Phthalide derivative CD21 regulates the platelet- neutrophil extracellular trap-thrombin axis and protects against ischemic brain injury in rodents.

Prothrombotic and proinflammatory properties of neutrophil extracellular traps (NETs) contribute to brain damage after ischemic stroke. CD21 is a novel phthalide neuroprotectant against cerebral ischemia in rodents. This study investigated effects of CD21 on the platelet-NET-thrombin axis and ischemic brain injury and the underlying mechanism. CD21 exerteddose-dependent neuroprotectionin rats that were subjected to2 h middle cerebral artery occlusion,dose-dependentlyinhibited adenosine diphosphate-mediatedplatelet aggregationin rats, and dose-dependentlyexertedanti-thrombotic activityin rodents that received a collagen-epinephrine combination, ferric chloride, or an arteriovenous shunt. Equimolar CD21 doses exerted stronger efficacy than 3-N-butylphthalide (NBP, natural phthalide for the treatment of ischemic stroke). CD21 dose-dependently improved regional cerebral blood flow, neurobehavioral deficits, and infarct volume in mice that were subjected to photothrombotic stroke (PTS). CD21 (13.79 mg/kg, i.v.) significantly decreased NET components (plasma dsDNA concentrations; mRNA levels of elastase, myeloperoxidase, and neutrophil gelatinase-associated lipocalin and protein level of citrullinated histone H3 in ischemic brain tissues), mRNA and protein levels of peptidyl-arginine deiminase 4 (PDA4, NET formation enzyme), and mRNA levels of NET-related inflammatory mediators (interleukin-1ß, interleukin-17A, matrix metalloproteinase 8, and matrix metalloproteinase 9) in ischemic brain tissues, despite no effect on mRNA levels of deoxyribonuclease I (NET elimination enzyme). Pretreatment with compound C (inhibitor of adenosine monophosphate-activated protein kinase [AMPK]) significantly reversed the inhibitory effects of CD21 on NETs, PDA4, and inflammatory mediators in PTS mice. These results suggest that CD21 might regulate the platelet-NET-thrombin axis and protect against ischemic brain injury partly through the induction of AMPK activation.

Clonal dynamics of tumor-infiltrating T-cell receptor beta-chain repertoires in the peripheral blood in response to concurrent chemoradiotherapy for Epstein-Barr virus-associated nasopharyngeal carcinoma.

The nasopharyngeal epithelium is highly susceptible to pathogenic infection. More than 95% of nasopharyngeal carcinomas (NPCs) are Epstein-Barr virus (EBV)-associated epithelial cancers densely infiltrated with EBV-free lymphocytes. It remains unknown whether the immune modulating effects of concurrent chemoradiotherapy (CCRT) on the tumor-infiltrating T-cell priming against EBV, tumor-associated antigens, and/or neoantigens can elicit systemic anti-tumor immunity and decrease recurrence or distant metastasis. Using matched EBV-associated NPCs, nasopharyngeal mucosal tissues, and longitudinal serial peripheral blood samples, we explored the spatiotemporal and quantitative changes in expansion and contraction of intratumoral T-cell clonotypes (ITCs) in peripheral blood samples from before, during, and after CCRT. The pre-treatment nasopharyngeal ITC repertoire contained unique mucosa-resident and commonly system-shared T-cell receptors (TCRs), portraying an individualized tumor-associated and/or metagenomic landscape. We found that the long-term disease-free patients had significantly more robust unique mucosa-resident ITCs that migrated into and expanded in the peripheral blood after CCRT than in the patients with recurrence or distant metastasis (Mann-Whitney U test, p = .0110). However, the system-shared productive ITC TCRs specific to the common viruses, such as EBV, cytomegalovirus, and influenzaA, in all the patients with and without recurrence demonstrated almost no expansion after CCRT. Thus, these findings underline the importance of determining the impact of unique intratumoral immune responses, reflected in the peripheral blood, on disease prognosis after treatment and challenge of mechanistically understanding the common systemic immune evasion of EBV in NPC patients.

The Effects of Neoadjuvant Treatment on the Tumor Microenvironment in Rectal Cancer: Implications for Immune Activation and Therapy Response.

