[Metastatic lung tumor from colon cancer with cavitation: report of a case].

A 55-year-old man underwent rectal amputation for rectal cancer in August 2005. A tiny thin-walled cavity lesion in his left S1+2 was found on computed tomography (CT) of the chest in November 2008. The cavity lesion in the left S1+2 gradually increased in size over 3 months and positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) showed FDG accumulation at the lesion. Videoassisted thoracoscopic (VATS) wedge resection was performed to make a definite diagnosis in March 2009. The pathological findings revealed a metastatic lung tumor from the rectal cancer. It is necessary to consider the possibility of metastatic lung tumors in a case with the cavity lesions especially in patients with a history of colon cancer.

Changes in blood circulation of the contralateral Achilles tendon during and after acupuncture and heating.

The purpose of this study was to investigate the effects of acupuncture and heating (application of hot pack) treatments on blood circulation in the contralateral Achilles tendon. During the treatments (10 min for acupuncture, 20 min for heating) and recovery period (40 min), the blood volume (THb) and oxygen saturation (StO2) of the treated and the non-treated tendons were measured using red laser lights. During both treatments, THb and StO2 of the treated tendon increased significantly from the resting level. The increased THb and StO2 of the treated tendon were maintained until the end of the recovery period after removal of the acupuncture needle, although these values decreased after removal of the hot pack. Although THb of the non-treated sides did not change during both acupuncture and heating treatments, it increased gradually after removal of the acupuncture needle or the hot pack. For both treatments, the amount of increase in THb of the non-treated tendon was significantly correlated to that of the treated tendon during the last phase of recovery period. These results obtained from the healthy subjects imply that blood circulation in the injured tendon in a plaster cast may be improved by applying acupuncture or heating treatments to the contralateral healthy limb.

Preferential proliferation of murine colony-forming units in culture in a chemically defined condition with a macrophage colony-stimulating factor-negative stromal cell clone.

The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. In this study, we investigated the possibility of using for this purpose a defined medium, mSFO2, which was developed for stromal cell-dependent bone marrow cultures. We found that a combination of epidermal growth factor (EGF), the OP9 stromal cell line, which lacks macrophage colony-stimulating factor, recombinant stem cell factor, and the chemically defined medium mSFO2 provides a microenvironment where c-Kit+ Thy-1+/lo Mac-1+/lo B220- TER119- common beta + IL-2R gamma + gp130+ cells are selectively propagated from normal, unfractionated bone marrow cells. This cell population produced an in vitro colony at a very high efficiency (50%), whereas it has only limited proliferative ability in the irradiated recipient. Thus, the cells selected in this culture condition might represent colony-forming units in culture (CFU-c) with short-term reconstituting ability. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelomonocytic and B cell lineages in vitro and in vivo. The fact that a similar culture condition was created by erb-B2-transduced OP9 in the absence of EGF indicated that EGF exerts its effect by acting on OP9 rather than directly on CFU-c. These results suggested that the balance between self-renewal and differentiation of CFU-c can be regulated by extra-cellular signals.

[Simultaneously treated thymoma and lung cancer; report of a case].

A 74-year-old man was admitted to our hospital in order to treat a mediastinal mass and 2 ground-glass attenuations in the right upper lobe detected by chest X-ray and computed tomography (CT). Partial resection of right lung and thymectomy were performed. The mediastinal mass and 2 ground-glass attenuations in the right upper lobe proved to be thymoma and bronchioloalveolar carcinomas, respectively by pathology.

PDGF mediates a neuron-astrocyte interaction in the developing retina.

Astrocytes invade the developing retina from the optic nerve head, over the axons of retinal ganglion cells (RGCs). RGCs express the platelet-derived growth factor A-chain (PDGF-A) and retinal astrocytes the PDGF alpha-receptor (PDGFR alpha), suggesting that PDGF mediates a paracrine interaction between these cells. To test this, we inhibited PDGF signaling in the eye with a neutralizing anti-PDGFR alpha antibody or a soluble extracellular fragment of PDGFR alpha. These treatments inhibited development of the astrocyte network. We also generated transgenic mice that overexpress PDGF-A in RGCs. This resulted in hyperproliferation of astrocytes, which in turn induced excessive vasculogenesis. Thus, PDGF appears to be a link in the chain of cell-cell interactions responsible for matching numbers of neurons, astrocytes, and blood vessels during retinal development.

