Biomedical subjects

Zhuo Chen

Publications and source records attributed to Zhuo Chen.

At least 37 records · Page 2Linked to original sources

PubMed2005

JNK and ERK MAP kinases mediate induction of IL-1beta, TNF-alpha and IL-8 following hyperosmolar stress in human limbal epithelial cells.

Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL) -1beta, tumor necrosis factor (TNF) alpha, and the C-X-C chemokine IL-8. Primary human limbal epithelial cultures in normal osmolar media (312 mOsM) were exposed to media with higher osmolarity (400-500 mOsM) by adding 50-90 mM NaCl, with or without SB202190, an inhibitor of c-Jun N-terminal kinases (JNK) pathway, PD 98059, an inhibitor of extracellular-regulated kinase (ERK) pathway, dexamethasone or doxycycline for different lengths of time. The conditioned media were collected after 24 hr of treatment for ELISA. Total RNA was extracted from cultures treated for 6 hr for semi-quantitative RT-PCR. Cells treated for 15-60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-ERK. The concentrations of IL-1beta, TNF-alpha and IL-8 proteins in 24 hr conditioned media of limbal epithelial cells progressively increased as the media osmolarity increased from 312 to 500 mOsM. Active p-JNK-1/p-JNK-2 and p-ERK-1/p-ERK-2 were detected by Western blot and peaked at 60 min in cells exposed to hyperosmolar media. The levels of p-JNK-1/p-JNK-2 and p-ERK1/p-ERK2 were positively correlated with the medium osmolarity. SB202190, PD98059 and doxycycline markedly suppressed the levels of p-JNK-1/p-JNK-2 and/or p-ERK1/p-ERK2, as well as IL-1beta, TNF-alpha and IL-8 mRNAs and proteins stimulated by hyperosmolar media. These findings provide direct evidence that hyperosmolarity induces inflammation in human limbal epithelial cells by increasing expression and production of pro-inflammatory cytokines and chemokines, a process that appears to be mediated through activation of the JNK and ERK MAPK signalling pathways. The efficacy of doxycycline in treating ocular surface diseases may be due to its ability to suppress JNK and ERK signalling activation and inflammatory mediator production in the limbal epithelium.

Adult↗

PubMed2005

Controllable interconnection of single-walled carbon nanotubes under ac electric field.

We demonstrated the controllable interconnection of single-walled carbon nanotubes (SWNTs) under alternating current (ac) electric field. The interconnected carbon nanotubes were found to be parallel with the electric flux and increased abruptly with deposition time following a self-accelerating process. Theoretical simulation indicates that the alignment and the interconnection of carbon nanotubes were induced by the dielectrophoresis force and the electric field redistribution at the nanotube apexes.

Letter↗

PubMed2005

Paroxetine is effective in desensitizing 5-HT1A receptor function in adult offspring exposed prenatally to cocaine.

RATIONALE: Desensitization of postsynaptic 5-HT(1A) receptors may be responsible for the therapeutic effectiveness of serotonin selective uptake inhibitors (SSRIs). As prenatal cocaine exposure produces long-term deficits in 5-HT neurons in offspring, it may alter the ability of postsynaptic 5-HT(1A) receptors to be desensitized by chronic paroxetine. OBJECTIVES: The aim of the study is to determine (1) prenatal cocaine-induced changes in 5-HT(1A) receptor function and (2) the effectiveness of chronic treatment with paroxetine to produce 5-HT(1A) receptor desensitization in adult offspring exposed to cocaine in utero. METHODS: Pregnant rats received saline or (-)cocaine (15 mg/kg, s.c.) twice daily from gestational days 13 through 20. Adult male offspring from each of prenatal groups were treated with saline or paroxetine (10 mg/kg/day; i.p.) for 14 days. Eighteen hours post-treatment, rats were challenged with saline or the 5-HT(1A) receptor agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.04 or 0.2 mg/kg, s.c.). Plasma oxytocin, adrenocorticotropic hormone (ACTH), corticosterone, renin and prolactin were determined. RESULTS: Prenatal cocaine exposure did not alter 5-HT(1A) receptor-mediated neuroendocrine responses. Paroxetine treatment desensitized 5-HT(1A) receptor-mediated increases in oxytocin, ACTH and corticosterone to a comparable extent in all offspring and reduced the E(max) for ACTH only in prenatal cocaine-exposed offspring. Cortical [(3)H]-8-OH-DPAT- or [(3)H]-WAY100635-labeled 5-HT(1A) receptors were unaltered by prenatal cocaine or subsequent paroxetine treatment. CONCLUSIONS: Postsynaptic 5-HT(1A) receptor function is unaltered by prenatal cocaine exposure and paroxetine can effectively desensitize 5-HT(1A) receptor function in adult cocaine-exposed offspring. These data suggest that paroxetine may be clinically effective in treating mood disorders in adults exposed in utero to cocaine.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2005

Electric-field-enhanced assembly of single-walled carbon nanotubes on a solid surface.

