Time-lapse observation of the dedifferentiation process in mouse chondrocytes using chondrocyte-specific reporters.

OBJECTIVE: When chondrocytes prepared from cartilage are expanded in monolayer culture, fibroblast-like cells gradually prevail. Although these prevailing fibroblast-like cells are believed to emerge because of the dedifferentiation of chondrocytes, the definite origin of the prevailing fibroblast-like cells has not been determined. We herein examined whether the prevailing non-chondrocytic cells observed after monolayer expansion culture arise from dedifferentiating chondrocytes or are the result of the overgrowth of fibroblasts that are present at the start of the culture. We also evaluated whether chondrocytes dedifferentiate because they proliferate or because they are cultured in monolayers. METHODS: Chondrocytes were prepared from Col11a2-EGFP transgenic mice and Col11a2-Cre; R26-stop(flox)-EYFP transgenic mice, which respectively express enhanced green fluorescent protein (EGFP) and Cre specifically in chondrocytes under the control of Col11a2 promoter/enhancer sequences. Col11a2-Cre; R26-stop(flox)-EYFP mice express enhanced yellow fluorescent protein (EYFP) only in cells which express or used to express Cre. We performed a time-lapse observation of the chondrocytes during monolayer expansion culture, and also observed the chondrocytes after treatment with mitomycin C. RESULTS: A time-lapse observation showed that Col11a2-EGFP chondrocytes underwent cell divisions, lost GFP fluorescence, increased cell numbers, and prevailed during the expansion culture. The observation of the Col11a2-Cre; R26-stop(flox)-EYFP chondrocytes confirmed that most of the cells after expansion in monolayer culture had been chondrocytes. Mitotically inactive chondrocytes generated by treatment with mitomycin C still underwent dedifferentiation, thus suggesting that chondrocyte dedifferentiation is not associated with cell division. CONCLUSION: The non-chondrocytic cells that prevail after the monolayer expansion culture of chondrocytes originate from chondrocytes, and are not generated by the overgrowth of fibroblasts that are present at the start of the culture. Chondrocyte dedifferentiation does not appear to be associated with cell division.

Influence of intramedullary stress on cervical spondylotic myelopathy.

STUDY DESIGN: A cross-sectional analysis. OBJECTIVE: To examine whether intramedullary stress is related to the appearance of symptoms in cervical spondylotic myelopathy (CSM). SETTING: Japan. METHODS: Thirty-three consecutive patients with CSM and 30 consecutive patients without CSM were enrolled. A total of 99 disc levels from C3 to C6 in 33 patients with CSM were divided into two groups: 33 disc levels with high signal intensity (HSI) on T2-weighted magnetic resonance image (HSI group) and 66 disc levels without HSI (Non-HSI group). Ninety disc levels from C3 to C6 in patients without CSM were set up in a control group. Intramedullary stress value at each level was analyzed using the finite element method. Stress was compared among the three groups. A cutoff value of stress to present HSI was investigated from receiver operator characteristics (ROC) curve. RESULTS: In all the patients with CSM, the disc level with HSI presented the highest stress among the three disc levels evaluated. The stress was 3.16 ± 0.86 kPa (mean ± s.d.) in the HSI group, 1.81 ± 0.72 kPa in the Non-HSI group and 1.01 ± 0.37 kPa in the control group. The stress differed significantly among the three groups (P<0.0001). The qualified cutoff value derived from the ROC curve was 2.30 kPa (sensitivity 78.8%, specificity 91.9%). None of the disc levels in the control group exceeded 2.30 kPa. CONCLUSION: HSI was strongly associated with intramedullary stress. Threshold of intramedullary stress to present HSI that related closely to the symptoms of myelopathy was revealed.

Enzastaurin has anti-tumour effects in lung cancers with overexpressed JAK pathway molecules.