Expression of angiogenic and neurotrophic factors in the progenitor cell niche of adult monkey subventricular zone.

The subventricular zone along the anterior horn (SVZa) of the cerebral lateral ventricle of adult mammals contains multipotent progenitor cells, which supposedly exist in an angiogenic niche. Numerous signals are known to modulate the precursor cell proliferation, migration or differentiation, in rodent models. In contrast, the data on signals regulating the primate SVZa precursors in vivo are scarce. We analyzed the expression at protein level of a panel of angiogenic and/or neurotrophic factors and their receptors in SVZa of adult macaque monkeys, under normal condition or after transient global ischemia which enhances endogenous progenitor cell proliferation. We found that fms-like tyrosine kinase 1 (Flt1), a receptor for vascular endothelial cell growth factor, was expressed by over 30% of the proliferating progenitors, and the number of Flt1-positive precursors was significantly increased by the ischemic insult. Smaller fractions of mitotic progenitors were positive for the neurotrophin receptor tropomyosin-related kinase (Trk) B or the hematopoietic receptor Kit, while immature neurons expressed Flt1 and the neurotrophin receptor TrkA. Further, SVZa astroglia, ependymal cells and blood vessels were positive for distinctive sets of ligands/receptors, which we characterized. The presented data provide a molecular phenotypic analysis of cell types comprising adult monkey SVZa, and suggest that a complex network of angiogenic/neurotrophic signals operating in an autocrine or paracrine manner may regulate SVZa neurogenesis in the adult primate brain.

Expression of angiopoietin-2 in human glioma cells and its role for angiogenesis.

In highly vascular malignant glioma, glioma cells themselves may express angiogenic factors and induce angiogenesis. Recent studies have shown that novel angiogenic factors, angiopoietin-1 (Ang1) and -2 (Ang2), play important roles in the modulation of vasculogenesis and angiogenesis. In this study, we determined Ang2 mRNA expression in cultured human malignant glioma cells (U105, U251, and U373 MG) by reverse transcriptase-PCR. Western blot analysis and immunocytochemical analysis with antihuman Ang2 antibody revealed that Ang2 protein was expressed and secreted by these cells. Furthermore, hypoxia increased the Ang2 protein level in cultured glioma cells. Serial sections of 32 human glioma tissues (14 glioblastomas, eight anaplastic astrocytomas, seven astrocytomas, and three pilocytic astrocytomas) were immunostained against Ang2, vascular endothelial growth factor, Tie2, von Willebrand factor, and alpha smooth muscle actin. The immunoreactivity of each angiogenic factor was higher in malignant gliomas than in low-grade gliomas. Ang2 protein was detected not only in endothelial cells but also in glioma cells, and its expression was prominent in both the area surrounding the necrosis and the periphery of glioblastomas. In the area surrounding necrosis, Ang2 was highly expressed and tumor vessels showed regression. In the tumor periphery, Ang2 was highly expressed and many small vessels stained positively for von Willebrand factor but not for alpha smooth muscle actin, suggesting angiogenesis. Statistical analysis revealed that the Ang2 expression was negatively correlated with vessel maturation in malignant gliomas and that vascular endothelial growth factor expression was positively correlated with vessel maturation in low-grade gliomas (P < 0.05). These results suggest that glioma cells themselves express Ang2 and that expression may be induced by hypoxic stimulation and may play a crucial role in the vessel maturation, angiogenesis, and vessel regression in malignant glioma.

Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in fibrous cap lesions in apolipoprotein E-deficient mice.

BACKGROUND: The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-alpha (PDGFR-alpha) and receptor-beta (PDGFR-beta) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-alpha and PDGFR-beta, respectively. METHODS AND RESULTS: Apolipoprotein E-deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced by 67% and 80%, respectively, compared with the control mice injected with irrelevant rat IgG. In contrast, the mice that received APA5 showed only minimal reduction of lesion size, and a large number of VSMCs were observed in the intima. In the intima of advanced lesions, APB5 immunolabeled VSMCs, whereas APA5 could detect VSMCs mainly in the media. CONCLUSIONS: These results indicate that PDGFR-beta plays a significant role in formation of fibrous atherosclerotic lesions and that regulation of the signal transduction through PDGFR-beta could affect atherogenesis in mice.

Frequent gene expression of granulocyte colony-stimulating factor (G-CSF) receptor in CD7+ surface CD3- acute lymphoblastic leukaemia.

