Roles of consultation organizations in the early detection of dementia: from the practices of the Community Consultation Center for Citizens with Mild Cognitive Impairment and Dementia, Nippon Medical School.

BACKGROUND: As society ages, early detection of dementia is becoming increasingly important. Many hospitals have opened memory-loss clinics, and various new approaches for early examination and appropriate diagnosis are being tried. However, these memory-loss clinics are ultimately part of the hospital establishment, implying that, in addition to the burdens of time and effort to undergo examinations and consultations, patients might have a certain psychological resistance. With a grant from the Ministry of Education, Culture, Sports, Science and Technology, the Institute of Development and Aging Science at Nippon Medical School has opened a center called the Community Consultation Center for Citizens with Mild Cognitive Impairment and Dementia, which is a dementia-related center outside the regular hospital. This center has been developing a system that makes consultations easier. We performed a retrospective follow-up study that aimed to determine how much this approach contributes to the early detection of dementia compared with outpatient visits to university hospitals. METHODS: Persons who were found to have organic brain syndrome (defined as organic diseases related to dementia, including mild cognitive impairment) after visiting the Consultation Center during the survey period were referred to as the Consultation Center group, and persons who were found to have organic brain syndrome after an initial visit to the Department of Neurology at Nippon Medical School Musashi Kosugi Hospital were referred to as the Hospital group. We compared the groups in terms of sex, age, Mini-Mental State Examination (MMSE) score, and subclassification by means of the t-test and χ(2) test. RESULTS: Both the mean MMSE score (p<0.001) and percentage of subjects with an MMSE score of 24 points of higher (p=0.007) were significantly higher in the Consultation Center group than in the Hospital group. CONCLUSIONS: Consultations can be made more casually at the Consultation Center than at hospitals. Our results suggest that more casual consultations contribute to the early detection of dementia.

RCAN1 regulates vascular branching during Xenopus laevis angiogenesis.

BACKGROUND/AIMS: The mechanisms that regulate the size-related morphologies of various blood vessels from the aorta to capillary vessels are still poorly understood. In this study, we evaluate the involvement of regulator of calcineurin 1 (RCAN1), a regulatory protein in the calcineurin/NFAT signal transduction pathway, in vascular morphology to gain further insight into these mechanisms. METHODS AND RESULTS: We first generated 2 types of vasculature in vitro from the same source of human umbilical vein endothelial cells by fibrin gel assay. We found that RCAN1 was significantly upregulated in large vessels with low branching frequencies when compared with small vessels with high branching frequencies. Next, to clarify whether RCAN1 regulates the branching of blood vessels in vivo, we injected RCAN1 mRNA into fertilized Xenopus laevis eggs. Overexpression of RCAN1 decreased the number of branching points that sprouted from intersomitic vessels during X. laevis angiogenesis. In addition, coexpression of calcineurin A, a target of RCAN1, could rescue RCAN1-suppressed vascular branching. CONCLUSIONS: These results provide in vivo evidence of RCAN1-regulated vascular branching which may play a role in the patterning of morphologically different vasculature.

Bronchoalveolar lavage fluid analysis provides diagnostic information on pulmonary Langerhans cell histiocytosis.

Histiocytes of Langerhans cell type are recovered from the bronchoalveolar lavage fluid (BALF) of patients with interstitial lung diseases in a nonspecific manner. Langerhans cells (LCs) can be identified through immunostaining for S-100, CD1a, and, more specifically, langerin. To evaluate the diagnostic value of BALF in pulmonary Langerhans cell histiocytosis (PLCH), we performed a retrospective clinicopathological study of 5 patients with biopsy-confirmed PLCH or Hand-Schüller-Christian disease involving the lung. As a control study, we examined BALF cells from 23 patients with various diseases, including sarcoidosis, hypersensitivity pneumonitis, collagen vascular disease, idiopathic pulmonary fibrosis, and adenocarcinoma of the lung. Cytospins obtained from BALF were stained with Giemsa or Papanicoloau and others were immunostained. In general, cytospins showed a monomorphous and dispersed cell population containing mononucleated or binucleated and occasionally multinucleated histiocytes. LCs recovered from BALF were characterized by clear and velvety cytoplasm; oval or kidney-shaped, vesicular nuclei with irregular shapes; nucleoli; and frequent grooves and indentations. Radiography and high-resolution computed tomography showed multiple bilateral nodular or cystic lesions in the middle and upper lung zones. The mean percentage of LCs in 9 lavages from the 5 patients was 8.0%, whereas that from the control group was only 0.3% (maximum, 1.6%). The percentage of cells positive for S-100 or CD1a was comparable to the percentage of Langerhans-like histiocytes stained with Giemsa stain. The present results indicate that the survey of LCs in BALF with the aid of immunocytochemical evaluation and corresponding clinical data could play a critical role in establishing the diagnosis of PLCH, thus providing a less invasive approach than lung biopsy, which carries a risk of complications.