BACKGROUND: Because more than one neoadjuvant treatment is available for advanced rectal cancer, the aim of this study was to compare the differential clinical and pathologic effects of different combinations of chemoradiation regimens, treatment sequencing, and timing to surgery on patient outcomes. PATIENTS AND METHODS: Between January 2015 and October 2018, 126 newly diagnosed patients with rectal cancer with magnetic resonance imaging-based cT3-4 or N+ rectal disease for curative-intent treatment received 1 of 4 neoadjuvant regimens, followed by immediate surgery or delayed surgery. Whole post-neoadjuvant surgical specimens were assessed by 3-dimensional digital whole-tumor microarray imaging and immunostaining in pathology to analyze the global tumor pathologic regression grades, residual tumor distribution patterns, the extent of lymphovascular permeation, lymph node positivity, and the overall density of lymphocyte infiltration in the tumor microenvironment. These factors were further examined to identify possible correlations with clinical outcomes. RESULTS: Among the 4 neoadjuvant treatment groups, including 2 conventional regimens, we found a significant increase of stromal CD3+ and CD8+ immune infiltrates in the postneoadjuvant tumor microenvironment in the 3 groups with delayed surgery after different chemoradiation regimens compared with the group with immediate surgery after a short course of RT alone. Independent of neoadjuvant chemoradiation regimens, the post-induction high-intermediate-low stromal-infiltrating CD8+ T-cell densities corresponded to tumor regression grades, distant metastasis rates, and disease-free survival and were prognostic factors for the further stratification of patients with American Joint Committee on Cancer stage III rectal cancer into different risk groups after surgery. CONCLUSION: The effectiveness of induction strategies on tumor remission and disease recurrence in advanced rectal cancer was significantly correlated with an enhanced cytotoxic immune response in the tumor microenvironment.

Sensory nerve-deficient microenvironment impairs tooth homeostasis by inducing apoptosis of dental pulp stem cells.

OBJECTIVES: The aim of this study is to investigate the role of sensory nerve in tooth homeostasis and its effect on mesenchymal stromal/stem cells (MSCs) in dental pulp. MATERIALS AND METHODS: We established the rat denervated incisor models to identify the morphological and histological changes of tooth. The groups were as follows: IANx (inferior alveolar nerve section), SCGx (superior cervical ganglion removal), IANx + SCGx and Sham group. The biological behaviour of dental pulp stromal/stem cells (DPSCs) was evaluated. Finally, we applied activin B to DPSCs from sensory nerve-deficient microenvironment to analyse the changes of proliferation and apoptosis. RESULTS: Incisor of IANx and IANx + SCGx groups exhibited obvious disorganized tooth structure, while SCGx group only showed slight decrease of dentin thickness, implying sensory nerve, not sympathetic nerve, contributes to the tooth homeostasis. Moreover, we found sensory nerve injury led to disfunction of DPSCs via activin B/SMAD2/3 signalling in vitro. Supplementing activin B promoted proliferation and reduced apoptosis of DPSCs in sensory nerve-deficient microenvironment. CONCLUSIONS: This research first demonstrates that sensory nerve-deficient microenvironment impairs tooth haemostasis by inducing apoptosis of DPSCs via activin B/SMAD2/3 signalling. Our study provides the evidence for the crucial role of sensory nerve in tooth homeostasis.

Calycosin Influences the Metabolism of Five Probe Drugs in Rats.

BACKGROUND: Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases. AIM: The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism. METHODS: Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group (T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P<0.001). CONCLUSION: Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment.

The Role of Promyelocytic Leukemia Protein in Steatosis-Associated Hepatic Tumors Related to Chronic Hepatitis B virus Infection.

