Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839)

Gefitinib (Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine kinase, has shown potent anti-tumor effects and improved symptoms and quality-of-life of a subset of patients with advanced non-small cell lung cancer (NSCLC). However, a large portion of the patients showed no effect to this agent. To establish a method to predict the response of NSCLC patients to gefitinib, we used a genome-wide cDNA microarray to analyze 33 biopsy samples of advanced NSCLC from patients who had been treated with an identical protocol of second to seventh line gefitinib monotherapy. We identified 51 genes whose expression differed significantly between seven responders and 10 non-responders to the drug. We selected the 12 genes that showed the most significant differences to establish a numerical scoring system (GRS, gefitinib response score), for predicting response to gefitinib treatment. The GRS system clearly separated the two groups without any overlap, and accurately predicted responses to the drug in 16 additional NSCLC cases. The system was further validated by the semi-quantitative RT-PCR, immunohistochemistry and ELISA for serological test. Moreover, we proved that the anti-apoptotic activity of amphiregulin, a protein that was significantly over-expressed in non-responders but undetectable in responders, leads to resistance of NSCLC cells to gefitinib in vitro. Our results suggested that sensitivity of a given NSCLC to gefitinib can be predicted according to expression levels of a defined set of genes that may biologically affect drug sensitivity and survival of lung cancer cells. Our scoring system might eventually lead to achievement of personalized therapy for NSCLC patients.     

Angiogenic endothelium-specific nestin expression is enhanced by the first intron of the nestin gene

Nestin is a member of intermediate filaments abundantly expressed in neural stem cells and glioblastomas. The nestin gene has four exons and three introns, and neural cell-specific expression is regulated by the second intron. We previously reported that nestin was invariably detected in the tumor endothelium in gliomas even though tumor cells were negative for nestin. In the present study, we further confirmed nestin immunostaining in tumor endothelium of a variety of common cancers, including lung, stomach, colon, and cervical carcinomas. We examined an endothelium-specific regulator using human umbilical vein endothelial cells (HUVECs) and human glioblastoma-derived U251 cells. In a luciferase reporter assay, the first intron plus 5' upstream promoter (5'UP) gave the highest activity, followed by 5'UP, and the second intron plus 5'UP. However, the assay values were much lower by HUVEC extracts than by U251 cell extracts. Although green fluorescent protein expression was positive over all U251 cells under either the first intron, second intron, or ubiquitously active CAG promoter, the fluorescence in HUVECs was limited to a few cells even under the first intron. This difference came from the growth feature of HUVECs which exhibit growth arrest by contact inhibition. We found that the nestin expression was specific to proliferative endothelium, by using proliferation markers in hemangioblastomas and in situ hybridization. Using an endothelial tube formation assay, tyrosine kinase domain-deleted VEGF receptor KDR effectively abolished the tube formation under the first intron. We suggest that the nestin expression in tumor endothelium is enhanced by the first intron.     

Preemptive analgesia by zaltoprofen that inhibits bradykinin action and cyclooxygenase in a post-operative pain model

The post-operative pain state results from a barrage of primary afferent inputs exposed to products of tissue damage such as bradykinin and prostaglandins and the central sensitization by the continuing inputs. This provides the rationale for preemptive analgesia, whereby the blockade of primary afferent inputs prior to injury may result in a reduction of post-operative pain. 2-(10,11-dihydro-10-oxo-dibenzo[b,f]thiepin-2-yl) propionic acid (zaltoprofen) is a unique compound that inhibits cyclooxygenase (COX) and exhibits anti-bradykinin activity. The present study evaluated the preemptive analgesic effect of zaltoprofen in a post-operative pain model produced by plantar incision. When orally, but no intrathecally, administered 30 min prior to incision, zaltoprofen significantly increased the withdrawal threshold 2 h and 1-3 days after incision at 10 mg/kg. While the bradykinin B1 antagonist des-Arg10-HOE-140, the selective COX-1 inhibitor SC-560, and the selective COX-2 inhibitor celecoxib did not affect post-operative pain, the B2 antagonist HOE-140 dose-dependently relieved the post-operative pain at 2-200 microg/kg with a time course similar to that of zaltoprofen. The B2 receptor mRNA was expressed in the hindpaw and the expression did not change before and 24 h after surgery. These results suggest that zaltoprofen produces the preemptive analgesic effect peripherally by blocking the B2 pathway.     

