B-1 cells, which constitute a predominant lymphocyte subset in serosal cavities and produce most of natural antibodies, are subdivided into the CD5+ B-1a and CD5- B-1b cell subpopulations, but the differential roles of B-1a and B-1b cells are not well understood. We report that B-1a cells preferentially migrate out of the peritoneal cavity and upregulate the expression of CXCR4 with heightened sensitivity to CXCL12 and CXCL13 upon LPS treatment compared to B-1b and B-2 cells. Whereas B-1a cells were slightly more abundant than B-1b and B-2 cells in the homeostatic condition, the number of B-1a cells preferentially decreased 48 hr after LPS treatment. The decrease in the peritoneal B-1a cell number was accompanied with increased migration of B-1a cells toward CXCL-12 and CXCL-13 in in vitro transmigration assay using peritoneal B cells from LPS treated mice. The expression level of CXCR4, but not of CXCR5, was also more prominently increased in B-1a cells upon LPS stimulation. LPS-stimulated B-1a cells did not accumulate in omental milky spots in contrast to B-2 cells. These results suggest that B-1a cells actively migrate out of the peritoneal cavity through the regulation of the migratory responsiveness to chemokines and actively participate in systemic immune responses.
B Lymphocyte Subsets; Chemokine CXCL12; Chemokine CXCL13; Chemotaxis; Receptor, CXCR4; Lipopolysaccharides
The microbiota plays a critical, weight-promoting role in diet-induced obesity (DIO), but the pathways that cause the microbiota to induce weight gain are unknown. We report that mice deficient in lymphotoxin (LT), a key molecule in gut immunity, were resistant to DIO. Ltbr−/− mice differed in microbial community composition compared to their heterozygous littermates, including an overgrowth of segmented filamentous bacteria (SFB). Furthermore, cecal transplantation conferred leanness to germ-free recipients. Housing Ltbr−/− mice with their obese siblings rescued weight gain, demonstrating the communicability of the obese phenotype. Ltbr−/− animals lacked interleukin 23 (IL-23) and IL-22 that can regulate SFB. Mice deficient in these pathways also resisted DIO, demonstrating that intact mucosal immunity guides diet-induced changes to the microbiota to enable obesity.
The purpose of this study was to investigate whether selective cyclooxygenase (COX) inhibitors promote paclitaxel-induced apoptosis in taxane-resistant ovarian cancer cells by suppressing MDR1/P-glycoprotein (P-gp) expression.
Taxane-resistant ovarian cancer cells were cultured with paclitaxel alone or combined with a selective COX inhibitors. The expression patterns of MDR1/P-gp and the ability of COX inhibitors to inhibit growth of taxane-resistant ovarian cancer cells were measured. The efficacy of prostaglandin E2 (PGE2) supplementation was measured to evaluate the mechanisms involved in suppressing MDR1 gene expression.
P-gp was upregulated in taxane-resistant ovarian cancer cells compared to paired paclitaxel-sensitive ovarian cancer cells. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that selective COX inhibitors significantly enhanced the cytotoxic effects of paclitaxel in taxane-resistant ovarian cancer cells via a prostaglandin-independent mechanism. These increased apoptotic effects were further verified by measuring an increased percentage of cells in sub-G1 stage using flow cytometry. Selective COX inhibitors suppressed MDR1 and P-gp expression. Moreover, combined treatment with paclitaxel and selective COX inhibitors increased poly (ADP-ribose) polymerase (PARP) cleavage in taxane-resistant ovarian cancer cells.
Selective COX inhibitors significantly promote paclitaxel-induced cell death in taxane-resistant ovarian cancer cells in a prostaglandin-independent manner. COX inhibitors could be potent therapeutic tools to promote paclitaxel sensitization of taxane-resistant ovarian cancers by suppressing MDR1/P-gp, which is responsible for the efflux of chemotherapeutic agents.
Chemosensitizer; Cyclooxygenase inhibitor; Ovarian cancer; Paclitaxel; P-glycoprotein
Pulmonary artery (PA) aneurysm is a rare finding in the thoracic cavity, accompanied by pulmonary hypertension. Clinical presentation of PA aneurysms is usually asymptomatic. The guideline for PA aneurysm treatment is unclear. We report an unusual case of low pressure PA aneurysm associated with atrial septal defect in a 69-year-old man.