We report a novel electric-field-enhanced chemical assembly approach for fabricating highly aligned SWNT arrays on a solid surface with remarkably improved efficiency and packing density, which is very important for the real applications of carbon nanotube arrays. With the enhancement of the electric field, the assembling kinetics of SWNTs is remarkably speeded up to effectively decrease the assembling time, and the packing density can even exceed the saturated density of conventional assembly method by four times within only half an hour. The molecular dynamics simulation results illustrated the alignment of SWNTs with their long axes along the electric flux in solution, leading to the increase of packing density and efficiency through overcoming the steric hindrance of the "giant" carbon nanotubes.

Journal Article↗

PubMed2005

Thermochemical hole burning on a triethylammonium bis-7,7,8,8-tetracyanoquinodimethane charge-transfer complex using single-walled carbon nanotube scanning tunneling microscopy tips.

The present article describes a thermochemical hole burning (THB) effect on a charge-transfer complex triethylammonium bis-7,7,8,8-tetracyanoquinodimethane (TEA(TCNQ)(2)) using single-walled carbon nanotube (SWNT) scanning tunneling microscopy (STM) tips, which demonstrates the possibility of optimizing the THB storage materials and the writing tips for ultrahigh-density data storage. TEA(TCNQ)(2) is proven to be a high-performance THB storage material, which gives deeper holes and larger hole depth-to-diameter ratio as compared to the previous materials dipropylammonium bis-7,7,8,8-tetracyanoquinodimethane and N-methyl-N-ethylmorpholinium bis-7,7,8,8-tetracyanoquinodimethane. Instead of conventional Pt/Ir STM tips, SWNT tips made by a unique chemical assembly technique we developed have been shown to be excellent writing tips for greatly decreasing the hole sizes and increasing the storage density. Possible reasons for the improvements on the storage performance were discussed.

Journal Article↗

PubMed2005

Fluoxetine treatment of prepubescent rats produces a selective functional reduction in the 5-HT2A receptor-mediated stimulation of oxytocin.

Various childhood mood disorders are being treated with serotonin selective reuptake inhibitors (SSRIs) such as fluoxetine (Prozac(R)), yet limited data are available on their effects on serotonergic systems prior to maturation. This study investigated the effects of chronic fluoxetine treatment on 5-HT2A serotonin receptor-mediated neuroendocrine responses in young male rats. Prepubescent male rats were treated with saline or fluoxetine (10 mg/kg/day, i.p.) for 14 days, a treatment regimen producing maximal changes in postsynaptic 5-HT2A function in adults. Eighteen hours post-treatment, the rats received saline or increasing doses (0.5, 2.0, or 5.0 mg/kg, i.p.) of the 5-HT2 receptor agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl ((+/-)-DOI). Trunk blood was obtained to determine changes in oxytocin, ACTH, corticosterone, and renin responses. Fluoxetine produced a small ( approximately 6%) but significant reduction in body weight gain, but no changes were observed in basal hormone levels. In both saline- and fluoxetine-treated rats, (+/-)-DOI increased plasma oxytocin levels in a dose-dependent manner. However, the magnitude of the oxytocin responses to all doses of (+/-)-DOI were markedly attenuated ( approximately 50%) in the fluoxetine-treated rats, indicating a functional reduction in the E(max) of 5-HT(2A) receptor-mediated oxytocin responses. In contrast, fluoxetine did not alter the (+/-)-DOI-induced increases in plasma ACTH, corticosterone, or renin. These data provide the first demonstration of selective neuroadaptive responses in 55-HT2A serotonin receptor function due to prepubescent treatment with fluoxetine. These data may be clinically relevant with respect to the use of selective serotonin reuptake inhibitors in children and adolescents.

