Retrovirus-Derived RTL9 Plays an Important Role in Innate Antifungal Immunity in the Eutherian Brain.

Retrotransposon Gag-like (RTL) genes play a variety of essential and important roles in the eutherian placenta and brain. It has recently been demonstrated that RTL5 and RTL6 (also known as sushi-ichi retrotransposon homolog 8 (SIRH8) and SIRH3) are microglial genes that play important roles in the brain's innate immunity against viruses and bacteria through their removal of double-stranded RNA and lipopolysaccharide, respectively. In this work, we addressed the function of RTL9 (also known as SIRH10). Using knock-in mice that produce RTL9-mCherry fusion protein, we examined RTL9 expression in the brain and its reaction to fungal zymosan. Here, we demonstrate that RTL9 plays an important role, degrading zymosan in the brain. The RTL9 protein is localized in the microglial lysosomes where incorporated zymosan is digested. Furthermore, in Rtl9 knockout mice expressing RTL9ΔC protein lacking the C-terminus retroviral GAG-like region, the zymosan degrading activity was lost. Thus, RTL9 is essentially engaged in this reaction, presumably via its GAG-like region. Together with our previous study, this result highlights the importance of three retrovirus-derived microglial RTL genes as eutherian-specific constituents of the current brain innate immune system: RTL9, RTL5 and RTL6, responding to fungi, viruses and bacteria, respectively.

A New Drug-Free Cancer Therapy Using Ultraviolet Pulsed Irradiation. PDT (PhotoDynamic Therapy) to PPT (Pulsed Photon Therapy).

BACKGROUND: Pulsed ultraviolet (UV) irradiation can be used to generate a broad UV-C spectrum. The pulsing nature of such a spectrum helps increase the damage to cancer cells, leading to their injury and death. In contrast, non-tumor cells repair the damage and survive the same pulsed UV irradiation energy. Herein, we describe the development of a pulsed UV irradiation method for cancer cell dysfunction that irradiates cells with pulsed light by generating tremendous instantaneous UV energy-tens of thousands of times greater than that generated by UV lamps-to cause specific cell injury and dysfunction of cancer cells. METHODS: A newly developed pulsed ultraviolet irradiation device was used. Features of the device used in this study. This device employs a quartz discharge xenon lamp. Cultured tumor cells and non-tumor cells were irradiated with pulsed light at different irradiation doses, and their reactions were observed using optical, electron, and laser microscopes. RESULTS: Cancer cells have more FAS (CD95) receptor domains than non-cancer cells, and pulsed UV irradiation stimulates the production of reactive oxygen species (ROS) and OH, which exceeds the oxidative stress removal function, resulting in cell injury and death. That is, at low UV doses, only cancer cells underwent cell death, whereas non-cancer cells did not. The pulsed UV irradiation technique directly destroys cancer cells and minimizes the number of residual cancer cells while allowing minimum invasion into non-tumor cells, thereby improving their survival. This suggests the possibility of activating the host's local immune response to eliminate residual cancer cells. CONCLUSIONS: A newly developed pulsed UV radiation system shows potential for use in the development of a drug-free cancer treatment system that selectively kills tumor cells by irradiating them with high-intensity pulsed UV rays over a broad UV-C range of 230-280 nm.

Retrovirus-derived RTL5 and RTL6 genes are novel constituents of the innate immune system in the eutherian brain.

Retrotransposon Gag-like 5 [RTL5, also known as sushi-ichi-related retrotransposon homolog 8 (SIRH8)] and RTL6 (also known as SIRH3) are eutherian-specific genes presumably derived from a retrovirus and phylogenetically related to each other. They, respectively, encode a strongly acidic and extremely basic protein, and are well conserved among the eutherians. Here, we report that RTL5 and RTL6 are microglial genes with roles in the front line of innate brain immune response. Venus and mCherry knock-in mice exhibited expression of RTL5-mCherry and RTL6-Venus fusion proteins in microglia and appeared as extracellular dots and granules in the central nervous system. These proteins display a rapid response to pathogens such as lipopolysaccharide (LPS), double-stranded (ds) RNA analog and non-methylated CpG DNA, acting both cooperatively and/or independently. Experiments using Rtl6 or Rtl5 knockout mice provided additional evidence that RTL6 and RTL5 act as factors against LPS and dsRNA, respectively, in the brain, providing the first demonstration that retrovirus-derived genes play a role in the eutherian innate immune system. Finally, we propose a model emphasizing the importance of extra-embryonic tissues as the origin site of retrovirus-derived genes. This article has an associated 'The people behind the papers' interview.