Low pressure; Pulmonary artery; Aneurysm; Atrial septal defect
We have designed a five-year multicentre prospective cohort study in women who are both human papillomavirus (HPV)-positive with either atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) of cervix. This study aimed to analyze the risk of developing a high-grade squamous intraepithelial lesion (HSIL) from either ASCUS or LSIL in HPV-positive women, so called 'progression' rate, to investigate differences in the progression rates according to HPV type-specific infection, and to evaluate the various factors associated with the persistence or clearance of HPV infection in the Korean population. At present, the study protocol composed of cervical cytology, HPV DNA testing, and questionnaire have been conducted actively since the first participant was enrolled in 2010. This study is the first nationwide Korea HPV cohort study. Our data will provide valuable information about not only the ambiguous cytology results of ASCUS and LSIL but also the effect of the specific HPV type and other various factors on the progression to HSIL. Finally, the results of our study will be helpful and applicable to determine the primary cervical cancer prevention strategies.
Cervical intraepithelial neoplasms; Cohort studies; Human papillomavirus; Uterine cervical neoplasms
The possible beneficial effects of chronic low-dose irradiation (LDR) and its mechanism of action in a variety of pathophysiological processes such as cancer are a subject of intense investigation. While animal studies involving long-term exposure to LDR have yielded encouraging results, the influence of LDR at the cellular level has been less well defined. We reasoned that since natural killer (NK) cells constitute an early responder to exogenous stress, NK cells may reveal sentinel alterations in function upon exposure to LDR. When purified NK cells received LDR at 4.2 mGy/h for a total of 0.2 Gy in vitro, no significant difference in cell viability was observed. Likewise, no functional changes were detected in LDR-exposed NK cells, demonstrating that LDR alone was insufficient to generate changes at the cellular level. Nonetheless, significant augmentation of cytotoxic, but not proliferative, function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation. This enhancement of NK cytotoxicity was not due to alterations in NK-activating receptors, NK1.1, NKG2D, CD69 and 2B4, or changes in the rate of early or late apoptosis. Therefore, LDR, in the presence of suboptimal cytokine levels, can facilitate anti-tumor cytotoxicity of NK cells without influencing cellular proliferation or apoptosis. Whether these results translate to in vivo consequences remains to be seen; however, our data provide initial evidence that exposure to LDR can lead to subtle immune-enhancing effects on NK cells and may explain, in part, the functional basis underlying, diverse beneficial effects seen in the animals chronically exposed to LDR.
Low-dose radiation; natural killer cells; natural cytotoxicity; innate immunity
We tested the efficacy of dual targeting of vascular endothelial growth factor (VEGF) and the alphaVbeta3 integrin in orthotopic mouse models of ovarian cancer.
In the SKOV3ip1 model, both single-agent bevacizumab and etaracizumab reduced tumor growth by 52–63% (p < 0.05), while combined therapy reduced growth by 63–74% compared to either agent alone (p < 0.05). Furthermore, bevacizumab/paclitaxel was superior to paclitaxel alone (weight reduction by 53%, p < 0.05), but etaracizumab/paclitaxel was not. Combining all three agents was more effective than either agent with paclitaxel (p < 0.05). Significantly, both bevacizumab and etaracizumab each sensitized the taxane-resistant SKOV3TRip2 cells to paclitaxel, reducing growth by 56–73% (p < 0.05). Both agents decreased proliferation and microvessel density, and increased apoptosis, alone and in combination with paclitaxel. In the HeyA8 model, there was significantly reduced growth with bevacizumab treatment, but not with etaracizumab, and combination therapy was not superior to bevacizumab alone.
In vivo therapy experiments were conducted in chemo-sensitive (SKOV3ip1, HeyA8) and -resistant (SKOV3TRip2) ovarian cancer models. VEGF was targeted with bevacizumab and alphaVbeta3 with etaracizumab. Mice were treated with each agent alone, together, or in combination with paclitaxel for assessment of tumor growth. Tumor specimens were tested for proliferative index, microvessel density and apoptosis.
Bevacizumab and etaracizumab are more effective in combination than individually in some ovarian cancer models, but not all. Both can sensitize taxane-resistant ovarian cancer cells to paclitaxel, though bevacizumab was superior to etaracizumab in this regard. Further study of this dual anti-angiogenic therapy is warranted.