Increased effect of fucoidan on lipoprotein lipase secretion in adipocytes.

AIMS: Fucoidan, a sulfated polysaccharide extracted from brown seaweed (F. vesiculosus) is recognized as an effective anticoagulant but its anti-lipidemic potency has not been well defined. We investigated the effect of fucoidan on lipoprotein lipase (LPL) secretion by human adipocytes. MAIN METHODS: LPL mRNA and protein expressions were measured using semi-quantitative RT-PCR, ELISA and immunohistochemistry in cultured adipocytes with or without fucoidan treatment. LPL enzyme activity was determined by a fluorometric assay. KEY FINDINGS: In cultured adipocytes, fucoidan induced LPL secretion in a dose- and time-dependent manner. An initial increase in LPL was maintained at a significant level but much slower than that in heparin-treated cells. Fucoidan also dose-dependently induced a cofactor of LPL, the apolipoprotein C-II (ApoC-II) secretion. In fucoidan-treated cells, LPL mRNA was time-dependently increased and LPL protein expression was also inceased. Treatment with both heparin and fucoidan showed no further increase in media LPL activity compared to heparin alone. In the conditioned medium from fucoidan-treated cells followed for 4 h, LPL activity decayed exponentially with half-life of about 180 min. In addition, the extracellular LPL mass in cycloheximide (a protein synthesis inhibitor) and fucoidan-treated cells did not change markedly, but LPL shifted significantly from active to inactive form. SIGNIFICANCE: These results suggest that fucoidan acts like heparin by releasing LPL in addition to increasing the intracellular transport and decreasing the degradation of LPL in the medium. Furthermore, LPL and ApoC-II secretion induced by fucoidan may be involved in regulating plasma triglyceride lowering clearance.

Evaluation of fractional analysis of bronchoalveolar lavage combined with cellular morphological features.

BACKGROUND: The value of bronchoalveolar lavage (BAL) still remains controversial, prompting a need for further improvement. The purpose of this study was to develop and evaluate a sequential analysis of cell content in fractional BAL (FBAL) from the airways and alveolar sacs with incorporation of the cellular morphologic features. METHODS: Initially, 30 ml saline was infused into a subsegmental lobe of the lung and the recovered fluid was assigned as FBAL-I being mainly originated from whole airways. The second and third lavages (FBAL-II and FBAL-III) each were performed using 50 ml saline being from more distal portions of airways and alveolar sacs respectively in the same lobe. Total cell number/ml and percentages of macrophages, lymphocytes, neutrophils, and eosinophils in each fraction together with their morphological alterations and mast cells, basophils and Masson bodies were assessed. RESULTS: In the 12 controls, percentage of neutrophils was high and lymphocytes and macrophages were low in FBAL-I while in FBAL-III, neutrophils decreased to nearly nil and lymphocytes and macrophages were increased. Analysis of FBAL from 76 patients with sarcoidosis and 14 with hypersensitivity pneumonitis (HP) revealed that a predominance of small, round and well-differentiated lymphocytes with relative absence of neutrophils, basophils and Masson bodies correlated best with sarcoidosis. In contrast, neutrophil predominance and presence of lymphocytes having deep nuclear indentations and abundant cytoplasm with a process resembling a "hand-mirror" correlated well with HP. CONCLUSIONS: Evaluation of FBAL together with cellular morphological features especially characteristics of lymphocytes provides valuable information for establishing the diagnosis in interstitial lung diseases.

Interaction between beta-amyloid protein and heparan sulfate proteoglycans from the cerebral capillary basement membrane in Alzheimer's disease.