Adrenocorticotropic Hormone↗

PubMed2005

Spermicidal effect of Jieze No. 1 in combination with nonoxynol-9 in vitro.

Spermicidal effect of Jieze No. 1 (JZ1) in combination with nonoxynol-9 (N-9) was examined in vitro. The minimum spermicidal concentration of JZ1 decoction, N-9 and their mixture solution in 20 s and 3 min were examined by improved spermicidal test of Sander-cramer in vitro. The percentages of progressively moving spermatozoa, moving spermatozoa and viable spermatozoa were also observed 20 s, 3 min and 30 min after the addition of the liquid medicine. Our results showed that sperms did not recover their activities in a revival test when the minimum spermicidal concentration of either JZ1 decoction, or N-9, or the mixed solution of the two agents, was used. N-9 (JZ1 in the mixed group) showed significant differences in the percentages of progressively moving spermatozoa, moving spermatozoa, and visible spermatozoa in 20 s, 3 min, and 30 min, when compared with N-9 alone (P < 0.01). We are led to conclude that JZ1 decoction can improve N-9 spermicidal action in vitro, and when used in combination with N-9, it has synergic effect.

Adult↗

PubMed2005

Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness.

The concept that corneal epithelium stem cells reside in limbus has been recognized for more than a decade, but isolation of these stem cells has not been accomplished. This study was an initial attempt to isolate a population of human limbal epithelial cells enriched for certain putative stem cell properties based on their phenotype. Epithelial cells harvested from fresh human limbal rings and their primary cultures were allowed to adhere to collagen IV-coated dishes for 20 min and 2 hr, sequentially. The rapidly adherent cells (RAC), slowly adherent cells and non-adherent cells were evaluated for certain stem cell properties: (a) BrdU-label retention, (b) expression of basal cell (integrin beta1, p63, ABCG2) and differentiation (involucrin, keratin 12) markers, and (c) colony forming efficiency (CFE) and growth capacity on a 3T3 fibroblast feeder layer. Among unfractionated cells and the three selected populations, the RAC, accounting for about 10% of whole population, were enriched 5-fold in BrdU label-retaining cells, displayed the highest number of integrin beta1 and p63 positive and involucrin negative cells, expressed high levels of DeltaNp63 and ABCG2 mRNA, and lacked involucrin and K12 expression, and possessed the greatest CFE and growth capacity. These findings demonstrated for the first time that human limbal epithelial cells with stem cell properties can be partially enriched by their adhesiveness to collagen IV. The RAC population enriched for certain putative stem cell properties may prove useful in the future for transplantation to diseased and damaged corneas with limbal stem cell deficiency.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2005

Effect of experimental dry eye on tear sodium concentration in the mouse.

PURPOSE: To examine the changes in tear volume, sodium concentration, and osmolarity in BALB/c and C57BL/6J mice in response to experimental dry eye (EDE). METHODS: Tear volumes were determined at baseline and after 2 days of EDE using phenol red-impregnated cotton thread. Tear fluid was collected by instilling 1 microl of distilled water into the conjunctival sac and collecting the diluted tear fluid in a 0.5-microL glass capillary tube. CoroNa Red in dimethyl sulfoxide DMSO was added, and the fluorescent intensity was measured, which was compared to a standard curve of sodium concentrations. The resulting concentration was then corrected by the proper dilution factor. The tear osmolarity owing to sodium was calculated by comparing the calculated tear sodium concentration to a standard curve established by a vapor pressure osmometer. Serum osmolarity was also determined using a vapor pressure osmometer. RESULTS: After 2 days of EDE, tear volume significantly decreased from 0.093 microL to 0.028 microL in BALB/c mice and from 0.066 microL to 0.026 microL in C57BL/6J mice. There was a concomitant significant increase in tear osmolarity from 177 mOsm/L to 300 mOsm/L in C57BL/6J mice. Tear osmolarity nearly doubled from 285 mOsm/L to 559 mOsm/L in BALB/C mice with EDE, approaching statistical significance (P=0.12). No change in the serum osmolarity was observed in either mouse strain. CONCLUSIONS: These experiments show the ability of these new techniques for determining tear volume and estimating tear osmolarity in mice. By mimicking the findings of human dry eye disease, these findings validate the relevance of the mouse model for studying the pathogenesis of human keratoconjunctivitis sicca.

Animals↗

PubMed2005

Correlation of human papillomavirus type 16 and human papillomavirus type 18 e7 messenger RNA levels with degree of cervical dysplasia.