VEGF; alphaVbeta3; bevacizumab; etaracizumab; ovarian cancer
The cell–microenvironment interaction is critical for cells to perceive environmental cues and co-ordinate signaling cascades to regulate physiological functions. Herein, a soft-lithography technique, micropattern via micromolding in capillaries (MIMIC), is explored to create cell-adhesive micropatterns on glass coverslips. Genetically encoded Src and RhoA fluorescence resonance energy transfer (FRET) biosensors are used to monitor the Src and RhoA activities in nonpatterned cells (stochastically migrating cells, SMCs) and those constrained to grow on micropatterned surfaces (restrictedly migrating cells, RMCs). The results reveal that epidermal growth factor (EGF) induces a decrease of RhoA and an increase of Src activities with biphasic time courses in RMCs. In contrast, the time courses of such activities in SMCs upon EGF stimulation are relatively monophasic. The inhibition of Src activity, actin network, or myosin machinery abolishes the biphasic RhoA response upon EGF stimulation in RMCs. The results indicate that this microenvironment effect on the biphasic RhoA activation in RMCs is mediated by Src and actomyosin machinery. Through the integration of FRET and micropatterning technologies, it is demonstrated that the microenvironment impacts significantly on cell shapes and subsequently the spatiotemporal signaling network of RhoA and Src in living cells. The results help to advance mechanistic understanding of how cells perceive and interpret microenvironments to co-ordinate intracellular molecular signals and ultimately physiological responses.
biosensors; cells; fluorescence resonance; energy transfer; lithography; pattern formation
In the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Our goal is to develop computer methods for aiding in this process. Towards that end, we created the RNAJunction database, which is a repository of RNA junctions, i.e. internal, multi-branch and kissing loops with emanating stem stubs, extracted from the larger RNA structures stored in the PDB database. These junctions can be used as building blocks for nanostructures. Two programs developed in our laboratory, NanoTiler and RNA2D3D, can combine such building blocks with idealized fragments of A-form helices to produce desired 3D nanostructures. Initially, the building blocks are treated as rigid objects and the resulting geometry is tested against the design objectives. Experimental data, however, shows that RNA accommodates its shape to the constraints of larger structural contexts. Therefore we are adding analysis of the flexibility of our building blocks to the full design process. Here we present an example of RNA-based nanostructure design, putting emphasis on the need to characterize the structural flexibility of the building blocks to induce ring closure in the automated exploration. We focus on the use of kissing loops (KL) in nanostructure design, since they have been shown to play an important role in RNA self-assembly. By using an experimentally proven system, the RNA tectosquare, we show that considering the flexibility of the KLs as well as distortions of helical regions may be necessary to achieve a realistic design.
RNA; Nanostructure; Design; Modeling; Flexibility; Molecular dynamics
Platelet-rich plasma (PRP) is a concentrate extract of platelets from autologous blood, and represents a possible treatment option for the stimulation and acceleration of soft-tissue healing and regeneration in orthopedics. Currently, the availability of devices for outpatient preparation and delivery contributes to the increase in the clinical use of PRP therapy in practical setting of orthopedic fields. However, there is still paucity of scientific evidence in the literature to prove efficacy of PRP therapy for the treatment of ligament or tendon problems around the knee joint. Moreover, strong evidence from well-designed clinical trials to support the PRP therapy for osteoarthritis of the knee joint is yet scanty in the literature. Scientific studies need to be performed to assess clinical indications, efficacy, and safety of PRP, and this will require high powered randomized controlled trials. Nonetheless, some hospitals exaggeratedly advertise PRP procedures as the ultimate treatment and a novel technology with abundant scientific evidence for the treatment of knee problems. As a matter of fact, PRP protocols are currently approved only for use in clinical trials and research, and are not allowed for treatment purpose by any institutions in Korea. At present, clinical use of PRP therapy for ligament or tendon problems or osteoarthritis of knee joint is defined as illegal medical practice, regardless of whether it is performed as a sole procedure or as a part of prolotherapy, because the safety and validity are not yet approved by the Ministry of Health and Welfare and Health Insurance Review and Assessment Service. Practicing physicians should remember that injection of PRP to patients by imposing medical charge is still illegal as per the current medical law in Korea.