Gene expression of various cytokine receptors in CD7+ acute lymphoblastic leukemia (ALL) cells in relation to responsiveness to these cytokines was examined by reverse transcription polymerase chain reaction and Northern blot studies. Leukemic cells from all of seven CD7+ ALL patients examined fulfilled the criteria for ALL according to the FAB classification; surface CD3 was absent in all of these patients, while cytoplasmic CD3 and/or CD3 epsilon mRNA were found in all of them. Samples from six of the seven patients at initial disease expressed the granulocyte colony-stimulating factor receptor (G-CSFR) gene. Leukemic cells with G-CSFR transcripts from one patient at initial disease showed growth response to G-CSF in vitro, and those from two other patients became responsive to G-CSF at relapse. Neither in vitro nor in vivo myeloid differentiation was observed in any samples that responded to G-CSF. Interleukin 3R alpha (IL-3R alpha) gene was expressed in samples from one patient at initial disease and from two patients at relapse. GM-CSFR beta gene mRNA was detected in two patients with IL-3R alpha mRNA. Our results show that the leukemic cells in these CD7+ ALL patients frequently expressed G-CSFR as a functional property, thus calling attention to the appropriate clinical application of G-CSF for ALL patients.

In vitro analysis of potency restriction during epiblast differentiation.

During mammalian gastrulation, posterior part of primitive ectoderm differentiates into subsets of mesoderm cells, the most proximal portion of which subsequently gives rise to vascular endothelial cells and hematopoietic cells. To analyze this process in which the potency of multipotent epiblast cells is restricted progressively into hematopoietic cell lineages, we have established a culture system that allows differentiation of epiblast cells as well as embryonic stem cells into hematopoietic cells. Using this culture system, we showed that all parts of epiblast tissues at early streak and late bud stages preserved hematogenic potency, while it was lost from anterior region at early head fold stages. However, this loss of hematogenic potency in the anterior region of the head fold stage embryo was reinduced by addition of activin in the culture. Moreover, in order to detect transitory stages of mesoderm cells that are the direct progeny of multipotent epiblast cells, we developed three monoclonal antibodies that are able to define distinct subsets of mesoderm cells in the gastrulating embryo. These results are discussed from a view of cell-mass based commitment.

Evaluation of PSF1 as a prognostic biomarker for prostate cancer.

BACKGROUND: Partner of SLD5 1 (PSF1) is an evolutionarily conserved DNA replication factor. Previous studies have suggested that transcriptional activity of the PSF1 gene correlated with malignancy of cancer cells. The objective of the current study was to evaluate the relationship between PSF1 expression and the clinical features of prostate cancer. METHODS: We determined the expression of PSF1 in 120 needle biopsy samples of prostate cancer by immunohistochemistry. We divided patients into PSF1-positive or -negative groups and analyzed the relationships between the expression of PSF1, the Gleason score, PSA level, TNM classification and prognosis. RESULTS: Our results showed that the PSF1 expression correlated significantly with PSA values at diagnosis (P=0.0028), with tumor grade (P<0.0001), and with clinical stage (P=0.0005). Moreover, the PSF1 expression correlated significantly with overall survival (hazard ratio (HR) 5.5; 95% confidence interval (CI) 2.17-15.8; P=0.003) and progression-free survival in 99 consecutive patients with prostate cancer. Noteworthy, the prognosis of PSF1-positive cases was also worse in patients with a Gleason score of 8-10 (HR 3.7; 95% CI 1.28-13.43; P=0.0143). Limitations include that this study had a retrospective design, that patients in the study were heterogeneous and included those with early and advanced cancer, and that small tumor fragments may not be representative of the entire carcinoma. CONCLUSIONS: PSF1 is expressed in high-grade prostate cancer and may be a useful biomarker to identify patients with a poor prognosis at the time of diagnosis.

Involvement of platelet-derived growth factor receptor-alpha in hair canal formation.