The persistence of hepatitis B surface antigen (HBsAg) is a risk factor for the development of steatosis-associated tumors in chronic hepatitis B virus (HBV) infection, yet little is known about the metabolic link with this factor. We correlated HBV-related pathogenesis in genetically engineered mice and human carriers with metabolic proteomics and lipogenic gene expression profiles. The immunohistochemistry showed that the promyelocytic leukemia protein (PML, a tumor suppressor involved in genome maintenance and fatty acid oxidation), being inversely influenced by the dynamic HBsAg levels from acute phase to seroclearance, appeared as a lipo-metabolic switch linking HBsAg-induced steatosis (lipogenesis) to HBsAg-lost fat-burning hepatocarcinogenesis (lipolysis). Knockdown of PML in HBsAg-transgenic mice predisposed to obesity and drove early steatosis-specific liver tumorigenesis. Proteome analysis revealed that the signaling pathways corresponding to energy metabolism and its regulators were frequently altered by suppression or depletion of PML in the HBsAg-transgenic mice, mainly including oxidative phosphorylation and fatty acid metabolism. Expression profiling further identified upregulation of stearoyl-CoA desaturase 1 (Scd1) and epigenetic methylation of NDUFA13 in the mitochondrial respiratory chain and the cell cycle inhibitor CDKN1c in concordance to the increased severity of lipodystrophy and neoplasia in the livers of HBsAg-transgenic mice with PML insufficiency. The defect in lipolysis in PML-deficient HBsAg-transgenic mice made the HBsAg-induced adipose-like liver tumors vulnerable to synthetic lethality from toxic saturated fat accumulation with a Scd1 inhibitor. Our findings provide mechanistic insights into the evolution of steatosis-associated hepatic tumors driven by reciprocal interactions of HBsAg and PML, and a potential utility of lipid metabolic reprogramming as a treatment target.

Spatiotemporal homogeneity and distinctness of the T-cell receptor ß-chain repertoires in Epstein-Barr virus-associated primary and metastatic nasopharyngeal carcinomas.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated lymphoepithelioma. The aim of this study was to characterize the homogeneity and distinctness of the T-cell repertoires within and between primary and metastatic NPCs. We used ultra-deep sequencing of the hypervariably rearranged antigen-binding CDR3 regions of T-cell receptor beta (TCRbeta ) to comprehensively profile the T-cell repertoires in NPC patients receiving definitive chemoradiotherapy with long-term follow-up. We observed not only various spatially heterogeneous patient-specific TCRbeta clone compositions that changed with time but also several commonly enriched TCRbeta subclones that were constantly shared between primary NPCs in the head and neck regions, locally recurrent tumors after treatment and later-developed distant metastatic tumors in the liver, lung and bone. Comparison of the overlap frequency of the T-cell clonality between TCRbeta repertoires enabled us to calculate the pairwise genetic distance between primary NPCs of different patients and different sites of metastatic or recurrent NPCs. The constructed NPC phylogeny clearly differentiated the low-risk patients without relapse from the high-risk patients with distant metastasis after chemoradiotherapy. In contrast to the rather low frequency of nonsilent somatic mutations in NPC cells, the degrees of similarity and divergence of NPC-infiltrating lymphocyte TCRbeta repertoires among different patients showed prognostication. Moreover, the persistent presence of commonly NPC-shared in-frame TCRbeta CDR3 gene sequences spatiotemporally identified in the NPC-infiltrating lymphocytes within varied EBV-positive NPCs and their metastases suggest the existence of frequently shared epitopes of neoantigens virally or nonvirally displayed on cancer cells, thereby providing opportunities for the development of precisely tumor-targeted immunotherapy for distant metastasis.

Generation of induced pluripotent stem cells from a patient with spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by a trinucleotide repeat (CAG) expansion in the coding region of ATXN3 gene resulting in production of ataxin-3 with an elongated polyglutamine tract. Here, we generated induced pluripotent stem cells (iPSCs) from the peripheral blood mononuclear cells of a male patient with SCA3 by using the Sendai-virus delivery system. The resulting iPSCs had a normal karyotype, retained the disease-causing ATXN3 mutation, expressed pluripotent markers and could differentiate into the three germ layers. Potentially, the iPSCs could be a useful tool for the investigation of disease mechanisms of SCA3.

Dual oncogenic and tumor suppressor roles of the promyelocytic leukemia gene in hepatocarcinogenesis associated with hepatitis B virus surface antigen.