Platelet-rich plasma; Osteoarthritis; Ligament; Tendon; Knee joint
Src, a nonreceptor tyrosine kinase, is a key mediator for multiple signaling pathways that regulate critical cellular functions and is often aberrantly activated in a number of solid tumors, including ovarian carcinoma. The purpose of this study was to determine the role of activated Src inhibition on tumor growth in an orthotopic murine model of ovarian carcinoma. In vitro studies on HeyA8 and SKOV3ip1 cell lines revealed that Src inhibition by the Src-selective inhibitor, AP23846, occurred within 1 hour and responded in a dose-dependent manner. Furthermore, Src inhibition enhanced the cytotoxicity of docetaxel in both chemosensitive and chemoresistant ovarian cancer cell lines, HeyA8 and HeyA8-MDR, respectively. In vivo, Src inhibition by AP23994, an orally bioavailable analogue of AP23846, significantly decreased tumor burden in HeyA8 (P = 0.02), SKOV3ip1 (P = 0.01), as well as HeyA8-MDR (P < 0.03) relative to the untreated controls. However, the greatest effect on tumor reduction was observed in combination therapy with docetaxel (P < 0.001, P = 0.002, and P = 0.01, for the above models, respectively). Proliferating cell nuclear antigen staining showed that Src inhibition alone (P = 0.02) and in combination with docetaxel (P = 0.007) significantly reduced tumor proliferation. In addition, Src inhibition alone and in combination with docetaxel significantly down-regulated tumoral production of vascular endothelial growth factor and interleukin 8, whereas combination therapy decreased the microvessel density (P = 0.02) and significantly affected vascular permeability (P < 0.05). In summary, Src inhibition with AP23994 has potent antiangiogenic effects and significantly reduces tumor burden in preclinical ovarian cancer models. Thus, Src inhibition may be an attractive therapeutic approach for patients with ovarian carcinoma.
To evaluate the clinicopathologic characteristics and prognostic factors of ovarian granulosa cell tumors.
Medical records of 113 patients presenting between January 1995 and December 2007 were retrospectively reviewed.
One-hundred two patients had adult type disease, with a mean age of 46.2 years (range, 18 to 83 years) and a mean follow-up period of 54.7 months (range, 1 to 155 months). The distribution of FIGO stages was 86 patients at stage I, 11 at stage II, and 5 at stage III. During follow-up, ten patients recurred at a mean time of 48 months (range, 4 to 109 months). Among them, three patients died after a mean of 57 months (range, 25 to 103 months). In recurrence analysis, advanced stage (p=0.032) and presence of residual disease (p=0.012) were statistically significant, and age<40 years, premenopause and positive washing cytology were marginally significant (p<0.1). In multivariate analysis, stage was the only factor associated with recurrence; adjuvant chemotherapy and fertility-sparing surgery were not statistically significant. Among 36 patients with fertility-sparing operations, eight patients had nine pregnancies and delivered seven babies. Eleven patients had juvenile type tumors; the mean age was 20.0 years (range, 8 to 45 years) and the mean follow-up period was 69.8 months (range, 20 to 156 months). The distribution of FIGO stage was nine patients at stage I and two at stage III. There were no recurrences or deaths reported. Four patients had seven pregnancies and delivered six babies.
Stage is the only factor associated with disease-free survival, and fertility-sparing surgery may be a treatment option for women with early-stage disease who want to retain fertility.
Clinical study; Granulosa cell tumor; Ovary; Pregnancy; Recurrence
Whether the metabolic syndrome (MetS) has prognostic value for coronary artery disease (CAD) beyond its individual components is controversial. We compared the relationship between the number of MetS components and CAD severity as assessed by angiography in non-diabetic and diabetic subjects. We consecutively enrolled 527 patients who underwent their first coronary angiography. Patients were divided into four groups according to the number of MetS components: 0/1, 2, 3, and 4/5. A coronary atherosclerosis score was used to quantify the extent of atherosclerotic involvement. The relationship between the MetS score and angiographic CAD severity or clinical presentation was compared between non-diabetic and diabetic subjects. Individuals with the MetS (n = 327) had a higher prevalence of CAD (60% vs 32%, P < 0.001), multi-vessel disease (34% vs 16%, P < 0.001), and acute coronary syndromes (49% vs 26%, P < 0.001) than those without the MetS. In the non-diabetic group, atherosclerosis score increased with the MetS score (1.0 ± 2.1, 2.0 ± 2.9, 2.8 ± 2.9, and 3.6 ± 3.9, P < 0.001) whereas there was no significant difference in the diabetic group (0.5 ± 1.0, 5.2 ± 4.7, 4.2 ± 2.9, and 4.4 ± 3.5, P = 0.102). The MetS score is related to CAD severity in non-diabetic patients but the association between the MetS score and angiographic CAD severity may be obscured in the presence of diabetes.