Proteoglycans are important in the pathogenesis of senile dementia of Alzheimer type (SDAT) by participating in amyloidogenesis. Knowledge about specific proteoglycan subtypes in SDAT may be of therapeutic advantage. In this study, we examined proteoglycan constituents of SDAT brains with reference to hyaluronic acid, heparan sulfate (HS), dermatan sulfate and chondroitin sulfate subtypes. Total proteoglycans showed a 1.6-fold increase in the hippocampus and 4.3-fold increase in the gyrus frontalis superior compared to non-demented elderly subjects. The HS subtype showed a 9.3-fold increase in hippocampus and a 6.6-fold increase in gyrus frontalis superior. Immunohistochemical studies of senile plaques revealed the expression of heparan sulfate proteoglycan (HSPG) in a portion of the core of typical plaques. beta-amyloid expression was positive in senile plaques and the degenerated neuronal processes and capillary basement membrane, but was negative in endothelial cells. Microglial cells adjacent to senile plaques were positive for HLA-DR expression, and astroglial cells positive for glial fibrillary acidic protein were scattered around the microglial cells. Immunoelectron microscopic examination showed an electron-dense reaction for HSPG in the thickened basement membrane adjacent to the endothelial cells of capillary vessels, but not inside the endothelial cells. These findings suggest that a markedly increased HSPG in SDAT brains is most likely caused by HSPG from the blood capillary basement membrane and that the degenerated processes around senile plaques may arise from microglial or astroglial cells.

Induction of pulmonary thromboembolism by neutrophil elastase in collagen-induced arthritis mice and effect of recombinant human soluble thrombomodulin.

We previously reported that during total knee arthroplasty in rheumatoid arthritis (RA) patients, the use of tourniquet might promote local release of neutrophil elastase (NE) from neutrophils, which may contribute to the development of pulmonary thromboembolism (PTE) and tissue injury. The aim of this study was to develop PTE by the use of NE in a mouse model of collagen-induced arthritis (CIA) and investigate the relationship between thrombus and endothelial cells as well as the effect of recombinant human soluble thrombomodulin (rhs-TM) in reducing the risk of PTE. Male DBA/1J mice were injected intracutaneously at several sites with an emulsion containing bovine collagen and later a booster shot to produce CIA mice. Subsequently, NE was injected intravenously 2 times a day for 3 days and after a further 4 days, mice were sacrificed. A group of mice received rhs-TM injections prior to NE injections. We divided the mice into four groups of normal, CIA control, CIA + NE, and CIA + rhs-TM + NE mice and evaluated thrombus formation status. All CIA + NE mice developed PTE. In contrast, no thrombosis was found in normal control, CIA control and CIA + rhs-TM + NE mice. Plasma thrombin level, fibrinogen expression and neutrophil count were increased in CIA + NE mice. Double staining for anticoagulant TM and procoagulant von Willebrand factor (vWF) in pulmonary endothelial cells in normal mice showed a TM-dominant expression while in both CIA control and CIA + NE mice a vWF-dominant expression compatible with coagulant status was observed. Injection of rhs-TM into CIA + NE mice resulted in a phenotypic conversion of endothelial cells from vWF-dominant to TM-dominant expression and a reduction in fibrinogen deposition. These findings demonstrate that by repeated use of NE in CIA mice, it is feasible to produce PTE and to study its pathogenesis and that rhs-TM reduces the risk of PTE. We suggest that in surgical operations of upper and lower extremities in RA patients, the use of a tourniquet should be avoided as it may trigger NE release.

A novel technique for observing the internal ultrastructure of human chromosomes with known karyotype.

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has frequently been attempted in spite of the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. In this study we have used a method in which metaphase chromosome spreads were prepared on a flexible thermoplastic membrane (ACLAR) film. To assess chromosome identity, a diamidino-phenylindole staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then fixed with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and carried whole chromosome spreads. By this method, TEM observation of chromosomes from normal human lymphocytes allowed a thorough examination of the ultrastructure of centromeres, telomeres, fragile sites, and other chromosomal regions. Various ultrastructural patterns including thick electron dense boundaries, less dense internal regions, and extended chromatin loops at the periphery of the chromosomes were discernible. Application of the present method to chromosome research is expected to provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.

Spatial and phenotypic characterization of vascular remodeling in a mouse model of asthma.