High PTX3 expression is associated with a poor prognosis in diffuse large B-cell lymphoma.

Tumor-associated macrophages (TAMs) are associated with a poor prognosis of diffuse large B-cell lymphoma (DLBCL). As macrophages are heterogeneous, the immune polarization and their pathological role warrant further study. We characterized the microenvironment of DLBCL by immunohistochemistry in a training set of 132 cases, which included 10 Epstein-Barr virus-encoded small RNA (EBER)-positive and five high-grade B-cell lymphomas, with gene expression profiling in a representative subset of 37 cases. Diffuse large B-cell lymphoma had a differential infiltration of TAMs. The high infiltration of CD68 (pan-macrophages), CD16 (M1-like), CD163, pentraxin 3 (PTX3), and interleukin (IL)-10-positive macrophages (M2c-like) and low infiltration of FOXP3-positive regulatory T lymphocytes (Tregs) correlated with poor survival. Activated B cell-like DLBCL was associated with high CD16, CD163, PTX3, and IL-10, and EBER-positive DLBCL with high CD163 and PTX3. Programmed cell death-ligand 1 positively correlated with CD16, CD163, IL-10, and RGS1. In a multivariate analysis of overall survival, PTX3 and International Prognostic Index were identified as the most relevant variables. The gene expression analysis showed upregulation of genes involved in innate and adaptive immune responses and macrophage and Toll-like receptor pathways in high PTX3 cases. The prognostic relevance of PTX3 was confirmed in a validation set of 159 cases. Finally, in a series from Europe and North America (GSE10846, R-CHOP-like treatment, n = 233) high gene expression of PTX3 correlated with poor survival, and moderately with CSF1R, CD16, MITF, CD163, MYC, and RGS1. Therefore, the high infiltration of M2c-like immune regulatory macrophages and low infiltration of FOXP3-positive Tregs is associated with a poor prognosis in DLBCL, for which PTX3 is a new prognostic biomarker.

HERV-Derived Ervpb1 Is Conserved in Simiiformes, Exhibiting Expression in Hematopoietic Cell Lineages Including Macrophages.

(1) Background: The ERVPb1 gene in humans is derived from an envelope (Env) gene of a human endogenous retrovirus group, HERV-P(b). The ERVPb1 gene reportedly has a conserved open reading frame (ORF) in Old World monkeys. Although its forced expression led to cell-fusion in an ex vivo cell culture system, like other Env-derived genes such as syncytin-1 and -2, its mRNA expression is not placenta-specific, but almost ubiquitous, albeit being quite low in human tissues and organs, implying a distinct role for ERVPb1. (2) Methods: To elucidate the cell lineage(s) in which the ERVPb1 protein is translated in human development, we developed a novel, highly sensitive system for detecting HERV-derived proteins/peptides expressed in the tissue differentiation process of human induced pluripotent stem cells (iPSCs). (3) Results: We first determined that ERVPb1 is also conserved in New World monkeys. Then, we showed that the ERVPb1 protein is translated from a uniquely spliced ERVPb1 transcript in hematopoietic cell lineages, including a subset of macrophages, and further showed that its mRNA expression is upregulated by lipopolysaccharide (LPS) stimulation in primary human monocytes. (4) Conclusions: ERVPb1 is unique to Simiiformes and actually translated in hematopoietic cell lineages, including a subset of macrophages.

High TNFRSF14 and low BTLA are associated with poor prognosis in Follicular Lymphoma and in Diffuse Large B-cell Lymphoma transformation.