BACKGROUND: Enzastaurin, an oral serine-threonine kinase inhibitor, was initially developed as an ATP-competitive selective inhibitor against protein kinase Cß. However, the mechanism by which enzastaurin contributes to tumourigenesis remains unclear. METHODS: We analysed the anti-tumour effects of enzastaurin in 22 lung cancer cell lines to ascertain the potential for enzastaurin-based treatment of lung cancer. To identify molecules or signalling pathways associated with this sensitivity, we conducted a gene, receptor tyrosine kinases phosphorylation and microRNA expression profiling study on the same set of cell lines. RESULTS: We identified eight genes by pathway analysis of molecules having gene-drug sensitivity correlation, and used them to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. Pathway analysis revealed that the JAK/STAT signalling pathway was one of the main ones involved in sensitivity to enzastaurin. Overexpression of JAK1 was observed in the sensitive cells by western blotting. Simultaneous administration of enzastaurin and JAK inhibitor inhibited enzastaurin-induced cell growth-inhibitory effect. Furthermore, lentiviral-mediated JAK1-overexpressing cells were more sensitive to enzastaurin than control cells. CONCLUSION: Our results suggested that the JAK1 pathway may be used as a single predictive biomarker for enzastaurin treatment. The anti-tumour effect of enzastaurin should be evaluated in lung cancer with overexpressed JAK pathway molecules.

Quantitative bioassays for measuring biologically functional gonadotropins based on eel gonadotropic receptors.

Significant declines in eel stocks have been noted in many parts of the world. Because eel aquaculture is dependent on wild-caught juveniles, there is a need to achieve artificial reproduction. Adult eel maturation is currently induced by repeated injections of purified gonadotropin (human chorionic gonadotropin [hCG]) or pituitary extract. Thus the determination of the biological efficacy and quantification of internal levels of gonadotropic hormones is important for optimizing artificial reproduction protocols. To quantify the plasma levels of biologically functional gonadotropic hormones, we developed a bioassay for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) based on the stable expression of receptors in HEK293 cells of the Japanese eel Anguilla japonica LH (ajLHR) and the European eel Anguilla anguilla FSH (aaFSHR), respectively. Such cells also contain a firefly luciferase reporter gene driven by a cAMP-responsive element (CRE-Luc). We found that the obtained stable cells, with ajLHR, responded linearly to a more than 100,000-fold concentration range of hCG diluted in saline. The cells with aaFSHR showed a linear response to a 1000-fold concentration range of salmon pituitary extract mixed with saline. The biological functionality of the LH and FSH bioassays was validated using hCG, human FSH, and pituitary extracts from salmon, carp and eel. Since the toxins in eel plasma damaged the HEK293 cells, the protocol was adapted to selectively inactivate the toxins by heating at 37°C for 24h. This process successfully enabled the monitoring of hormone levels in blood plasma sampled from hCG-injected eels. In this paper, we describe the development of gonadotropin bioassays that will be useful for improving reproduction protocols in eel aquaculture.

Mutations in the human lambda5/14.1 gene result in B cell deficiency and agammaglobulinemia.

B cell precursors transiently express a pre-B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of lambda5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igalpha and Igbeta. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine lambda5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for lambda5/14.1. The maternal allele carried a premature stop codon in the first exon of lambda5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the lambda5/14. 1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant lambda5/14.1. These findings indicate that expression of the functional lambda5/14.1 is critical for B cell development in the human.

An essential role for BLNK in human B cell development.

The signal transduction events that control the progenitor B cell (pro-B cell) to precursor B cell (pre-B cell) transition have not been well delineated. In evaluating patients with absent B cells, a male with a homozygous splice defect in the cytoplasmic adapter protein BLNK (B cell linker protein) was identified. Although this patient had normal numbers of pro-B cells, he had no pre-B cells or mature B cells, indicating that BLNK plays a critical role in orchestrating the pro-B cell to pre-B cell transition. The immune system and overall growth and development were otherwise normal in this patient, suggesting that BLNK function is highly specific.

Heating applicator based on reentrant cavity with optimized local heating characteristics.

PURPOSE: A reentrant-cavity-based applicator can produce a concentrated electric field between reentrant electrodes for localized heating. However, this field is inadequate for treating early small tumors localized in the head and neck. In order to safely heat such well-localized lesions, the electric field distribution should be more localized. MATERIALS AND METHODS: In order to achieve localized heating, four parameters of the reentrant cavity (applicator height, outer diameter, reentrant diameter, and reentrant gap size), which influence the distribution of the electric field produced in the reentrant gap, are optimized using the Taguchi method. The variation in the heating characteristics affected by the size of the heating object is estimated using the signal-to-noise ratio (SNR) index. In this study, the electromagnetic field distributions in a cylindrical phantom and an oblate sphere phantom are analyzed by the three-dimensional finite element method, and the full width at half height (FWHH) of the specific absorption rate (SAR) distribution in the reentrant gap is evaluated. RESULTS: It is shown that the optimized applicator yields both the maximum SNR and minimum mean FWHH, and the sizes of the heating region in the phantom expressed using the averaged FWHH values of the SAR distribution are 60 and 80 mm along the radial and long-axis directions of the applicator, respectively. CONCLUSIONS: A heating region can be robustly and optimally localized by using the Taguchi method and considering the variation in the size of the heating object.