Proteasome-mediated degradation of promyelocytic leukemia tumor suppressor (PML) is upregulated in many viral infections and cancers. We previously showed that PML knockdown promotes early-onset hepatocellular carcinoma (HCC) in hepatitis B virus surface antigen (HBsAg)-transgenic mice. Here we report the effects of PML restoration on late-onset HBsAg-induced HCC. We compared protein expression patterns, genetic mutations and the effects of pharmacologically targeting PML in wild-type, PML-/-, PML+/+HBsAgtg/o and PML-/-HBsAgtg/o mice. PML-/- mice exhibited somatic mutations in DNA repair genes and developed severe steatosis and proliferative disorders, but not HCC. PML-/-HBsAgtg/o mice exhibited early mutations in cancer driver genes and developed hyperplasia, fatty livers and indolent adipose-like HCC. In PML+/+HBsAg-transgenic mice, HBsAg expression declined over time, and HBsAg-associated PML suppression was concomitantly relieved. Nevertheless, these mice accumulated mutations in genes contributing to oxidative stress pathways and developed aggressive late-onset angiogenic trabecular HCC. PML inhibition using non-toxic doses of arsenic trioxide selectively killed long-term HBsAg-affected liver cells in PML+/+HBsAgtg/o mice with falling HBsAg and rising PML levels, but not normal liver cells or early-onset HCC cells in PML-/-HBsAgtg/0 mice. These findings suggest dual roles for PML as a tumor-suppressor lost in early-onset HBsAg-induced hepatocarcinogenesis and as an oncogenic promoter in late-onset HBsAg-related HCC progression.

Amniotic fluid stem cells with low γ-interferon response showed behavioral improvement in Parkinsonism rat model.

Amniotic fluid stem cells (AFSCs) are multipotent stem cells that may be used in transplantation medicine. In this study, AFSCs established from amniocentesis were characterized on the basis of surface marker expression and differentiation potential. To further investigate the properties of AFSCs for translational applications, we examined the cell surface expression of human leukocyte antigens (HLA) of these cells and estimated the therapeutic effect of AFSCs in parkinsonian rats. The expression profiles of HLA-II and transcription factors were compared between AFSCs and bone marrow-derived mesenchymal stem cells (BMMSCs) following treatment with γ-IFN. We found that stimulation of AFSCs with γ-IFN prompted only a slight increase in the expression of HLA-Ia and HLA-E, and the rare HLA-II expression could also be observed in most AFSCs samples. Consequently, the expression of CIITA and RFX5 was weakly induced by γ-IFN stimulation of AFSCs compared to that of BMMSCs. In the transplantation test, Sprague Dawley rats with 6-hydroxydopamine lesioning of the substantia nigra were used as a parkinsonian-animal model. Following the negative γ-IFN response AFSCs injection, apomorphine-induced rotation was reduced by 75% in AFSCs engrafted parkinsonian rats but was increased by 53% in the control group after 12-weeks post-transplantation. The implanted AFSCs were viable, and were able to migrate into the brain's circuitry and express specific proteins of dopamine neurons, such as tyrosine hydroxylase and dopamine transporter. In conclusion, the relative insensitivity AFSCs to γ-IFN implies that AFSCs might have immune-tolerance in γ-IFN inflammatory conditions. Furthermore, the effective improvement of AFSCs transplantation for apomorphine-induced rotation paves the way for the clinical application in parkinsonian therapy.

Promyelocytic leukaemia protein links DNA damage response and repair to hepatitis B virus-related hepatocarcinogenesis.

DNA damage response and repair pathways are important barriers to carcinogenesis. Here, we show that promyelocytic leukaemia (PML, also known as TRIM19), involved in sensing DNA damage and executing homologous recombination repair, is down-regulated in non-tumour liver cells surrounding hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). No PML mutation or deletion was found in HBV-infected liver or HCC cells. Immunohistochemical analysis of liver biopsies from patients with breast or liver cancer and HBV reactivation after chemotherapy revealed PML up-regulation and HBV exacerbation in normal liver tissue in response to DNA damage (functional PML), PML down-regulation in HCC peritumour cells associated with high HBsAg accumulation and low HBV replication activity (suppressive PML), and heterogeneous nuclear PML expression in HCC cells that lost HBV DNA and HBsAg and were non-reactive to DNA damage (dysregulated PML). Loss of PML in HBsAg-transgenic mice promoted chromosome breaks in liver cells and accelerated the accumulation of body and liver fat and the development of a liver steatosis-dysplasia-adenoma-carcinoma sequence in an inflammation-independent and male-predominant manner, compared to PML knock-out or HBsAg-transgenic mice during the same time period. These results indicate that PML deficiency facilitates genomic instability and promotes HBsAg-related hepatocarcinogenesis, which also involves androgen and lipid metabolism. These findings uncover a novel PML link between HBV-related tumourigenesis, DNA repair, and metabolism.