The microenvironment influences the behavior of follicular lymphoma (FL) but the specific roles of the immunomodulatory BTLA and TNFRSF14 (HVEM) are unknown. Therefore, we examined their immunohistochemical expression in the intrafollicular, interfollicular and total histological compartments in 106 FL cases (57M/49F; median age 57-years), and in nine relapsed-FL with transformation to DLBCL (tFL). BTLA expression pattern was of follicular T-helper cells (TFH) in the intrafollicular and of T-cells in the interfollicular compartments. The mantle zones were BTLA+ in 35.6% of the cases with similar distribution of IgD. TNFRSF14 expression pattern was of neoplastic B lymphocytes (centroblasts) and "tingible body macrophages". At diagnosis, the averages of total BTLA and TNFRSF14-positive cells were 19.2%±12.4STD (range, 0.6%-58.2%) and 46.7 cells/HPF (1-286.5), respectively. No differences were seen between low-grade vs. high-grade FL but tFL was characterized by low BTLA and high TNFRSF14 expression. High BTLA correlated with good overall survival (OS) (total-BTLA, Hazard Risk=0.479, P=0.022) and with high PD-1 and FOXP3+Tregs. High TNFRSF14 correlated with poor OS and progression-free survival (PFS) (total-TNFRSF14, HR=3.9 and 3.2, respectively, P<0.0001), with unfavorable clinical variables and higher risk of transformation (OR=5.3). Multivariate analysis including BTLA, TNFRSF14 and FLIPI showed that TNFRSF14 and FLIPI maintained prognostic value for OS and TNFRSF14 for PFS. In the GSE16131 FL series, high TNFRSF14 gene expression correlated with worse prognosis and GSEA showed that NFkB pathway was associated with the "High-TNFRSF14/dead-phenotype".In conclusion, the BTLA-TNFRSF14 immune modulation pathway seems to play a role in the pathobiology and prognosis of FL.

A diagnostic marker for superficial urothelial bladder carcinoma: lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression.

BACKGROUND: Pathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma. METHODS: Seven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation. RESULTS: ATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P = 0.021) and intravesical recurrence-free survival (P = 0.013) were detected between ATBF1+ (n = 110) and ATBF1- (n = 7) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P = 0.008). CONCLUSIONS: Cleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.

Inositol Hexakisphosphate Kinase 2 Promotes Cell Death in Cells with Cytoplasmic TDP-43 Aggregation.

TAR DNA-binding protein 43 (TDP-43) has been identified as a major component of ubiquitin-positive inclusions in the brains and spinal cords of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) or amyotrophic lateral sclerosis (ALS). The phosphorylated C-terminal fragment of TDP-43 forms aggregates in the neuronal cytoplasm, possibly resulting in neuronal cell death in patients with FTLD-U or ALS. The inositol pyrophosphate known as diphosphoinositol pentakisphosphate (InsP7) contains highly energetic pyrophosphate bonds. We previously reported that inositol hexakisphosphate kinase type 2 (InsP6K2), which converts inositol hexakisphosphate (InsP6) to InsP7, mediates cell death in mammalian cells. Moreover, InsP6K2 is translocated from the nucleus to the cytosol during apoptosis. In this study, we verified that phosphorylated TDP-43 co-localized and co-bound with InsP6K2 in the cytoplasm of anterior horn cells of the spinal cord. Furthermore, we verified that cell death was augmented in the presence of cytoplasmic TDP-43 aggregations and activated InsP6K2. However, cells with only cytoplasmic TDP-43 aggregation survived because Akt activity increased. In the presence of both TDP-43 aggregation and activated InsP6K2 in the cytoplasm of cells, the expression levels of HSP90 and casein kinase 2 decreased, as the activity of Akt decreased. These conditions may promote cell death. Thus, InsP6K2 could cause neuronal cell death in patients with FTLD-U or ALS. Moreover, InsP6K2 plays an important role in a novel cell death pathway present in FTLD-U and ALS.