Metabolic Syndrome; Coronary Atherosclerosis; Coronary Angiography
Polarized molecular activities play important roles in guiding the cell toward persistent and directional migration. In this study, the polarized distributions of the activities of phosphatidylinositol 3-kinase (PI3K) and the Rac1 small GTPase were monitored using chimeric fluorescent proteins (FPs) in cells constrained on micro-patterned strips, with one end connecting to a neighboring cell (junction end) and the other end free of cell-cell contact (free end). The recorded spatiotemporal dynamics of the fluorescent intensity from different cells was scaled into a uniform coordinate system and applied to compute the molecular activity landscapes in space and time. The results revealed different polarization patterns of PI3K and Rac1 activity induced by the growth factor stimulation. The maximal intensity of different FPs, and the edge position and velocity at the free end were further quantified to analyze their correlation and decipher the underlying signaling sequence. The results suggest that the initiation of the edge extension occurred before the activation of PI3K, which led to a stable extension of the free end followed by the Rac1 activation. Therefore, the results support a concerted coordination of sequential signaling events and edge dynamics, underscoring the important roles played by PI3K activity at the free end in regulating the stable lamellipodia extension and cell migration. Meanwhile, the quantification methods and accompanying software developed can provide a convenient and powerful computational analysis platform for the study of spatiotemporal molecular distribution and hierarchy in live cells based on fluorescence images.
Mechanical forces play important roles in the regulation of cellular functions, including polarization, migration and stem cell differentiation. Tremendous advancement in our understanding of mechanotransduction has been achieved with the recent development of imaging technologies and molecular biosensors. In particular, genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) technology have been widely developed and applied in the field of mechanobiology. In this article, we will provide an overview of the recent progress of FRET application in mechanobiology, specifically mechanotransduction. We first introduce fluorescent proteins and FRET technology. We then discuss the mechanotranduction processes in different cells including stem cells, with a special emphasis on the important signalling molecules involved in mechanotransduction. Finally, we discuss methods that can allow the integration of simultaneous FRET imaging and mechanical stimulation to trigger signalling transduction. In summary, FRET technology has provided a powerful tool for the study of mechanotransduction to advance our systematic understanding of the molecular mechanisms by which cells respond to mechanical stimulation.
fluorescence resonance energy transfer; mechanotransduction; stretch; shear stress; substrate stiffness
Regulation of B cell receptor (BCR)-induced Ca2+ signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to α-IgM antibody (0.5~48 hr), the initial transient decrease in BCR-induced [Ca2+]i was followed by spontaneous recovery to control level within 24 hr. The recovery of Ca2+ signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of PLCγ2 and IP3R-3. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced Ca2+ signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of PLCγ2 and IP3R-3. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of Ca2+ signaling. In contrast to immature WEHI-231 cells, identical long-term α-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced [Ca2+]i, regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced Ca2+ signaling.
B cell receptor; Ca2+; CD40; Reactive oxygen species; WEHI-231
To evaluate the occurrence of residual or recurrent disease after conization for adenocarcinoma in situ (AIS) of the uterine cervix.
Medical records of 99 patients with a histologically diagnosis of AIS of the uterine cervix by conization between 1991 and 2008 were reviewed retrospectively.
Seventy eight of 99 patients (78.8%) had negative and 18 (18.2%) had positive resection margins of the conization specimen, and 3 (3.0%) had unknown margin status. Of the 78 patients with negative margins, 45 underwent subsequent hysterectomy and residual AIS were present in 4.4% (2/45) of patients. Ten of the 18 patients with positive margins received subsequent hysterectomy and 3 patients (30%) had residual AIS. Twenty-eight patients had conservative treatment and during the median follow-up time of 23.5 months (range, 7 to 124 months), only one patient (3.6%) had recurrent AIS and was treated with a simple hysterectomy. Eight patients became pregnant after conization, 4 of them delivered healthy babies, one had a spontaneous abortion and 3 were ongoing pregnancies.