Infection with certain types of human papillomavirus (HPV) is a necessary event in the development of cervical carcinoma; however, not all women who become infected with HPV will progress to cancer. Much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation. Little is known about the additional factors needed to drive the process. Quantitative real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, low-grade squamous intraepithelial lesions (LSIL), and high-grade squamous intraepithelial lesions (HSIL). Prevalence of mRNA transcripts was lower among normal women (27%) than for women with LSIL (40%) and HSIL (37%). Mean levels ranged from 2.0 (ln scale per 20 ng cDNA) among normal women to 4.2 among those with HSIL, with a significant trend (P=0.008). This trend was only significant for HPV 18 transcripts if separately analyzed by HPV type. The transcriptional activity of HPV 18 is higher than that of HPV 16 and increases with increasing level of dysplasia. This is in concert with the findings of other studies, and reinforces the notion that HPV 18 is a more aggressive viral type. Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from a cervical swab, thus providing a way to better screen women who may progress to higher grade lesions or invasive carcinoma from those who will spontaneously regress.

Adult↗

PubMed2005

ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells.

ABCG2, a member of the ATP binding cassette (ABC) transporters, has been identified as a molecular determinant for bone marrow stem cells and proposed as a universal marker for stem cells. This study investigates ABCG2 expression and its potential as a marker that identifies human limbal epithelial stem cells. ABCG2 expression was evaluated by immunofluorescent and immunohistochemical staining, laser scanning confocal microscopy, flow cytometry, and semiquantitative reverse transcription-polymerase chain reaction. Cells selected from primary limbal epithelial cultures by flow cytometry with ABCG2 monoclonal antibody (mAb) or Hoechst 33342 dye staining were evaluated for their gene expression and colony-forming efficiency (CFE). ABCG2 protein was mainly located in the basal cells of limbal epithelia but not in the limbal suprabasal and corneal epithelia. ABCG2 staining was also observed in primary limbal epithelial cultures. Limbal epithelia express higher levels of ABCG2 and DeltaNp63 mRNAs than corneal epithelia. Labeling with ABCG2 mAb yielded 2.5%-3.0% positive cells by flow cytometry. The ABCG2-positive cells exhibited greater CFE on a 3T3 fibroblast feeder layer than ABCG2-negative cells. A side population (SP) was detected by the Hoechst 33342 exclusion assay. SP cells displayed stronger expression of ABCG2 and DeltaNp63 mRNA and greater CFE than the non-SP cells. In conclusion, these findings demonstrate that ABCG2 transporter was exclusively expressed by limbal basal cells and that the ABCG2-positive and SP cells possess enriched stem cell properties, suggesting for the first time that ABCG2 could serve as a marker to identify the putative limbal epithelial stem cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2005

[Identification of human corneal epithelial stem cells].

OBJECTIVE: To investigate the molecular markers of corneal epithelial stem cells. METHODS: The anatomy structure of corneal limbus was analyzed by histological examination. Fluorescence microscopy and laser scan confocal microscopy (LSCM) were used to evaluate the expression of un differentiation markers as p63, ABCG2, alpha6, alpha9 and beta1 integrin, alpha-enolase, EGFR, cytokeratin 19 (CK19) and CD71 in cornea-limbus sections and whole mount cornea tissue using immunohistochemistry staining. Semiquantitative RT-PCR and in situ hybridization (ISH) were used to evaluate gene expression of corneal and limbal epithelia. RESULTS: The limbal area in horizontal cut direction showed that limbal epithelial cells were as papilloma-form to correspond to the Palisades of Vogt environment. The expressions of beta1 integrin, K19, alpha-enolase and EGFR were much stronger in limbal basal cells than superficial cells. Un differentiation markers p63, ABCG2, alpha9 integrin were detected only in the basal limbal epithelial cells. LSCM and RT-PCR results showed that the protein and mRNA of p63, ABCG2 and alpha9 integrin were only detected in limbal epithelia. ISH showed that p63 mRNA only expressed in the limbal basal epithelial layer. CONCLUSIONS: The corneal limbal epithelial area is papilloma-form. Limbal stem cells have complex markers as p63 expressing in the nuclei, ABCG2 in the cytoplasm and alpha9 integrin expressing in the membrane. With these markers complex, it is easy to distinguish limbal stem cells from other epithelial cells in cornea.