A novel mutation (P236S) in the succinate dehydrogenase subunit B gene in a Japanese patient with a posterior mediastinal paraganglioma.

Succinate dehydrogenase subunit B gene (SDHB) is associated with the development of hereditary paraganglioma (PGL) and pheochromocytoma (PCC). Here we describe a novel germline mutation in SDHB in a 69-year-old Japanese woman with a posterior mediastinal PGL. We summarize the clinical presentation, diagnostic work-up, and pathological features of a patient with a posterior mediastinal PGL and review the pertinent literature. Direct sequencing of SDHB and SDHD was performed. The patient presented with a posterior mediastinal tumor and was normotensive. She underwent abdominal tumor resection at the age of 38 years, but clinical and pathological diagnoses were unknown. She had no family history of hypertension, PGL, or PCC. Imaging studies suggested that the tumor was neurogenic. Endocrinological examinations showed normal plasma catecholamine levels. The tumor was completely removed without metastasis. Pathological findings confirmed PGL. Immunohistochemical staining showed that the tumor cells were positive for chromogranin A, synaptophysin, and CD56, and the Ki67 index was low (<1 %). The patient has not experienced recurrence or metastasis for the last 5 years. DNA sequencing revealed a novel P236S (c.843 C > T) mutation in SDHB. The P236S germline mutation in SDHB was associated with posterior mediastinal PGL. Strict follow-up of the patient is necessary because the SDHB mutation may be related to malignancy.

Syndrome of inappropriate secretion of antidiuretic hormone caused by pituitary macroadenoma with hemangiomatous stroma.

A 55-year-old Japanese man was referred to our hospital because of disturbance of consciousness and hyponatremia. He had been aware of general fatigue, nausea, and headache for two weeks. Tests revealed hyponatremia, plasma hypoosmolarity with urine hyperosmolarity, an elevated level of urine sodium excretion, and normal functions of the kidney, adrenal gland, and thyroid. These findings were compatible with syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Head magnetic resonance imaging (MRI) demonstrated a pituitary tumor measuring 20 x 22 x 21 mm that pushed the pituitary stalk upward. Endocrinological evaluations suggested that the pituitary adenoma was non-functional. The pituitary adenoma was surgically removed, and histological examination revealed a biphasic appearance characterized by endocrine cells and a hemangiomatous stroma. After surgery, the patient developed pituitary hypothyroidism, pituitary adrenal insufficiency, and pituitary gonadal failure. Therefore, levothyroxine sodium, 50 µg per day, and hydrocortisone, 10 mg per day, were administered orally. Androgen depot, 250 mg every two months, was also injected intramuscularly. The hyponatremia did not recur, and the patient has done well for the last five years. The pituitary adenoma in this case showed two features: one was the cause of SIADH, and the other was a biphasic histological picture of endocrine cells with a hemangiomatous stroma.

Matrix metalloproteinase-13 promotes recovery from experimental liver cirrhosis in rats.

OBJECTIVE: To evaluate the role of matrix metalloproteinase (MMP)-13 gene expression in the early phase of recovery from liver fibrosis/cirrhosis. METHODS: Liver fibrosis was induced in male Wistar rats by administration of carbon tetrachloride (CCl(4)) for 10 weeks. Recombinant adenovirus-mediated human MMP-13 gene transfer (RAdMMP-13) was performed via the femoral vein on day 3 after the last CCl(4) injection. The role of MMP-13 in stably expressing cell lines was also analyzed. RESULTS: Fibrous deposition in the liver was decreased in RAdMMP-13-injected rats by day 3 after gene transfer compared with empty vector RAd66-injected rats. Furthermore, MMP-2 and MMP-9 enzymatic activity was markedly enhanced in the liver of RAdMMP-13 injected rats. Hepatocyte growth factor (HGF) induction was also increased in RAdMMP-13 injected rats. In established stable HT-1080 cells transfected with MMP-13, HGF-α expression and MMP-2 and MMP-9 enzymatic activity were increased. The conversion of precursor HGF into mature HGF was also increased in the MMP-13 expressing cell lines. CONCLUSION: Forced MMP-13 expression effectively accelerated recovery from liver cirrhosis via the effects of MMP-13-mediated HGF, MMP-2, and MMP-9 expression, which induced the degradation of collagen fibers and promoted hepatic regeneration.