Patients with positive resection margins after conization for AIS of the uterine cervix are significantly more likely to have residual disease. However, negative resection margin carries a lower risk for residual AIS, therefore conservative management with careful surveillance seems to be feasible in women who wish to preserve their fertility.
Adenocarcinoma in situ; Uterine cervix cancer; Conization; Conservative treatment
This study was conducted to examine recent trends in ovarian cancer incidence and mortality and secular trends in demographic factors in Korea.
With the data from Korea Central Cancer Registry, International Agency for Research on Cancer, Korean Death Registry, and World Health Organization's Statistical Information System, we calculated age-standardized incidence and mortality rates for ovarian cancer. Also we estimated future incidence of ovarian and cervical cancer using linear regression model. To assess the demographic trend, data from national surveys in Korea or results from published papers were searched.
Ovarian cancer incidence rate was similar to that in women worldwide but lower than those in Western countries, and the trend has been increased steadily. Ovarian cancer-related mortality rates have been increasing in Korea, even though those in western and some Asian countries, such as China, have been decreasing. Age-specific incidence rate and mortality rate showed steep increases with advancing age. The incidence rate of ovarian cancer was estimated to surpass that of uterine cervix cancer in 2015. Korea showed rapid changes in nutritional, reproductive, and anthropometric factors.
These recent trends in ovarian cancer incidence and mortality may be partly attributed to gradual westernizing of life styles and to changes in socio-demographic behavior factors. In particular, the increasing trend in ovarian cancer mortality in Korea may be attributed to a real rise in mortality as well as, in part, a decline in misclassification bias related to an increase in the proportion of deaths confirmed by physician diagnosis.
Ovarian neoplasms; Epidemiology; Incidence; Mortality
The aim of this study is to evaluate the clinical feature and pregnancy outcome in patients with ovarian cancer diagnosed during pregnancy. We retrospectively analyzed the medical records of 27 patients diagnosed with ovarian cancer during pregnancy at Cheil General Hospital & Women's Healthcare Center from January 1996 to December 2006. Mean age of the patients was 29.1 yr (range 23-40), and a mean follow-up period was 57 months (range 7-112 months). Of 27 patients, 15 (55.5%) had borderline malignancies, 7 (25.9%) had epithelial malignancies and 5 (18.6%) had germ cell tumors. A total of 26 patients received a conservative surgery preserving pregnancy. The mean time for surgical intervention during pregnancy was 20 weeks of gestational age. Of the 27 patients, 26 had full term delivery of a healthy baby without any congenital malformation. Only one patient with epithelial ovarian cancer had a relapse at 19 months after the first conservative operation with adjuvant chemotherapy. There were few data for managing patients with ovarian cancer diagnosed during pregnancy. This study results could help establish a guideline for management of ovarian malignancy complicating pregnancy.
Ovarian Neoplasms; Pregnancy; Conservative Surgery
Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly-sensitive Ca2+ biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca2+ oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca2+ oscillation in comparison to stiffer or rigid substrate. This Ca2+ oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca2+ oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca2+ oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity-regulated Ca2+ oscillation, which determines the physiological functions of HMSCs.
FRET; Calcium; RhoA; signal transduction; substrate stiffness; mesenchymal stem cells
The MHC region of the chromosome contains a lot of genes involved in immune responses. Here we have investigated the mouse NG29/Cd320 gene in the centrometrically extended MHC region of chromosome 17.
We cloned the NG29 gene by RT-PCR and confirmed the tissue distribution of its gene expression by northern blot hybridization. We generated the NG29 gene expression constructs and polyclonal antibody against the NG29 protein to perform the immunofluorescence, immunoprecipitation and flow cytometric analysis.
The murine NG29 gene and its human homologue, the CD320/8D6 gene, were similar in the gene structure and tissue expression patterns. We cloned the NG29 gene and confirmed its expression in plasma membrane and intracellular compartments by transfecting its expresssion constructs into HEK 293T cells. The immunoprecipitation studies with rabbit polyclonal antibody raised against the NG29-NusA fusion protein indicated that NG29 protein was a glycoprotein of about 45 kDa size. A flow cytometric analysis also showed the NG29 expression on the surface of Raw 264.7 macrophage cell line.