Hair follicles develop and are maintained by multiple rounds of inductive events involving interactions among various cell types within the follicles and the adjacent mesenchyme. Although evidence suggests that several growth factors, cell adhesion molecules, and transcriptional regulators are involved in those cell-cell interactions, the molecular mechanisms regulating each pivotal step of hair follicle development, such as formation of the hair germ, root sheath, sebaceous gland, and hair canal, remain largely unknown. In this study, we established the antagonistic monoclonal antibody APA5 against platelet-derived growth factor (PDGF) receptor-alpha (PDGFR-alpha) and used it to investigate the role of PDGFR-alpha in neonatal skin development. In addition to the dermal mesenchyme, a known site of PDGFR-alpha expression, immunohistologic staining of neonatal skin detected transient expression of PDGFR-alpha in the perinatal epidermis for several days. On the other hand, ligands for PDGFR-alpha were detected in epithelial cells and sebaceous glands of hair follicles. To determine whether this contiguous expression of PDGF and PDGFR-alpha in neonatal skin plays a functional role, we injected APA5 into neonates to block the function of PDGFR-alpha. Consistent with the PDGF/PDGFR-alpha expression in the neonatal skin, two defects were induced by this procedure. First, hair canal formation in the epidermis was severely suppressed. Second, the growth of dermal connective tissues and of hair follicles of pelage hairs was suppressed. These results indicate that PDGF signals are involved in both the epidermis-follicle interaction and the dermal mesenchyme-follicle interaction required for hair canal formation and the growth of the dermal mesenchyme, respectively.

PDGFR alpha expression during mouse embryogenesis: immunolocalization analyzed by whole-mount immunohistostaining using the monoclonal anti-mouse PDGFR alpha antibody APA5.

We investigated the cells that express platelet-derived growth factor receptor alpha (PDGFR alpha) during mouse embryogenesis. PDGFR alpha expression has been identified by in situ hybridization or immunohistochemistry using polyclonal antibodies on tissue sectins. Because no immunostaining study using whole-mount specimens has been published to date, we established a new monoclonal antibody (MAb), APA5, for this purpose. Our results differed in that APA5 stained only the paraxial mesoderm, whereas other investigators concluded that most if not all mesodermal cells expressed PDGFR alpha. Moreover, we did not find PDGFR alpha expression in embryonic erythrocytes, which have been previously suggested to express PDGFR alpha. On the basis of our present results, we wish to revise the proposed PDGFR alpha expression as follows. At the pregastrulation stage, PDGFR alpha is expressed only in primitive endoderm, particularly that in the ectoplacental cone. On gastrulation, it is expressed at high levels in the paraxial mesoderm. This expression continues after its differentiation into the somite. Along with the differentiation and migration of the sclerotome, PDGFR alpha + cells begin to become distributed throughout the embryonal mesenchyme. During organogenesis, particularly intense staining is detected in regions of epithelial and mesenchymal interaction, such as the tooth bud and bronchi. In addition to mesodermal derivatives, the developing lens, apical ectodermal ridge, glial precursor, cardiac valves, and choroid plexus express PDGFR alpha. Our results with whole-mount immunostaining show that PDGFR alpha is abundantly expressed and may play important roles during embryogenesis.

Stromal cell-dependent bone marrow culture with a nearly protein-free defined medium.

We examined the ability of a defined medium mSFO2 to support stromal cell-dependent B lymphopoiesis and myelopoiesis. Both ST2 and PA6 stromal cell lines were able to form monolayers in the presence of mSFO2 alone. While the ST2 monolayer could last for long period of time, either bFGF or EGF were required for the maintenance of the PA6 monolayer with mSFO2. mSFO2 did support the generation of distinct stages of B precursors on the ST2 layer and this culture condition was efficient enough to be used for limiting dilution assay of in vitro clonable B progenitors. While ST2 cell line failed to support long-term myelopoiesis with mSFO2, PA6 in combination of bFGF was able to support sustained generation of various hematopoietic progenitors. Indeed, 20 times increase of IL-3 reactive colony-forming cells was induced upon culturing the normal bone marrow cells on the PA6 layer for 8 days with mSFO2. Because total protein concentration of mSFO2 is only 10 micrograms/ml consisting of transferrin and insulin, the present result that mSFO2 is able to support the proliferation of normal hematopoietic progenitor cells would make an important step towards the standardization of bone marrow culture.

Hematopoietic tissues, as a playground of receptor tyrosine kinases of the PDGF-receptor family.

Three receptor tyrosine kinases of the PDGF receptor family (RTKP) that clustered within 1000 Kb of the mouse chromosome 5 constitute an interesting unit that are expressed in three distinct cell lineages essential for constructing hematopoietic tissues. Namely, the c-kit gene that is expressed in hematopoietic stem cells is flanked by pdgfr alpha and flk genes expressed respectively in stromal cells and vascular endothelial cells. In this article, we review our results on their expression in the embryonic hematopoietic tissues. We found that co-expression of Flkl and c-Kit was frequently detected either in vascular endothelial cells or hematopoietic cells in the early hematopoietic tissues. On the other hand, the three RTKPs are expressed in different cell lineages in the fetal liver. On the basis of this finding, we propose two modes of embryonic hematopoiesis; hematogenic angiopoiesis and hematopoiesis.

