CD105 and did not express neural crest markers P75 and Nestin. After monolayer culture in predifferentiation medium and transdifferentiation medium with a mixture of glial growth factors and chemical regents for 14 days, the differentiated hTMSCs exhibited a spindle-like morphology similar to that of SCs. RT-PCR, immunocytochemical staining, and western blotting analysis indicated that SC-like cells expressed the glial markers S100ß, P75, and glial fibrillary acidic protein at the gene and protein level. Compared with hTMSCs, differentiated hTMSCs secreted more neurotrophins, and significantly enhanced the neurite length when cocultured with dorsal root ganglia neuronal cells. Our data indicated that hTMSCs can differentiate into functional SC-like cells and have the ability to facilitate the neurite growth of dorsal root ganglia neuronal cells in vitro, representing a promising source of cells for nerve repair.

Influence of border disease virus (BDV) on serological surveillance within the bovine virus diarrhea (BVD) eradication program in Switzerland.

BACKGROUND: In 2008, a program to eradicate bovine virus diarrhea (BVD) in cattle in Switzerland was initiated. After targeted elimination of persistently infected animals that represent the main virus reservoir, the absence of BVD is surveilled serologically since 2012. In view of steadily decreasing pestivirus seroprevalence in the cattle population, the susceptibility for (re-) infection by border disease (BD) virus mainly from small ruminants increases. Due to serological cross-reactivity of pestiviruses, serological surveillance of BVD by ELISA does not distinguish between BVD and BD virus as source of infection. RESULTS: In this work the cross-serum neutralisation test (SNT) procedure was adapted to the epidemiological situation in Switzerland by the use of three pestiviruses, i.e., strains representing the subgenotype BVDV-1a, BVDV-1h and BDSwiss-a, for adequate differentiation between BVDV and BDV. Thereby the BDV-seroprevalence in seropositive cattle in Switzerland was determined for the first time. Out of 1,555 seropositive blood samples taken from cattle in the frame of the surveillance program, a total of 104 samples (6.7%) reacted with significantly higher titers against BDV than BVDV. These samples originated from 65 farms and encompassed 15 different cantons with the highest BDV-seroprevalence found in Central Switzerland. On the base of epidemiological information collected by questionnaire in case- and control farms, common housing of cattle and sheep was identified as the most significant risk factor for BDV infection in cattle by logistic regression. CONCLUSION: This indicates that pestiviruses from sheep should be considered as a source of infection of domestic cattle and might well impede serological BVD surveillance.

[Effect of Amphotericin B on human nasal ciliary beat frequency].

OBJECTIVE: To investigate the effects of Amphotericin B on ciliary beat frequency (CBF) in in vitro cultured human nasal epithelial cells. METHODS: Human nasal epithelial cells derived from uncinate process or inferior turbinate of six patients were cultured in vitro, and treated with different concentrations of Amphotericin B(0.25、0.5、1.0、2.0、4.0 µg/ml)over a peroid of 7~10 d; normal saline(NS)was used as a negative control. CBF was detected before treatment and recorded continuously for 20 min after treatment. RESULTS: CBF of epithelial cells in normal saline (NS) control group showed no significant changes in 20 min(F=0.351, P>0.05); Amphotericin B at 0.25 µg/ml(F=0.286, P>0.05), 0.5 µg/ml(F=0.468, P>0.05) or 1.0 µg/ml(F=0.383, P>0.05) did not affect epithelial CBF in 20 min measurement period; Amphotericin B at 2.0 µg/ml decreased epithelial CBF with time(F=1.908, P<0.05), significant changes were observed after 7 min of drug treatment; Amphotericin B at 4.0 µg/ml decreased epithelial CBF with time(F=8.223, P<0.05), significant changes were observed after 6 min of drug treatment. CONCLUSIONS: Amphotericin B caused a dose-dependent decrease of human nasal CBF. Therefore, the influence of this drug on ciliary function should be considered when nasal topically applied.

Altered G Protein Coupling in Olfactory Neuroepithelial Cells From Patients With Schizophrenia.

Increasing evidence suggests that olfactory dysfunction is an endophenotype of schizophrenia, and thus the olfactory system can be studied both in relation to this sensory dysfunction and also as a means of examining pathophysiologic mechanisms of schizophrenia. In this study, we examined human olfactory neuroepithelial (ON) biopsy tissues and their in vitro culture cells for ligand-induced guanine nucleotide-binding protein (G protein) activation and downstream signaling. We assessed the binding of a nonhydrolyzable GTP analogue [(35)S]GTPγS binding to specific G protein subtypes in response to odorants, dopamine, or serotonin in ON cell membranes from matched schizophrenia-control subjects. In response to odorant mixtures, we found decreased [(35)S]GTPγS binding to Gαs/olf in schizophrenia patients. These changes were not mediated by mRNA expression of key molecules of G protein coupling, including adenylate cyclase III (ACIII), protein kinase A (PKA), protein kinase Cγ (PKCγ), or Gαs or Gαolf in ON cells or ON biopsy tissues. In contrast, dopamine (DA)- and serotonin (5HT)-induced S(35)-GTPγS binding to Gαs/olf and Gαq/11 were significantly increased in schizophrenia cases, while these parameters were strikingly reduced by in vitro treatment with antipsychotics. Patients with schizophrenia exhibit increases in electrolfactogram (EOG) recordings, suggesting enhanced odorant-induced activation. Our results of decreased odorant-induced G protein activation may point further downstream for underlying mechanisms for increased EOG measures. Increased G protein activation in response to DA and 5HT may suggest increased postreceptor DA or 5HT signaling as an additional mechanism of dopaminergic or serotonergic dysregulation in schizophrenia.