Establishment of immortalized mesenchymal stromal cells with red fluorescence protein expression for in vivo transplantation and tracing in the rat model with traumatic brain injury.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSC) play a crucial role in tissue engineering and regenerative medicine, and have important clinical potential for cell therapy. However, many hMSC studies have been restricted by limited cell numbers and difficult detection in vivo. To expand the lifespan, hMSC are usually immortalized by virus-mediated gene transfer. However, these genetically modified cells easily lose critical phenotypes and stable genotypes because of insertional mutagenesis. METHODS: We used a non-viral transfection method to establish human telomerase reverse transcriptase-immortalized cord blood hMSC (hTERT-cbMSC). We also established red fluorescent protein (RFP)-expressing hTERT-cbMSC (hTERT/RFP-cbMSC) by the same non-viral transfection method, and these cells were injected into a rat model with traumatic brain injury for in vivo detection analysis. RESULTS: The hTERT-cbMSC could grow more than 200 population doublings with a stable doubling time and maintained differentiation capacities. hTERT/RFP-cbMSC could proliferate efficiently within 2 weeks at the injury location and could be detected easily under a fluorescent microscope. Importantly, both hTERT-cbMSC and hTERT/RFP-cbMSC showed no chromosomal abnormalities by karyotype analysis and no tumor formation in severe combined immunodeficient (SCID) mice by transplantation assay. CONCLUSIONS: We have developed immortalized cbMSC with hTERT expression and RFP expression, which will be useful tools for stem cell research and translational study.

Liver receptor homolog-1 localization in the nuclear body is regulated by sumoylation and cAMP signaling in rat granulosa cells.

Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the nuclear receptor superfamily, mainly expressed in endoderm-derived tissues and in the ovary. In ovarian granulosa and luteal cells, LRH-1 regulates the expression of genes associated with ovarian steroidogenesis. LRH-1 can be transported to transcriptionally inactive nuclear bodies after conjugation with small ubiquitin-related modifier (SUMO). In the present study, we investigated the effects of SUMO modification at five lysine residues of LRH-1 in rat granulosa cells. Lysine 289 could be conjugated with SUMO-1 in vitro, and the mutation K289R increased transcriptional activity of LRH-1, suggesting that SUMO conjugation is associated with transcription repression. Coexpression of SUMO-1 targets LRH-1 to the dot-like nuclear bodies, but the effect of lysine mutations on blocking subnuclear localization depended on the cell type. In COS-7 cells, mutation of either K173 or K289 prevented SUMO-1-mediated translocation of LRH-1 into nuclear bodies and also reduced the conjugation by SUMO-1, suggesting that K289 and K173 are two important sites involved in SUMO-1 modification. In granulosa cells, three or more altered lysine residues were required for nucleoplasm retention. This result suggests that multiple lysine residues are targets for SUMO conjugation in vivo and granulosa cells are more sensitive to SUMO-1-mediated LRH-1 localization to nuclear bodies. Nuclear body localization of LRH-1 was suppressed by forskolin and cholera toxin. Forskolin treatment obviously influences the expression of members involved in the SUMO pathway. The results obtained in the present study suggest that cAMP signaling could change the dynamic process of sumoylation and repress LRH-1 targeting to nuclear speckles in rat granulosa cells.

[Culture of porcine mesenchymal stem cells in vitro and developmental capacity of reconstructed embryos by nuclear transfer].

The developmental potential of reconstructed embryos varied according to the source of donor cells, it was thought that the donor cells capabilities to be reprogrammed were different. We established the method of culturing porcine bone marrow mesenchymal stem cells (pMSCs), identified and observed the growth characteristics of pMSCs, and determined pMSCs reprogramming potential as donor cells for nuclear transfer (SCNT). We found that the method of gradient centrifugation to isolate pMSCs from porcine bone marrow was better than the method of anchoring culture; the number of pMSCs achieved peak at day 6, the adhesive rate of cultured cells was 78.50% at 10h and the division index of cultured cells was 24.00 per thousand at day 4. The developmental competence were compared among three kinds of embryos, reconstructed embryos with PF and pMSCs, Parthenogenetic. The blastocysts rate and total cell number of blastocysts were 15.07%, 14.63% vs 30.91% and 24.1 +/- 6.5, 30.67 +/- 17.7 vs 25.8 +/- 11.4. These results indicated that pMSCs could be high proliferation and stable growth characters in vitro, and were suitable donor cells type for nuclear transfer.