Asthma is a chronic inflammatory disease characterized by airway wall remodeling in which vascular remodeling is thought to be a main contributor. Vascular endothelial growth factor (VEGF) is known as a major regulator of angiogenesis and enhancer of vascular permeability. Here, we define the spatial nature of vascular remodeling and the role of VEGF and its receptors (Flt-1 and Flk-1) in the allergic response in mice (A/J) susceptible to the development of allergen-induced airway hyperresponsiveness using morphometric and quantitative approaches. Increased vascularity, vasodilatation, and endothelial cell proliferation were found in the tracheal and bronchial walls in the early and late phases of asthma. Vascular changes were observed not only in small vessels but also in larger vessels. In contrast to normal control, lung tissue from the asthma model showed dual expression for CD31 and von Willebrand factor in the endothelial cells and alpha-smooth muscle actin and desmin in the mural cells of the vessels, suggesting a phenotypic and functional transformation. The mRNA levels of VEGF isoforms, VEGF(164) and VEGF(188), were significantly increased in the tracheal and lung tissue, respectively. In addition, the mRNA level of VEGF receptor Flk-1 was significantly increased in the trachea. These results establish the existence of vascular remodeling in the airways in a mouse model of allergic asthma and support a key role for the expression of unique VEGF isoform genes as mediators of structural changes.

Anti-apoptotic PTD-FNK protein suppresses lipopolysaccharide-induced acute lung injury in rats.

The present study was aimed at clarifying the effects of an anti-apoptotic protein for modulating symptoms in acute lung injury (ALI). From Bcl-x(L), a Bcl-2 family member, we constructed an artificial protein (FNK) and fused it with the protein transduction domain (PTD) of the HIV/Tat protein (PTD-FNK) to facilitate its permeation into cells. ALI was induced by intratracheal infusion of lipopolysaccharide (LPS) into Sprague-Dawley male rats. PTD-FNK was injected into the peritoneal cavity of the animals either 2 h before, or 3 h or 6 h after LPS challenge. All rats were sacrificed 24 h after the last treatment. Cell differential ratios and albumin concentration were estimated in bronchoalveolar lavage fluid. We examined histological change, myeloperoxidase activity, TUNEL assay, caspase-3/caspase-3-like activity and immunohistochemical reaction for caspase 3 (active form). In animals with PTD-FNK treatment, the albumin leakage was significantly attenuated with protection of tissue damage. Also, the apoptosis of alveolar wall cells was reduced by PTD-FNK treatment, while a total cell number and the neutrophil ratio were not changed. Human umbilical vein endothelial cells (HUVEC) and cells of an alveolar epithelial cell line (A549) were exposed to LPS or TNF-alpha with or without PTD-FNK treatment in vitro. Cell survival rates examined by trypan-blue exclusion assay were increased by PTD-FNK treatment in a concentration-dependent manner. Thus, PTD-FNK could play a protective role in ALI by suppressing apoptosis of alveolar epithelial cells and capillary endothelial cells despite of some effect on neutrophil activity.

Angiogenic switching in the alveolar capillaries in primary lung adenocarcinoma and squamous cell carcinoma.

The status of angiogenic switching was examined in alveolar capillaries of primary lung adenocarcinoma (ADC) from 10 patients and primary squamous cell carcinoma (SCC) from 11 patients, using immunostaining for CD31, thrombomodulin, von Willebrand factor (vWF), collagen types IV and VII, and alpha-smooth muscle actin (alpha-SMA). We applied the TdT-mediated dUTP nick-end labeling assay and the reverse transcription-polymerase chain reaction for vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). In bronchioloalveolar and papillary subtypes of ADC, the neoplastic cells, replacing the normal alveolar epithelial cells, had spread over alveolar walls and adhered firmly to alveolar interstitium as shown by the development of type IV collagen. Neoplastic cells of SCC were characterized by local proliferation in alveolar sacs without firm attachment to alveolar walls. Tumor lesions of SCC had often developed necrotic foci of various size. In ADC and SCC, alveolar capillary endothelial cells newly obtained reactivity to vWF. Such segments of endothelial cells lost surface thrombomodulin expression. CD31 was consistently expressed in normal and ADC tissues, but each endothelial cell marker was often attenuated or even lost in SCC, suggesting degeneration or necrosis of the alveolar capillaries. The capillary pericytes and interstitial fibroblasts were often hypertrophic and developed alpha-SMA in the cytoplasm in ADC, but they became atrophic in SCC. In ADC, apoptosis occurred in cells of alveolar capillaries more frequently in the peripheral zone than in the deeper zone of the tumor, whereas the frequency was not consistent in SCC. In microdissected alveolar wall tissues, mRNA expression patterns of VEGF isoforms and VEGFRs were similar in both ADC and SCC. In ADC, de novo angiogenic switching took place in cytoplasm as a unit of cells segments in alveolar capillary endothelium. Suppression of angiogenic switching in SCC implies that factors other than VEGF-VEGFR interaction, such as physical contact and compression of tumor cells, might play a critical role in alveolar capillaries.