Functional molecular morphology of anterior pituitary cells, from hormone production to intracellular transport and secretion.

Combined in situ hybridization (ISH) and immunohistochemistry (IHC) under electron microscopy (EM-ISH & IHC) has sufficient ultrastructural resolution to provide two-dimensional images of subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (Quantum dots; Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH & IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationship between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin revealed that both rab3B and SNARE system proteins play an important role and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. An experimental pituitary cell line, the GH3 cell, in which growth hormone (GH) is linked to enhanced yellow fluorescein protein (EYFP), has been developed. This stable GH3 cell secretes GH linked to EYFP upon being stimulated by Ca(2+) influx or Ca(2+) release from storage. This GH3 cell is useful for real-time visualization of the intracellular transport and secretion of GH. These three methods enable us to visualize consecutively the processes of transcription, translation, transport, and secretion of pituitary hormone.

Inositol hexakisphosphate kinases induce cell death in Huntington disease.

Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.

Methodological development of a clonogenic assay to determine endothelial progenitor cell potential.

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133(+) cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133(+) cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.

Molecular morphology of pituitary cells, from conventional immunohistochemistry to fluorescein imaging.

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca²+ influx or Ca²+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone.

Generation of a neutralizing human monoclonal antibody Fab fragment to surface antigen 1 of Toxoplasma gondii tachyzoites.

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 × 10(-9) M for Tox203 and 2.01 × 10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.

Cytoplasmic expression of the JM403 antigen GlcA-GlcNH3+ on heparan sulfate glycosaminoglycan in mammary carcinomas--a novel proliferative biomarker for breast cancers with high malignancy.

The expressions of heparan sulfate glycosaminoglycans (HSGAGs) in breast carcinoma specimens from 60 patients were immunohistochemically investigated using monoclonal antibodies (mAbs) that recognized different epitopes of the glycan structure. Cytoplasmic expression of GlcA-GlcNH(3)(+) on HSGAG was detected in carcinomas at high frequency (58.3%) using mAb JM403, whereas it was almost undetectable in normal breast ducts. This cytoplasmic expression was confirmed using confocal laser scanning microscopy. The expression of JM403 antigen in invasive carcinomas significantly correlated with nuclear atypia score (p = 0.0004), mitotic counts score (p = 0.0018), nuclear grade (p = 0.0061) and the incidence of metastasis to axillary lymph nodes (p = 0.0061). Furthermore, its expression was significantly correlated with the Ki67-labeling index in 55 invasive carcinomas (p < 0.05) as well as in 26 non-invasive carcinomas (5 non-invasive carcinomas and 21 non-invasive carcinomas that were observed in individual invasive carcinomas) (p < 0.005). Interestingly, the JM403 antigen GlcA-GlcNH(3)(+) was also expressed in the cytoplasm of normal crypt epithelial cells where Ki67 protein was expressed in the cell nuclei in the proliferative compartment of the human small intestines. To date, HSGAGs have generally been found to exist on cell surface membranes and in extracellular matrices as components of HS proteoglycans, and the negatively-charged sulfated domains on HSGAGs are considered to be important for their functions. However, our present findings indicate that the cytoplasmic expression of the JM403 antigen GlcA-GlcNH(3)(+) on positively charged, non-sulfated HSGAG may be involved in cell proliferation and associated with increased degrees of malignancy. The unordinary carbohydrate antigen of GlcA-GlcNH(3)(+) on HSGAGs recognized by mAb JM403 may represent a novel proliferative biomarker for highly malignant mammary carcinomas.

L157X nonsense mutation of the succinate dehydrogenase subunit B gene in a Japanese patient with right paraaortic paraganglioma.