Biomarkers↗

PubMed2004

Chronic mild stress induces behavioral and physiological changes, and may alter serotonin 1A receptor function, in male and cycling female rats.

RATIONALE: Interactions among stress, serotonin 1A (5-HT(1A)) receptors, and the hypothalamic-pituitary-adrenocortical (HPA) system have been proposed to influence the development of depression in humans. The investigation of depression-relevant behaviors and physiological responses to environmental stressors in animal models of depression may provide valuable insight regarding these mechanisms. OBJECTIVES: The purpose of these experiments was to investigate the interactions among central 5-HT(1A) receptors, endocrine function, and behavior in an animal model of depression, chronic mild stress (CMS). METHODS: The current study examined behavioral responses to a pleasurable stimulus (sucrose), estrous cycle length (in female rats), and plasma hormone levels following systemic administration of a selective 5-HT(1A) receptor agonist [(+)8-hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT); 40 mug/kg, s.c.; administered 15 min prior to sacrifice], in male and female rats exposed to 4 weeks of CMS. RESULTS: Four weeks of CMS produced a reduction in the intake of 1% sucrose (anhedonia), as well as attenuated adrenocorticotropic hormone (ACTH) responses to 8-OH-DPAT in both male and female rats (22 and 18% lower than the control groups, respectively). Corticosterone and oxytocin responses to 8-OH-DPAT were not altered by exposure to CMS. In female rats, CMS induced a lengthening of the estrous cycle by approximately 40%. CONCLUSIONS: CMS produces minor HPA disruptions along with behavioral disruptions. Alterations in 5-HT(1A) receptor function in specific populations of neurons in the central nervous system may be associated with the CMS model. The current findings contribute to our understanding of the relations that stress and neuroendocrine function have to depressive disorders.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2004

Simultaneously targeting epidermal growth factor receptor tyrosine kinase and cyclooxygenase-2, an efficient approach to inhibition of squamous cell carcinoma of the head and neck.

PURPOSE: Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (Cox-2) contribute to development of squamous cell carcinoma of the head and neck (SCCHN). Simultaneously blocking both EGFR and Cox-2-mediated pathways may be an efficient means of inhibiting cancer cell growth in SCCHN. EXPERIMENTAL DESIGN: A combination of EGFR-selective tyrosine kinase inhibitors (TKIs) AG1478 or ZD1839 (Iressa or gefitinib) with a Cox-2 inhibitor (Cox-2I) celecoxib (Celebrex) was studied for its effects on cell growth, cell cycle progression, and apoptosis in SCCHN cell lines by cell growth assay, clonogenic assay, flow cytometric analysis, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. A potential effect of EGFR TKIs and Cox-2I on angiogenesis was examined by endothelial capillary tube formation assay. Primary and secondary targets of EGFR TKIs and Cox-2I were also examined using immunoblotting and immunoprecipitation after the combined treatment. RESULTS: The combination of AG1478 or ZD1839 with celecoxib either additively or synergistically inhibited growth of the five SCCHN cell lines examined, significantly induced G(1) arrest and apoptosis, and suppressed capillary formation of endothelium. Furthermore, the combination showed strong reductions of p-EGFR, p-extracellular signal-regulated kinase 1/2, and p-Akt in SCCHN cells as compared with the single agents. Both AG1478 and ZD1839 inhibited expression of Cox-2 protein, whereas celecoxib mainly blocked the production of prostaglandin E(2). CONCLUSIONS: These results suggest that cell growth inhibition induced by a combination of EGFR TKIs and Cox-2I is mediated through simultaneously blocking EGFR and Cox-2 pathways. This combination holds a great potential for the treatment and/or prevention of SCCHN.

Angiogenesis Inhibitors↗

PubMed2004

Expression pattern of chemokine receptor 6 (CCR6) and CCR7 in squamous cell carcinoma of the head and neck identifies a novel metastatic phenotype.