Generation of natural killer cells from serum-free, expanded human umbilical cord blood CD34+ cells.

Natural killer (NK) cells are important effectors of the innate immune system, which exhibits cytolytic activity against infectious agents and tumor cells. NK cells are derived from CD34(+) hematopoietic stem cells (HSCs). Human umbilical cord blood (UCB) has been recognized as a rich source of HSCs. Previously, we have reported an optimized serum-free medium for ex vivo expansion of CD34(+) cells from UCB. In this study, the serum-free, expanded CD34(+) cells were tested to differentiate into NK cells and their induction kinetics. After 5 weeks of induction, the induced NK cells were characterized by analysis of surface antigens, IFN-gamma secretion, and cytotoxicity against K562 cells. The results indicated that NK cells derived from the serum-free, expanded CD34(+) cells exhibited both characteristics and functions of NK cells. Furthermore, the serum-free, expanded CD34(+) cells showed a significantly higher NK cell differentiation potential than freshly isolated CD34(+) cells. NK cells induced from serum-free, expanded CD34(+) cells showed a higher concentration of IFN-gamma secretion and ability of cytotoxicity than those from freshly isolated CD34(+) cells. Therefore, ex vivo-expanded CD34(+) cells in optimized serum-free medium could differentiate into NK cells and provided a promising cell source for immunotherapeutic approaches.

Nasopharyngeal biopsy imprint cytology: a retrospective analysis of 191 cases.

Patients with nasopharyngeal carcinoma (NPC) are common in Taiwan. To provide efficient management to patients, the surgeons often perform cytological imprints immediately after biopsies of lesions suspicious for NPC. The results of cytological assessment of imprints usually are reported within 30 min after biopsies. The patients with positive cytological results can then be arranged for further examinations during the same visit. We reviewed 191 imprints and corresponding biopsies from 187 patients during 1997-2004 at Koo Foundation Sun Yat-Sen Cancer Center, Taipei. The cytological diagnoses were categorized into four groups: negative (62 cases), suspicious (8 cases), positive (116 cases), and inadequate specimen (5 cases). There were 18 false-negative and 1 false-positive diagnoses. All suspicious cases were positive histologically. Our results showed a sensitivity of 87.2% and a specificity of 97.8%. The accuracy was 89.8%. Therefore, nasopharyngeal imprint cytology is a sensitive and specific method for rapid diagnosis of nasopharyngeal cancer at an outpatient setting.

Effects of different intensities of extremely low frequency pulsed electromagnetic fields on formation of osteoclast-like cells.

Over the past 30 years, the beneficial therapeutic effects of selected low energy, time varying electromagnetic fields (EMF) have been documented with increasing frequency to treat therapeutically resistant problems of the musculoskeletal system. However, the underlying mechanisms at a cellular level are still not completely understood. In this study, the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on osteoclastogenesis, cultured from murine bone marrow cells and stimulated by 1,25(OH)(2)D(3), were examined. Primary bone marrow cells were cultured from mature Wistar rats and exposed to ELF-PEMF stimulation daily for 7 days with different intensities of induced electric field (4.8, 8.7, and 12.2 micro V/cm rms) and stimulation times (0.5, 2, and 8 h/day). Recruitment and authentication of osteoclast-like cells were evaluated, respectively, by determining multinuclear, tartrate resistant acid phosphatase (TRAP) positive cells on day 8 of culture and by the pit formation assay. During the experiments, cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and prostaglandin-E(2) (PGE(2)) were assayed using the enzyme linked immunosorbent assay (ELISA). These findings suggest that ELF-PEMF can both enhance (approximately 50%) and suppress (approximately 27%) the formation of osteoclast-like cells in bone marrow culture, depending on the induced electric field intensity. In addition, consistent correlations were observed between TNF-alpha, IL-1beta, and osteoclast-like cell number after exposure to different induced electric field intensities of ELF-PEMF. This in vitro study could be considered as groundwork for in vivo ELF-PEMF clinical applications on some osteoclast-associated bone diseases.