Functional implications of the IL-6 signaling pathway in keloid pathogenesis.

The molecular mechanism(s) behind keloid pathogenesis remains unclear. Previously by global gene expression analysis of keloid fibroblasts (KFs), we implicated the IL-6 signaling pathway in keloid pathogenesis. Here, we determine a functional role of IL-6 signaling in keloid scars. Primary cultures of KFs and surrounding nonlesional fibroblasts (NFs) were subjected to induction or inhibition of IL-6 or its specific receptor IL-6 receptor alpha (IL-6R alpha) and detection of their effects on extracellular matrix gene expression. The levels of gp130 and several downstream targets in IL-6 signaling were also examined. IL-6 secretion was significantly higher in KFs than NFs. Addition of IL-6 peptide to NFs culture or inhibition of IL-6 or its receptor IL-6R alpha by their corresponding antibodies in KFs culture revealed a dose-dependent increase or decrease in collagen type I alpha 2 and fibronectin 1 mRNAs, respectively. Induction of IL-6 by IL-1beta peptide and stimulation by IL-6 peptide in NFs, or inhibition of IL-6 or IL-6R alpha in KFs cultures demonstrated a dose-dependent increase or decrease in procollagen I synthesis, respectively. The mRNA and protein expressions of gp130 and several downstream targets in IL-6 signaling (JAK1, STAT3, RAF1, and ELK1) were upregulated in KFs versus NFs. Our results indicate that IL-6 signaling may play an integral role in keloid pathogenesis and provide clues for development of IL-6 receptor blocking strategies for therapy or prophylaxis of keloid scars.

Potential role of the Slit/Robo signal pathway in angiogenesis.

Intensive investigations on angiogenesis and vasculogenesis have increased our understanding of molecular mechanisms of blood vessel formation during pathologic and developmental conditions. However, endothelial cells (ECs), the main component of vasculature, are heterogeneous, as revealed by our phenotypic and molecular biological studies in the laboratory, and it is still hard to adequately understand the molecular mechanisms of angiogenesis and vasculogenesis. Indeed, there are several major ligand/receptor signal pathways: VEGF/VEGFR, Jagged-1/Notch, Wnt ligand/frizzled receptor, and ephrin/Eph; each of which having distinct and independent roles during vascular formation. In this review, we focus on the angiogenic effect of the Slit and Robo signal pathway that was formally known as neuronal axon guidance. Among the existing vascular signals, this pathway is the most recently found ligand/receptor vascular signal, and may play important physiological roles as other major receptor/ligand signals do. Here, we briefly address: (1) the background of Slit and Robo families; (2) expression patterns of Slit and Robo; (3) functional roles of the Slit/Robo pathway in vascular formation; and (4) confronting tasks of this novel vascular pathway in the near future. Together, a summary of these data suggest the essential role of the Slit/Robo pathway in angiogenesis, and may explain why multiple vascular signals exist in heterogenic endothelial cells.

Critical roles of capillary endothelial cells for alveolar remodeling in nonspecific and usual interstitial pneumonias.