Expression of mesenchymal stem cell phenotype in human nasal respiratory epithelial cells / Expresión del fenotipo de células troncales mesenquimales en células epiteliales nasales humanas

The respiratory epithelium is the first line of contact with the external hazards. Thus it can be damaged and need to be replaced to avoid healing by fibrosis. Tracheal tissue engineering is an alternative promising treatment modality. Mesenchymal stem cell markers are surface proteins, which are responsible for some of these cells unique properties. The objective of this study was to detect the mesenchymal stem cell phenotype among the human nasal respiratory epithelial cells via two immunophenotyping techniques. Respiratory epithelial cells were cultured using co-culture technique, fibroblasts was removed at confluence leaving respiratory epithelial cells, which were passage further to passage 4. Cells were evaluated for mesenchymal stem cell markers that were CD73, CD90, CD105 and the hematopoietic stem cell marker CD45 at passage 1 (P1) and passage 4 (P4) using Flow cytometry and Immunocytochemistry techniques. Respiratory epithelial cells expressed the mesenchymal stem cell markers at P1 and maintain the expression these markers until P4. Using both techniques, to compare the values of mesenchymal stem cell markers expression at P1 to P4 there was no significant difference. This study indicates that respiratory epithelial cells derived from nasal turbinate retain some of mesenchymal stem cells properties even after serial passages. Both methods of Immunophenotyping are comparable.

El epitelio respiratorio es la primera línea de contacto con los peligros externos. Por lo tanto, puede ser dañado y necesita ser reemplazado para evitar uan cicatrización por fibrosis. La ingeniería de tejidos traqueales es una modalidad de tratamiento alternativo prometedora. Los marcadores de células troncales mesenquimales son proteínas de superficie, que son responsables de algunas propiedades únicas de estas células. El objetivo fue detectar el fenotipo de células troncales mesenquimales entre las células epiteliales respiratorias nasales humanas a través de dos técnicas de inmunofenotipaje. Fueron cultivadas las células epiteliales respiratorias utilizando la técnica de co-cultivo; los fibroblastos se eliminaron en la confluencia dejando solo células epiteliales respiratorias, resultantes de los 4 pasajes. Las células fueron evaluadas para encontrar marcadores de células troncales mesenquimales mediante CD73, CD90, CD105 y el marcador de células troncales hematopoyéticas CD45 en el paso 1 (P1) y el paso 4 (P4), usando citometría de flujo y técnicas de inmunocitoquímica. Las células epiteliales respiratorias expresaron los marcadores de células troncales mesenquimales en P1 y mantuvieron la expresión de estos marcadores hasta P4. No hubo diferencias significativas en el uso de ambas técnicas al comparar los valores de los marcadores de células troncales mesenquimales expresadas desde P1 a P4. Este estudio indica que las células epiteliales respiratorias derivadas de la concha nasal retienen algunas de las propiedades de células troncales mesenquimales, incluso después de pases seriados. Ambos métodos de inmunofenotipificación son comparables.

Late production of CXCL8 in ruminant oro-nasal turbinate cells in response to Chlamydia abortus infection.

Chlamydia abortus is an obligate intracellular bacterium that is an important cause of ovine abortion worldwide. There are reports of abortions in cattle, but these are very rare compared to the reported incidence in sheep. The bacterium is transmitted oro-nasally and can establish a sub-clinical infection until pregnancy, when it can invade the placenta and induce an inflammatory cascade leading to placentitis and abortion. Early host-pathogen interactions could explain differential pathogenesis and subsequent disease outcome in ruminant species. In this study, we assessed the ability of sheep and cattle oro-nasal turbinate cells to sense and respond to C. abortus infection. The cells expressed toll like receptor (TLR) 2, TLR4, nucleotide oligomerization domain (NOD) 1 and NOD-like receptor pyrin domain containing 3 (NLRP3) mRNA. In response to C. abortus infection, both ovine and bovine turbinate cells produce CXCL8 mRNA and protein late in the bacterial developmental cycle, but do not produce IL-1ß or TNF-α. The UV-inactivated bacteria did not elicit a CXCL8 response, suggesting that intracellular multiplication of the bacteria is important for activating the signalling pathways. The production of innate immune cytokines from cattle and sheep turbinate cells in response to C. abortus infection was found to be largely similar.