Mutations in Igalpha (CD79a) result in a complete block in B-cell development.

Mutations in Btk, mu heavy chain, or the surrogate light chain account for 85-90% of patients with early onset hypogammaglobulinemia and absent B cells. The nature of the defect in the remaining patients is unknown. We screened 25 such patients for mutations in genes encoding components of the pre-B-cell receptor (pre-BCR) complex. A 2-year-old girl was found to have a homozygous splice defect in Igalpha, a transmembrane protein that forms part of the Igalpha/Igbeta signal-transduction module of the pre-BCR. Studies in mice suggest that the Igbeta component of the pre-BCR influences V-DJ rearrangement before cell-surface expression of mu heavy chain. To determine whether Igalpha plays a similar role, we compared B-cell development in an Igalpha-deficient patient with that seen in a mu heavy chain-deficient patient. By immunofluorescence, both patients had a complete block in B-cell development at the pro-B to pre-B transition; both patients also had an equivalent number and diversity of rearranged V-DJ sequences. These results indicate that mutations in Igalpha can be a cause of agammaglobulinemia. Furthermore, they suggest that Igalpha does not play a critical role in B-cell development until it is expressed, along with mu heavy chain, as part of the pre-BCR.

Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs)--severe combined immunodeficiency (SCID, n = 11), Wiskott-Aldrich syndrome (WAS, n = 11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n = 8)--who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n = 8). Since 1996, the donors have been HLA-matched related donors (MRD) (n = 8), unrelated BM (UR-BM) (n = 7) and unrelated cord blood (UR-CB) (n = 7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

Efficacy of droperidol in the prevention of cisplatin-induced delayed emesis: a double-blind, randomised parallel study.

We conducted a prospective, randomized, double-blind, parallel study comparing the antiemetic activity and tolerability of treatment with droperidol (2.5 mg d.i.v. twice daily for 5 days) and placebo, both combined with granisetron (3 mg d.i.v. on the first day) and dexamethasone (16 mg d.i.v. on the first day, 8 mg d.i.v. on days 2, 3, and 4 mg d.i.v. on days 4, 5). A total of 180 lung cancer patients receiving high-dose cisplatin (80 mg/m(2))-containing chemotherapy were enrolled in the study, and 171 of them were capable of being evaluated. The clinical characteristics of the patients in the two treatment arms were well balanced. Complete protection from nausea and vomiting was recorded in the acute phase in 97% of patients who treated with droperidol versus 98% of patients who given the placebo (P=0.920), and in 42% versus 38% in the delayed phase (P=0.615). The multiple logistic regression analysis showed that a history of motion sickness was a significant risk factor for cisplatin-induced delayed emesis (odds ratio [OR]=5.98; 95% CI=2.15 to 16.7, P=0.0006). Droperidol-containing treatment was well tolerated by most patients, however, the incidence of sleepiness in the droperidol group was higher than in the placebo group (69% versus 30%, P<0.0001). In conclusion, our data did not support the hypothesis that addition of droperidol to granisetron and dexamethasone reduces the delayed emesis induced by high-dose cisplatin.

T cell reconstitution by haploidentical BMT does not restore the diversification of the Ig heavy chain gene in patients with X-linked SCID.

We previously examined the Ig heavy (H) chain gene of pretransplant patients with X-linked SCID (XSCID), having defects in the gene of the IL-2 receptor (R) gamma chain. In the present study, we analyzed two post-transplant XSCID patients, in whom T cell-depleted haploidentical BMT resulted in lymphoid split chimeras, i.e., donor functional T cells coexisting with recipient B cells. Although the recipient B cells produced IgM, no isohemagglutinin or Ag-specific Ab was detected. To investigate the cause of failure to produce Ab in the patients, we sequenced the complementarity determining region 3 (CDR3) and adjacent region of Ig H chain gene, which govern Ab specificity. Among the 64 post-transplant CDR3 junctional sequences, combinatorial and junctional diversity were normal compared with those in age-matched controls. All of the post-transplant joining regions except one clone were equal to germline and the frequency of somatic mutation was significantly lower than that in age-matched controls. The results indicated that T cell reconstitution by BMT does not restore diversification of the Ig gene in the IL-2R gamma chain-deficient B cells, which might be associated with the defect in the Ag-specific Ab production.