To characterize the relationship between angiogenesis factors and alveolar remodeling in interstitial lung diseases, we examined alveolar capillary endothelial cells in the normal lung (n=5) and in lungs with nonspecific interstitial pneumonia (NSIP) (n=4) or usual interstitial pneumonia (UIP) (n=6) using immunofluorescence staining for thrombmodulin and von Willebrand factor (vWF). With three-dimensional images of alveolar capillaries, the diameter of capillary tubes and their branching frequency per unit length were determined to define rearrangement of the capillary meshwork. Alveolar capillary endothelial cells in normal lungs expressed surface thrombomodulin, and those in lungs with cellular NSIP often showed coexpression of surface thrombmodulin and cytoplasmic vWF. In the alveolar septa of fibrotic NSIP and UIP, capillary endothelial cells demonstrated vWF in only the cytoplasm. Capillary branching frequencies in NSIP and UIP were decreased to 45% and 22%, respectively, of the normal level (p<0.002). Compared with normal lungs, in NSIP and UIP lungs alveolar capillaries containing TUNEL-positive endothelial cells (p<0.05) showed increases of 3.6-fold and 4.3-fold, respectively, indicating a close correlation between endothelial cell apoptosis and remodeling of alveolar capillary frameworks. The analysis of mRNA expression of vascular endothelial growth factors (VEGF) and their receptors (VEGFR1 and VEGFR2) showed a significant decrease in each VEGF isoform and in VEGFR2 mRNA in representative alveolar wall tissues microdissected from the normal, NSIP, and UIP lungs. These results suggest that decreased expression of VEGF mRNA is associated with a reduction in the number of capillary tubes via endothelial cell apoptosis that possibly results in alveolar remodeling in NSIP and UIP. However, whether VEGF is related to fibroblastic activation in the interstitial matrix remains unclear.

Growth-suppressive function of phosphatidylethanolamine-binding protein in anaplastic thyroid cancer.

BACKGROUND: A cDNA microarray analysis of anaplastic thyroid cancer cell lines (ACL) was recently performed and the down-regulation of phosphatidylethanolamine-binding protein (PBP) [RAF kinase inhibitor protein (RKIP)] in ACL compared to normal thyroid tissues was identified. MATERIALS AND METHODS: The expression levels of PBP in primary anaplastic and papillary thyroid cancer, thyroid cancer cell lines (anaplastic, papillary and follicular) and several normal human organs were examined. To examine the function of PBP, cell-growth assays were performed. RESULTS: PBP expression was reduced in anaplastic thyroid cancers, compared to either normal thyroid tissues or differentiated thyroid cancers. PBP was expressed ubiquitously in normal human tissues. Exogenous PBP expression suppressed ACL growth, and suggested a tumor suppressive function of PBP in ACL. CONCLUSION: This is the first report demonstrating that PBP may be a tumor suppressor whose loss is associated with development of anaplastic thyroid cancer from differentiated thyroid cancer.

Activation of PAR4 induces a distinct actin fiber formation via p38 MAPK in human lung endothelial cells.

Protease-activated receptors (PARs) are multifunctional G protein-coupled receptors. Among the four existing PARs, PAR4 is preferentially expressed in the human lung tissue. However, the function of PAR4 has not been defined in the lung endothelial cells. Because PAR1-mediated cellular effects are deeply related to the morphological changes, we focused on the actin fiber and p38 mitogen-activated protein kinase (MAPK) signaling involved in actin polymerization to elucidate the role of PAR4. RT-PCR and Western blot analyses identified PAR4 expression in human pulmonary artery endothelial cells and in human microvascular endothelial cells from lung. We then examined the changes in actin fibers in endothelial cells treated with PAR4-activating peptide. PAR1-activating peptide was used for comparison. Activation of PAR4 and PAR1 by their corresponding peptides induced actin fiber formation; however, the actin filaments were broadly bundled in PAR4 as compared with the ringlike actin filaments in PAR1 activation. Correspondingly, the magnitude of p38 MAPK phosphorylation was different between cells treated with PAR4 and PAR1, with PAR4-activating peptide showing a significantly higher sensitivity to p38 MAPK inhibitor, SB203580. Taken together, these results demonstrate that activation of PAR4 results in the formation of actin fiber distinct from that by PAR1 activation, suggesting PAR4 may play specific roles in the lung endothelial cells.

Up-regulation of growth factor receptor-bound protein 10 in cervical squamous cell carcinoma.