These findings suggested that NG29 gene in mouse extended MHC class II region was the orthologue of human CD320 gene even though human CD320/8D6 gene was located in non-MHC region, chromosome 19p13.
CD320; NG29; 8D6; MHC; follicular dendritic cell; cell surface antigen
Although nickel hypersensitivity is known as a delayed-type hypersensitivity mediated by nickel-specific T cells, it is greatly influenced by other immune cells. Here we show that splenic natural killer cells (NK cells) directly or indirectly respond to nickel by secretion of IFN-γ. Using enzyme-linked immunosorbent spot (ELISPOT) assays, we found that nickel-reactive cells readily secreted IFN-γ when splenocytes were cultured in the presence of varying concentrations of nickel sulfate (NiSO4) for 24 h. However, nickel-reactive IL-2- or IL- 4-secreting cells were infrequent during the 24-h culture with NiSO4. Immune responses to nickel were innate, not adaptive, in nature since the frequency of nickel-reactive IFN-γ-secreting cells did not increase upon previous exposure to NiSO4 and recombination activating gene (RAG)-1-deficient mice contained nickel-reactive IFN-γ-secreting cells. The involvement of NK cells in the innate response to NiSO4 was confirmed since we could observe a significant reduction of the frequency of nickel-reactive cells in NK cell-depleted mice. Furthermore, the number of IFN-γ secreting cells was significantly reduced in the ELISPOT assays when NKG2D was blocked by anti-NKG2D antibody. These results suggest that there is an early and rapid innate immune response to nickel, which is mediated by NK cells and the NKG2D receptor. The significance of the innate response to nickel is that it may contribute to development of the late T cell-mediated delayed type hypersensitivity against nickel.
hypersensitivity; immunosorbent techniques; interferon-γ; killer cells, natural; nickel; NK cell lectin-like receptor subfamily K
To assess the role of fertility preservation in the treatment of patients with early epithelial ovarian cancer (EOC).
We retrospectively analyzed the medical records of 21 patients with early EOC from January 1995 to December 2006. All eligible patients with a strong desire to preserve fertility were younger than 35 years and underwent fertility-sparing surgery with or without adjuvant chemotherapy.
Twenty-one eligible patients with a median age of 26.7 years (range, 20 to 33 years) were identified, and the mean follow-up period was 43 months (range, 5 to 86 months). Only one patient with stage IC recurred 34 months after the first operation. A total of five patients were able to become pregnant at least once after the first fertility preserving treatment, with or without adjuvant chemotherapy. All five patients succeeded in full-term vaginal delivery with healthy infants. No patients died of their disease.
Fertility preserving treatment in patients with early EOC can be considered as a proper treatment strategy in patients with early EOC, who have the strong desire for fertility preservation.
Fertility preservation; Epithelial ovarian cancer; Staging operation
Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-α, IL-1β, and LPS resulted in significant ROS production and NF-κB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-α levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.
anti-inflammatory agents; asthma; lipoxygenase; macrophages; models, animal; NF-κB; reactive oxygen species
To evaluate the outcomes of controlled ovarian hyperstimulation (COH)-in vitro fertilization (IVF) such as clinical pregnancy rate (CPR), implantation rate (IR) and live birth rate (LBR) for infertile patients with borderline ovarian tumor (BOT) after conservative treatment, 10 IVF cycles in five patients from January 1999 to July 2005 were analyzed. At the time of diagnosis with BOT, the mean age of patients was 30.0 yr (range, 22-40). For 8 cycles out of 10 attempted IVF cycles, except for 2 cancellation cycles, the mean number of oocytes retrieved was 5.6 (range, 2-16) with a mean fertilization rate of 74.4%. The CPR, IR, and LBR were 50.0% (4/8 cycles), 31.6% (6/19) and 50.0% (4/8 cycles) respectively. The mean follow-up period after COH-IVF initiation was 29.6 (range, 14-61) months. A gynecological oncologist followed all patients every 3 months during the first year and every 6 months thereafter. There was no recurrence during the follow-up period. Our results suggest that COH-IVF may be acceptable for infertile patients with BOT, especially in patients with early-stage BOT after conservative treatment.
In Vitro Fertilization; Borderline Ovarian Tumor; Conservative Treatment