Direct fractionation of proteins in particle-containing feedstocks by a filter paper pieces-based DEAE-cellulose column chromatography. Rapid, robust and low-cost capturing procedure for protein.

Filter paper pieces-based (FPB) DEAE-celluloses was prepared for direct fractionation of proteins in particle-containing feedstocks. FPB DEAE-cellulose has a protein binding capacity equivalent to that of commercially available DEAE-cellulose. Crude extracts from porcine intestine and kiwi fruit pulp, which were unmanageable by commercially available chromatographic media due to rapid clothing, could be directly fractionated with FPB DEAE-cellulose column. In addition, effluents from an FPB DEAE-cellulose column were extensively clarified. The present approach can be used as a rapid, robust and low-cost capturing step for protein from particle-containing feedstocks.

[Effectiveness of docetaxel plus cisplatin in large cell lung cancer showing little response to prior chemotherapy with MVP].

The present patient was a 53-year-old male who had large cell lung cancer of c-T4N1M0. We administered multi-drug regimen including mitomycin C, vindesine and cisplatin (CDDP) because of cancer invasion into the great vessels seen on a chest CT. After 3 courses, the cancer showed no change in size. Therefore, we adopted chemotherapy of docetaxel (Taxotere: TXT) and CDDP. After 4 courses, the size of the mass had decreased (partial response). The only major toxic defect was grade 3 neutropenia. A good response to TXT and CDDP could lead to complete resection of lung cancer. It is suggested that TXT is effective in the treatment of large cell lung cancer.

FRS2beta, a potential prognostic gene for non-small cell lung cancer, encodes a feedback inhibitor of EGF receptor family members by ERK binding.

An adaptor protein FRS2beta inhibits epidermal growth factor-receptor (EGFR) tyrosine kinase without being phosphorylated at tyrosine residues after EGF stimulation. Although binding to ERK appears to be important for this inhibition, the precise molecular mechanisms and the role of FRS2beta in signal transduction mediated by other EGFR family members, as well as its role in human cancer, remain unclear. In this study, we demonstrate that FRS2beta inhibits anchorage-independent cell growth induced by oncogenic ErbB2, another member of EGFR family, and that it inhibits heterodimer formation between EGFR and ErbB2. We mapped the residues important for the FRS2beta and ERK interaction to two docking (D) domain-like sequences on FRS2beta and two aspartic acid residues in the common docking (CD) domain of ERK. Moreover, in response to EGF, ERK translocated to the plasma membrane in cells expressing FRS2beta but not an FRS2beta mutant in which four arginine residues in the D domains were replaced with alanines, suggesting that FRS2beta serves as a plasma membrane anchor for activated ERK. Finally, a low mRNA expression level of FRS2beta was significantly correlated with poor prognosis in a cohort of 60 non-small cell lung cancer patients. Therefore, we have identified the molecular mechanisms by which FRS2beta acts as a feedback inhibitor of EGFR family members and suggest a role for FRS2beta as a tumor suppressor.

Reduced Shh expression in TFF2-overexpressing lesions of the gastric fundus under hypochlorhydric conditions.

Expression of sonic hedgehog (Shh), a morphogen for the gastric fundic glands, is reduced in the atrophic mucosa that develops in association with Helicobacter pylori infection, resulting in impaired differentiation of the fundic gland cells, increased expression of trefoil factor family 2 (TFF2) and the formation of spasmolytic polypeptide (SP)-expressing metaplasia (SPEM), a preneoplastic lesion. However, it is still unresolved whether H. pylori-induced inflammation and the resultant reduction in parietal cell number or reduced parietal cell function per se reduces Shh expression. The present study was designed to clarify the expression of Shh and TFF2 in the context of parietal cell dysfunction in the absence of inflammation, using histamine H(2) receptor-knockout (H(2)R-null) mice and an acid exposure model. Age-matched H(2)R-null mice and wild-type (WT) mice were used. The expression of Shh and TFF2 mRNA was quantified by quantitative RT-PCR. Immunohistochemistry was also performed to detect the expression of Shh, TFF2 and cell markers. To study the effects of acid exposure, HCl solution was administered to the animals. The H(2)R-null mice exhibited higher gastric pH, increased TFF2 expression and reduced Shh expression. Impaired mucous neck-to-zymogenic cell differentiation was observed in the H(2)R-null mice. Furthermore, Shh expression increased in the presence of gastric acid and showed a significant correlation with gastric surface pH. In conclusion, our results suggest that persistent parietal cell dysfunction alone (suppressed gastric acid secretion), in the absence of inflammation or parietal cell loss caused by H. pylori infection, may be sufficient to down-regulate Shh expression in TFF2-overexpressing preneoplastic lesions of the gastric fundus. Since exposure to acid restored fundic Shh expression, appropriate gastric acid secretion may play an important role in the morphogen dynamics involved in the maintenance of gastric fundic gland homeostasis.