Nuclear genes succinate dehydrogenase B subunit and succinate dehydrogenase D subunit, which encode two mitochondrial complex II subunits, are associated with the development of familial paraganglioma (PGL). Succinate dehydrogenase B subunit gene mutation is highly associated with extraadrenal PGL and subsequent distant metastasis. We describe the case of a 29-year-old Japanese man with a 3-year history of hypertension, headache, and palpitation. Endocrinological examinations showed that the patient had elevated levels of catecholamines, and imaging studies revealed a right paraaortic PGL without distant metastases. The PGL was surgically removed. Genetic analysis of the patient showed a heterozygous thymine deletion at position 470 (c.470delT) in exon 5 of the succinate dehydrogenase B subunit gene complementary DNA. This thymine deletion changed TTG (leucine) to TGA (stop codon) at codon 157 (L157X). It remains unclear whether this mutation was associated with PGL malignancy because the patient has had no metastases for the past 3 years. It has been recently reported that L157X is associated with malignant paraaortic PGL. Thus, strict follow-up is required because this succinate dehydrogenase B subunit gene's nonsense mutation (L157X) may be related to the malignancy.

Inositol hexakisphosphate kinases promote autophagy.

We and other authors have previously reported that increasing cellular diphosphoinositol pentakisphosphate (InsP(7)) levels increases cell sensitivity to cell death. In the present study, we elucidated the relationship between inositol hexakisphosphate kinases (InsP(6)Ks), which form InsP(7), and autophagy using InsP(6)Ks overexpression and disruption systems. A large number of autophagosomes were induced in cells transfected with InsP(6)Ks, as revealed by the conversion of LC3-I to LC3-II, which was examined using immunoblotting, immunocytochemistry, and immuno-electron microscopy for LC3; consequently, the rate of cell death was higher among these cells than among cells transfected with a control vector, as shown using propidium iodide staining. However, the reduction of InsP(6)Ks levels using RNAi suppressed the formation of autophagosomes. Moreover, the number of autophagosomes and the rate of cell death were significantly higher among cells transfected with InsP(6)Ks subjected to staurosporine-induced stress than among cells transfected with InsP(6)Ks subjected to normal conditions. The cell death induced by InsP(6)Ks was not completely suppressed by z-VAD-fmk, a pan-caspase inhibitor. The phosphorylation of mammalian target of rapamycin (mTOR) was also depressed in cells overexpressing InsP(6)Ks, suggesting that the mTOR pathway regulates autophagosomes generated by InsP(6)Ks. These findings imply that InsP(6)Ks promote autophagy and induce caspase-independent cell death. This phenomenon opens a new pathway of autophagy via InsP(6)Ks.

Somatostatin analogue inhibits the mobility of prostate carcinoma cells: a new therapeutic method for advanced prostate carcinoma.

The somatostatin analogue (SA) Octreotide has been used as a therapeutic reagent for somatostatin receptor type 2a (SSTR2a)-positive cancers. The purpose of this study is to detect SSTR2a in human prostate carcinomas and to elucidate the effects of SA on SSTR2a-positive prostate carcinoma cells to determine the potential of this drug as a new therapeutic method for advanced prostate carcinoma. Immunohistochemical study of SSTR2a was performed on 95 prostate carcinoma cases, and the results showed expression of SSTR2a in 14 of the 95 cases (14.74%); the histological grade (Gleason) and capsular invasion of the prostate carcinoma were directly related to SSTR2a expression. Among the ten cases of lymph node metastasis, SSTR2a expression was markedly higher. In vitro studies were performed using SSTR2a-positive prostate cancer cells, DU145 and PC3. Migration and invasion abilities of DU145 and PC3 cells were inhibited by SA in a dose-dependent manner. This inhibition was reversed by Rho-kinase inhibitor Y-27632. Morphological changes of the prostate cancer cells treated with SA and Y27632 corroborate the migration and invasion assays, although SA had no effect on proliferation of DU145 and PC3 cells. In conclusion, the somatostatin analogue may be beneficial for patients with advanced prostate carcinoma or to protect from distal metastasis if they are positive for SSTR2a.