Fibroblast growth factor 2 induces differentiation and apoptosis of Askin tumour cells

Peripheral primitive neuroectodermal tumour (PNET)/Ewing's sarcoma (ES) and neuroblastoma (NB) are related tumours of neural crest origin with primitive neural characteristics. Fibroblast growth factor 2 (FGF2) is a critical signalling molecule for primitive neural crest cells. The treatment of NB cells with FGF2 variably affects biological characteristics such as growth and differentiation, while in PNET/ES, FGF2 predominantly induces apoptosis. The JK-GMS Askin tumour cell line can be induced to differentiate upon treatment with nerve growth factor (NGF), indicating the integrity of the cellular machinery necessary for differentiation. The present study assesses whether FGF2 can induce differentiation in JK-GMS cells. JK-GMS cells expressed high-affinity FGF receptors (FGFRs), and treatment with FGF2 induced phosphorylation of FGFR1 together with activation of extracellular signal-regulated kinases (ERK1/ERK2) and c-Jun N-terminal kinase (JNK). Subsequent biological effects were growth inhibition, neuronal differentiation, and apoptosis, and these changes were associated with increased expression of neurofilaments, reduction of c-myc and bcl-2 expression, and activation of caspase 3. Treatment of the cells with a specific inhibitor of the MAPK/extracellular signal-regulated kinase (MEK)-1, PD98059, predominantly inhibited the effects of FGF2 on growth, differentiation, and apoptosis, while an inhibitor of JNK reduced apoptosis, indicating that the ERK1/2 and JNK pathways are critical components of FGF2-mediated effects in JK-GMS cells. Additional comparative analyses of FGF2-mediated effects in two ES cell lines (CADO-ES, RD-ES) and a PNET cell line (SK-N-MC) showed pronounced differentiation in SK-N-MC, but not in CADO-ES or RD-ES cells. This study demonstrates that FGF2 can induce neuronal differentiation of PNET including Askin tumour. These findings clearly indicate that the FGF2-mediated signalling pathway plays a critical role in controlling the major properties of PNET cells and may provide a potential therapeutic target for PNET.     

Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves

Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4(+) T cells with a memory phenotype (CD45R0(+)) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8(+) T cells proliferated in response to antigens until 18 months p.i. gammadelta T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1(+) CD2(-) and a few WC1(-) CD2(+) gammadelta T cells expressed CD25 at time zero. By 18 months, however, subsets of gammadelta T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3(-) non-T non-B null cells, CD2(+) and CD2(-), proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.     

Norepinephrine transporter (NET) promoter and 5'-UTR polymorphisms: association analysis in panic disorder

Several biochemical and pharmacological studies suggest that the catecholaminergic system involving the norepinephrine transporter (NET) is relevant for the pathogenesis of panic disorder. Three single nucleotide polymorphisms in the promoter or untranslated 5' region of the NET gene were investigated by means of RFLP analysis in a sample of 115 German patients with panic disorder and 115 matched controls. Statistical analysis failed to show association with the overall diagnosis of panic disorder. In the subgroup of patients with panic disorder without agoraphobia, however, two polymorphisms were found to be associated with the disease (G/C (rs2397771): p < 0.05; T/C (rs2242446): p < 0.01). While our data do not support a major function of the NET gene in the development of panic disorder, it may play a role in the subgroup of panic disorder without agoraphobia.     

Loss of heterozygosity on chromosomes 3p,8p,9p and 17p in the progression of squamous cell carcinoma of the larynx

Previous molecular genetic studies of laryngeal squamous cell carcinoma (SCC)have shown certain chromosomal regions with recurring alterations. But studies of sequential molecular alterations and genetic progression model of laryngeal SCC have not been clearly defined. To identify the chromosomal alterations associated with the carcinogenesis of laryngeal SCC, we analyzed genomic DNA from microdissected squamous metaplasia, squamous dysplasia, invasive SCC, and metastatic carcinoma samples from 22 laryngeal SCC patients for loss of heterozygosity (LOH) at microsatellite loci. Ten microsatellite markers on chromosome 3p, 8p, 9p, and 17p were used. LOH at 9p21 was observed in the all stages including squamous metaplasia, squamous dysplasia, invasive SCC and metastatic carcinoma. LOH at 17p13.1, 3p25 and 3p14.2 was observed from the squamous dysplasia, invasive SCC and metastatic carcinoma. LOH at 8p21.3-p22 was observed mainly from the invasive SCC and metastatic carcinoma. The results suggest that 9p21 in the early event, 17p13.1, 3p25 and 3p14.2 in the intermediate event and 8p21.3- p22 in the late event may be involved in the laryngeal carcinogenesis.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Neointimal formation in the porcine aortic organ culture. I. Cellular dynamics over 1 month