The effect of zinc supplementation on ghrelin-immunoreactive cells and lipid parameters in gastrointestinal tissue of streptozotocin-induced female diabetic rats.

Zinc is an essential nutrient with a wide range of functions and closely involved in a variety of enzymatic processes of importance in glucose, protein and lipid metabolism. Ghrelin is the endogenous ligand of the G protein coupled growth hormone secretagogue receptor. The regulatory mechanism that explain the biosynthesis and secretion of ghrelin in the gastrointestinal tract has not been clarified. This study was undertaken to examine the effect of zinc supplementation on the streptozotocin (STZ)-induced diabetic rats, which exhibits ghrelin production and secretion, and lipid metabolism on the gastrointestinal tract. The animals were divided into four groups. Group I: Non-diabetic untreated animals. Group II: Zinc-treated non-diabetic rats. Group III: STZ-induced diabetic untreated animals. Group IV: Zinc-treated diabetic animals. Zinc sulfate was given to some of the experimental animals by gavage at a dose of 100 mg/kg body weight every day for 60 days. In the zinc-treated diabetic group, the blood glucose levels decreased and body weight increased as compared to the diabetic untreated group. Zinc supplementation to STZ-diabetic rats revealed the protective effect of zinc on lipids parameters such as total lipid, cholesterol, HDL-cholesterol and atherogenic index. There is no statistically change in ghrelin-immunoreactive cells in gastrointestinal tissue. But, it has found that zinc supplementation caused a significant reduction in densities of ghrelin-producing cells of fundic mucosa of zinc-treated diabetic animals as compared to untreated, non-diabetic controls. Zinc supplementation may contribute to prevent some complications of diabetic rats, biochemically.

Reduction of PTEN protein and loss of epidermal growth factor receptor gene mutation in lung cancer with natural resistance to gefitinib (IRESSA).

Gefitinib (IRESSA), an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, has antitumour activity in the advanced non-small-cell lung cancer (NSCLC) setting. However, in chemotherapy-naïve patients with advanced NSCLC, the addition of gefitinib to standard chemotherapy regimens failed to increase survival. These results suggest the need for improved patient selection and combination rationales for targeted therapies. We have identified subpopulations of an adenocarcinoma cell line that are naturally resistant to gefitinib, and have analysed the cDNA expression profiles, genomic status of EGFR gene and the effect of gefitinib on signalling pathways in these cell lines in order to identify key mechanisms for naturally acquired resistance to gefitinib. Gefitinib-resistant subpopulations demonstrated increased Akt phosphorylation (not inhibited by gefitinib), reduced PTEN protein expression and loss of the EGFR gene mutation when compared with parental cell lines. These differences in Akt and PTEN protein expression were not evident from the cDNA array profiles. These data suggests that (1) the EGFR gene mutation may be possibly lost in some cancer cells with other additional mechanisms for activating Akt, (2) reintroduction of PTEN or pharmacological downregulation of the constitutive PI3K-Akt-pathway activity may be an attractive therapeutic strategy in cancers with gefitinib resistance.

Negative selection at the pre-BCR checkpoint elicited by human mu heavy chains with unusual CDR3 regions.

Approximately 9% of in-frame mu heavy chain transcripts found in normal human pro-B cells encode proteins that can be expressed on the cell surface in the absence of surrogate or conventional light chains. These unusual mu heavy chains demonstrate preferential use of certain VH genes (VH3-23), frequent expression of DH regions in underrepresented reading frames, and an increased number of positively charged amino acids within the CDR3 region. Transcripts for these proteins are not found in pre-B cells or in mature B cells. When expressed in Jurkat T cells with the Ig(alpha)/Ig(beta) signal transduction module, these aberrant mu heavy chains induce cell activation and apoptosis. These results suggest that some mu heavy chains elicit negative selection at the pro-B cell to pre-B cell transition.