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes predictably to cervical lymph nodes, with low rates of distant metastases. Tumor cells can express various receptors that facilitate such metastatic spread to lymph nodes and other nonlymphoid organs. Chemokine receptors (CCR), normally expressed on lymphocytes, control immune and inflammatory cell migration, providing a link between innate and adaptive immunity. Chemokine receptor expression was evaluated in SCCHN, using paired primary and metastatic tumors cell lines, and paired primary and metastatic biopsies from the same patients. Quantitative reverse transcription-PCR showed a consistent pattern of CCR6 down-regulation and up-regulation of CCR7 in metastatic cells and tissues. Chemotaxis assays, ligand-induced receptor down-regulation, and specific antibody blocking experiments supported the quantitative reverse transcription-PCR results, indicating that these surface receptors were functional on metastatic tumor cells. Cells derived from a highly metastatic mouse model of SCCHN were used to confirm CCR7 up-regulation in tumor cells with higher metastatic potential. CCR6 down-regulation is consistent with its decreased expression in cells emigrating from peripheral mucosal sites, whereas CCR7, important for homing of immune cells to secondary lymphoid organs, was significantly up-regulated. Thus, CCR6, CCR7, and their ligands, normally important in controlling immune cell trafficking in response to inflammatory stimuli, may have an important role in determining the metastasis of SCCHN cells in vivo.

Carcinoma, Squamous Cell↗

PubMed2004

Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures.

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.

Adolescent↗

PubMed2004

TGF-beta1 stimulates production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) by human corneal epithelial cells.

Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of ocular surface diseases. This study investigated the regulated expression of gelatinases (MMP-2 and -9), collagenases (MMP-1 and -13) and stromelysins (MMP-3, -10, and -11) by TGF-beta1 in cultured human corneal epithelial cells. Primary human corneal epithelial cell cultures were grown to confluence and treated with different concentrations (0.1, 1.0, 10 ng ml(-1)) of TGF-beta1 in serum-free medium for 6-24 hr. Total RNA was isolated from cultured cells with or without TGF-beta1 treatment for 6 hr and subjected to semi-quantitative RT-PCR and Northern hybridization. Conditioned media were collected from cultures with or without TGF-beta1 treatment for 24 hr to evaluate the MMP production by ELISA and activity assays. Semi-quantitative RT-PCR revealed that the expressions of MMP-9, -1, -13, -3, -10 and -11 mRNA were up-regulated by TGF-beta1 in a concentration-dependent fashion, while MMP-2 and MMP-14 production did not change. Northern hybridization confirmed these findings. Gelatin zymography, MMP ELISA and activity assays showed concentration-dependent stimulated production and activity of MMP-9, -1, -13, -3 and -10 protein in the conditioned media of cultures treated for 24 hr with TGF-beta1. In conclusion, our results demonstrate that TGF-beta1 stimulates the expression and production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) in human corneal epithelial cells. These findings suggest that TGF-beta1 may play a role in the pathogenesis of MMP mediated ocular surface diseases, such as sterile corneal ulceration.

Cells, Cultured↗

PubMed2004

Prenatal cocaine exposure potentiates paroxetine-induced desensitization of 5-HT2A receptor function in adult male rat offspring.

This study investigated the ability of prenatal exposure to cocaine to alter serotonin(2A) (5-HT(2A)) receptor function and paroxetine-induced desensitization of 5-HT(2A) receptor function in rat offspring. Following exposure to saline or (-)cocaine (15 mg/kg, s.c., b.i.d.), during gestational days 13-20, adult male offspring were treated with either saline or paroxetine (10 mg/kg/day, i.p. 14 days). Eighteen hours post-treatment, changes in the stimulation of oxytocin, adrenocorticotropic hormone (ACTH) and corticosterone by (-)4-iodo-2,5-dimethoxyphenylisopropylamine (DOI, 0.5 or 2.0 mg/kg, s.c.) and in 5-HT(2A) receptor densities were determined. Prenatal cocaine exposure did not alter 5-HT(2A) receptor-mediated neuroendocrine responses or 5-HT(2A) receptor densities. In contrast, paroxetine treatment reduced cortical 5-HT(2A) receptors (18-25%) and desensitized 5-HT(2A) receptor-mediated oxytocin responses in both offspring groups. Furthermore, in cocaine offspring, paroxetine produced an inhibition of 5-HT(2A) receptor-mediated increase in plasma ACTH levels and a greater attenuation of the oxytocin responses to (-)DOI. Paroxetine-induced reductions in body weight gain (-8.8%) were comparable in both offspring groups. These data, demonstrating that prenatal exposure to cocaine potentiates paroxetine-induced desensitization of 5-HT(2A) receptor function, may be clinically relevant with respect to treating mood disorders in adults exposed in utero to cocaine.

Adrenocorticotropic Hormone↗