Structural alterations of the p53 gene in human leukemias.

We have investigated alterations of the p53 gene in human leukemias by polymerase chain reaction-mediated restriction fragment length polymorphism analysis. This method detects the codon 72 polymorphism of the p53 gene, allowing identification of loss of heterozygosity (LOH) of the p53 gene. In this study, at least two specimens were obtained from each patient to compare the allele status at different points of clinical course. Of 21 cases examined 7 were heterozygous at this polymorphic site and were evaluable for LOH study. Only one patient of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) lost the heterozygosity in the specimen of her last relapse. Leukemic cells from her repeated relapses including the last one revealed to have the clonally rearranged immunoglobulin H chain gene of the same size, indicating that alteration of the p53 gene in this patient might account for the lethal relapse as a clonal evolution event. Northern-blot analysis of the p53 gene showed that one case of CD7(+) ALL had altered p53 mRNA. Overall, it is demonstrated that alterations of the p53 gene might be involved in the pathogenesis or progression of at least some human leukemias, although the alterations in leukemias seemed to be not as frequent as in solid tumors.

Allelic imbalance and microsatellite instability in plasma DNA released from polyclonal pancreatic adenocarcinoma.

Evidence of circulating soluble DNA in blood-stream of cancer patients has emerged. Because the plasma DNA is largely derived from cancer cells, genetic analysis of plasma DNA is important to understand the molecular events occurred in cancer patient. Seven microsatellite markers in the soluble plasma DNA from patients with pancreatic adenocarcinoma and other pancreato-biliary malignant tumors were examined for microsatellite instability (MSI) and allelic imbalance (AI). A variety of genetic alterations including MSI and AI were detected in the plasma DNA. Some alterations were detected before recurrence of the tumor was verified. Analysis of five primary pancreatic adenocarcinomas by microdissection revealed that the heterogeneous nature of pancreatic tumors is associated with both MSI and AI in the same tumor. The presence of altered plasma DNA including MSI and/or AI from the same pancreatic cancer patient may be important evidence for the presence of these alterations in heterogeneous primary tumors. Analysis of plasma DNA could become one of the diagnostic or therapeutic measures for this type of pancreatic adenocarcinoma.

The p53 gene is a potent determinant of chemosensitivity and radiosensitivity in gastric and colorectal cancers.

We previously reported that introduction of the wild-type p53 gene into human cancer cells with deleted p53 enhanced apoptosis induced by chemotherapy [Fujiwara et al. (1994) Cancer Res 54:2287]. This suggests that p53 status could be a potent determinant of the therapeutic efficacy of DNA-damaging cancer therapy. We analyzed 24 patients with gastric or colorectal cancer for p53 mutations and apoptotic changes in surgical specimens. Out of 11 patients with gastric cancer, 3 were treated with chemotherapeutic drugs before resection; 5 of 13 patients with colorectal cancer had 30 Gy radiation prior to surgery. p53 mutations were detected in 4 cases of gastric cancer (36.4%) and in 6 cases of colorectal cancer (46.2%) by immunohistochemical staining. The preoperative DNA-damaging therapies increased the number of apoptotic cells in wild-type-p53-expressing tumors; tumors with mutant p53, however, significantly showed fewer apoptotic cells compared with those expressing wild-type p53. The p53-inducible WAF1/CIP1 protein was immunohistochemically observed in wild-type-p53-containing tumors, whereas mutant-p53-expressing tumors expressed no detectable WAF1/CIP1. Taken together, we conclude that p53 mutations are associated with the poor response of chemotherapy and radiotherapy.

Study on PDGF receptor beta pathway in glomerular formation in neonate mice.

The assembly of vascular endothelial cells (ECs) and smooth muscle cells is a critical event in the development of the cardiovascular system. Although the role of ECs in this event has been studied intensively, the cross-talk between the two cell components remains poorly understood. In this study, we blocked platelet-derived growth factor receptor (PDGFR) pathways in mice by antagonistic rat monoclonal antibody APB5 against murine PDGFR-beta and examined glomerular capillary formation.