The Grb10 gene on chromosome 7p11.2-p12 belongs to a family of adapter proteins known to interact with a number of receptor tyrosine kinases, such as EGF, ErbB2/Her2, platelet-derived growth factor (PDGF), IGF-I receptors and vascular endothelial growth factor (VEGF) receptor, KDR (kinase insert domain containing receptor). In addition to receptor tyrosine kinases, Grb10 has also been found to interact with non-receptor tyrosine kinases such as Tec and Bcr-Abl, other cellular signaling molecules such as Raf-1, and the mitogen-activated protein (MAP) kinase, MEK. We demonstrated increased expression of Grb10 mRNA in more than one half of primary cervical squamous cell cancers (12 of 15 cases) when compared to corresponding non-cancerous uterine squamous cell tissues. In addition, immunohistochemical staining demonstrated that the Grb10 protein was prominent in the cytoplasm of cancer cells, whereas it was unreactive in the surrounding normal cervical squamous cells. In addition, its interruption by siRNA exhibited marked cell growth inhibition. These data indicate that amplification and increased expression of the Grb10 gene may play a role in the development of a portion of human cervical squamous cell cancer.

Global gene expression analysis of keloid fibroblasts in response to electron beam irradiation reveals the involvement of interleukin-6 pathway.

Keloid is a dermal fibroproliferative lesion of unknown etiology that commonly recurs after surgical excision. Post-operative adjuvant electron beam (EB) irradiation has been successfully used to reduce keloid recurrences. To provide new insights into the molecular mechanism behind the effect of EB irradiation, we used a cDNA microarray screening of more than 5000 genes to assess early changes in gene expression between EB-irradiated and non-irradiated keloid and non-lesional dermal fibroblasts. Primary fibroblast cultures from keloid and associated non-lesional dermis obtained from five patients were exposed to 15 Gy EB irradiation and analyzed after 15 min incubation. Early response to EB irradiation showed that 96 (1.8%) genes were modulated 2-fold or more in keloid fibroblasts. Upregulated genes accounted for 29.2% (28 genes), whereas downregulated genes comprised 70.8% (68 genes), indicating a silencing of many genes in keloid fibroblasts after EB irradiation. Many of the downregulated genes play roles in the enhancement of cell proliferation and extracellular matrix production, whereas several of the upregulated genes involves in the promotion of apoptosis and extracellular matrix (ECM) degradation. Using emerging bioinformatic tools and further corroboration, the interleukin 6 (IL-6) signaling pathway was found to be mainly involved in EB irradiation response. We also showed co-expression of IL-6 and its specific receptor (IL-6Ralpha) in keloid fibroblasts that points to the existence of an IL-6 autocrine loop in these cells. These results suggested that at the molecular level, EB irradiation might hinder keloid formation by regularizing disturbances in the homeostatic equilibrium between inducer and inhibitor activities in the matrix system most likely through the IL-6 pathway. Our study provides clues for the molecular mechanism(s) behind the beneficial effect of EB irradiation in reducing keloid recurrences and may help develop alternative strategies for the therapy and prophylaxis of this lesion.

Role of cdk4, p16INK4, and Rb expression in the prognosis of bronchioloalveolar carcinomas.

BACKGROUND: The p16(INK4) protein has been identified as a potent inhibitor of cyclin-dependent kinase (cdk)4 by blocking cdk4-mediated phosphorylation of the tumor suppressor retinoblastoma (Rb) protein, thus allowing Rb-mediated growth suppression. OBJECTIVES: Loss of p16(INK4) has been associated with a poor cancer prognosis, but its potential significance in bronchioloalveolar carcinomas (BACs) has not been explored. METHODS: We examined immunohistochemical expression of p16(INK4), cdk4, and Rb proteins in 38 BACs and correlated their expression levels with known clinicopathological features of the disease. RESULTS: All BACs expressed cdk4, while 89 and 82% expressed p16(INK4) and Rb proteins, respectively. None of the clinicopathological factors correlated with p16(INK4), cdk4, or Rb expression separately. A low p16(INK4)/cdk4 ratio was significantly associated with a high disease stage (p = 0.04), and the ratio tended to be lower in mucinous than nonmucinous tumors. BACs with a low p16(INK4)/cdk4 ratio showed significantly higher Rb expression levels (p = 0.02). Univariable survival analyses showed a significantly lower 5-year survival probability in patients with a high stage (p = 0.002) or low p16(INK4)/cdk4 ratio (p = 0.01). CONCLUSIONS: The results suggest a role of the cdk4/p16(INK4) pathway in the prognosis of BACs. Further studies are warranted to clarify whether a low p16(INK4)/cdk4 ratio may identify tumors that are destined to behave unfavorably.