Since intimal smooth muscle cells (SMC) are an important feature of atherosclerotic fibrofatty plaques, we tested the hypothesis that endothelial cells (EC) regulate SMC proliferation in the process of neointimal formation. Using a porcine thoracic aortic organ culture (OC) system, we previously showed in a preliminary study that short-term porcine aortic explants incubated in 5% fetal bovine serum for 7 days promote neointimal formation only in the presence of endothelium or in conditioned media collected from proliferating OC with endothelium present. We now report that these organ cultures can be maintained in culture for up to 4 weeks. The surface cells stained positively for dil-acetylated-low-density lipoprotein throughout the 4-week period indicating the continued presence of EC while the neointimal cells stained positively for the smooth muscle actin-specific monoclonal antibodies alpha-SM1 and HHF-35 indicating their smooth muscle nature. The number of EC did not change during the 4-week incubation period, however, EC turnover peaked at 5 days and remained elevated but constant throughout. The number of intimal SMC doubled between day 0 (18.4 +/- 0.2 cells/field) and day 7 (41.5 +/- 0.9) and between day 7 and day 14 (75.8 +/- 10.1). The intimal SMC number then stabilized thereafter (day 21: 79.5 +/- 7.8, day 28: 73.8 +/- 12.1). Cell proliferation played a large part since autoradiography studies using nondenuded OC showed that intimal SMC thymidine index on day 0 was 1.4% +/- 0.4, peaked at the end of day 5 (25.7% +/- 4.1) and gradually decreased thereafter. The peak in the intimal SMC thymidine index occurred during a period of high EC turnover (maximum EC thymidine index on day 5: 21.8% +/- 2.1), and the stabilization of intimal SMC number observed beyond 2 weeks of incubation occurred when EC replication was reduced (day 14: 6.7% +/- 0.21). Incubation of organ cultures denuded of their endothelium in 5% fetal bovine serum showed a marked decrease in neointimal formation. However, denuded OC when incubated in conditioned medium collected from 4-day-old nondenuded OC (4DCM) enhanced neointimal formation of denuded OC. In contrast, the neointima of denuded OC incubated in 24-day-old nondenuded OC conditioned medium was similar to that of controls. The thymidine index of intimal SMC of denuded OC treated with 4DCM was markedly higher than that found with the control treatment at all time points.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.     

Infected infradiaphragmatic retroperitoneal extralobar pulmonary sequestration: a case report

Infradiaphragmatic extralobar pulmonary sequestration is an extremely rare congenital malformation. It is more frequently diagnosed in the antenatal period due to routine ultrasonic examination of the fetus or in the first 6 months of life, though on rare occasions it is discovered incidentally in adults. A 32-yr-old man presenting with epigastric discomfort and fever was referred. Computed tomographic scanning showed that a 16-cm, multiseptated, dumbbell-shaped, huge cystic tumor was located beneath the diaphragm. On the next day, 850 mL of thick yellowish pus was drained by sonography-guided fine needle aspiration for the purpose of infection control and diagnosis, but no microscopic organisms were found in repeated culture studies. Surgical removal of the cyst was performed through thoracoabdominal incision and most of these pathologic lesions were removed but we could not find the feeding arteries or any fistulous tract to surrounding structures. Histopathologic study revealed that it was extralobar pulmonary sequestration and culture study showed that many WBC and necrotic materials were found but there were no microorganisms in the cystic contents. We report the first case of an infected infradiaphragmatic retroperitoneal extralobar sequestration which was administered a staged management and achieved an excellent clinical course.     

Prognostic Significance of p27, Ki-67, and Topoisomerase lla Expression in Clinically Nonfunctioning Pancreatic Endocrine Tumors

Nonfunctioning islet cell tumors or pancreatic endocrine tumors are the most common type of malignant islet cell tumor. Although previously detected usually at an advanced stage because of mass effect, the early detection rate of small localized disease has been increasing. To date it has been difficult to predict the clinical behavior in localized regional nonfunctioning tumors. To investigate potential markers predicting malignancy and poor prognosis in nonfunctioning pancreatic endocrine tumors, we analyzed the expression of Ki-67, topoisomerase IIα (Topollα), and p27, as well as a variety of clinicopathologic parameters in 76 cases of nonfunctioning islet cell tumors (23 benign cases and 53 malignant cases). Ki-67, Topollα, and p27 labeling indices were significantly different between benign and malignant tumors. Expression of Ki-67, Topollα, and p27 were associated with survival in patients with a malignant tumor in a univariate setting. However, only p27 and Topollα were jointly associated with survival in multivariate analysis. Immunohistochemical staining for p27, Topollα, and Ki-67 can be helpful in the diagnosis of nonfunctioning pancreatic endocrine tumor. Analysis of p27 and Topollα may also have potential utility as prognostic factors for malignant tumors.