Biotin deficiency in amino acid formula nutrition for an infant with milk protein allergy]

We reported a 4-month-old girl with biotin deficiency caused by amino acid formula. Two weeks after birth, she was diagnosed as having a milk protein allergy. After switching to amino acid formula from usual formula, her symptoms and laboratory findings became normal. About three weeks after the beginning of amino acid formula, she developed intractable skin erosions around the eyes, mouth, neck, and anogenital area. By measuring concentrations of some trace elements, she was diagnosed as having a biotin deficit, because of the organic aciduria and undetectable serum biotin concentration. Her serum biotinidase level was normal. Upon administration of oral biotin supplementation, all her symptoms and laboratory findings were dramatically improved. Since amino acid formula contains very few biotin, we should pay attention to biotin deficiency when infants receiving amino acid formula.     

Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung

A variety of pathological changes are seen in lymphoproliferative disorders of the lung but the histogenesis of these abnormalities is not yet fully understood. We previously showed that adenovirus vector-mediated transient expression of both the human interleukin-6 (IL-6) and IL-6 receptor (IL-6R) genes, but not the IL-6 gene alone, in the rat lung induced lymphocytic alveolitis. In the present study, we explored the lung pathology of human IL-6 and IL-6R double transgenic mice to elucidate the effects of prolonged IL-6 signalling on the lung. The transgenic animals developed mononuclear cell accumulation in peribronchovascular regions, but little infiltration into alveolar spaces. Immunohistochemical analysis revealed that the cellular accumulations contained not only mixtures of inflammatory cells but also lymphoid tissue-like structures. As the expression of CXCL13/BLC, the indispensable chemokine for lymphoid organogenesis, was recognized in the B cell follicles of the pulmonary lesions, we speculate that this chemokine plays an inductive role in the development of the lymphoid tissue-like structures. These structures were distinguished from bronchus-associated lymphoid tissues (BALTs) by their location and by the lack of lymphoepithelium, which is a characteristic of BALT. These findings imply that IL-6 signalling may play a role in the pathogenesis of lymphoproliferative disorders of the lung.     

Freeze-fracture studies of muscle plasma membranes in myopathic patients with hypo-and hyperthyroidism

To examine the influence of thyroid hormone on the skeletal muscle plasma membrane, we analyzed the changes in ultrastructural architecture and membrane area complexed with digitonin of muscle plasma membrane in myopathic patients with hypo-and hyperthyroidism by the conventional freeze-fracture (F-F) technique and F-F cytochemistry using the sterol-specific ligand digitonin. The densities of flask-shaped invaginations, which are mainly thought to correspond to caveolae, intramembranous particles, and orthogonal arrays, and the changes of digitoninreacted membrane areas in the muscle plasma membranes in three patients with hypothyroid myopathy and one patient with both myasthenia gravis and hyperthyroidism were compared with those in age-matched controls. In the conventional F-F study, the muscle plasma membrane of hypothyroid patients showed increased invagination density, whereas that of the hyperthyroid patient was normal ultrastructurally. In the F-F cytochemistry study, however, the ratio of digitonin-reacted membrane areas versus fractured membrane areas was not different between hypothyroid patients and controls, whereas that of the hyperthyroid patient was lowered in comparison with that of control. These results suggest that thyroid hormone may alter the biochemical properties and ultrastructural architecture of muscle plasma membrane.This study was presented at the 29th annual meeting of the Clinical Electron Microscopy Society of Japan, Sapporo, October 2–3, 1997     

Cell surface laminin-like substances and laminin-related carbohydrates of rat ascites hepatoma AH7974 and its variants with different lung-colonizing potential

Rat ascites hepatoma AH7974 cells strongly expressed antilaminin antibody-reactive substances (laminin-like substances) andGriffonia simplicifolia isolectin B4 (GS)-reactive carbohydrate (alpha)-d-galactose; alpha-Gal) on their cell surface. The alpha-Gal expression was not apparently influenced by the pretreatment of cells with methanol. The cell membrane laminin-like substances had approximate molecular weights of 150, 62 and 56 kDa in denaturating reducing conditions, of which the 62 and 56 kDa bands were stained with GS. The cell membrane molecules bearing alpha-Gal were 62 and 56 kDa and were stained with antilaminin antibody. Therefore, the major molecules bearing alpha-Gal residues of AH7974 cell membrane are considered to be laminin-like substances. To determine the role of the substances in metastasis, we selected four cell lines (74AD, 74AD-f, 74FL, 74FL-a) from AH7974 in culture. 74AD and 74FL-a are adherent lines and 74AD-f and 74FL are floating lines. All of these cell lines strongly expressed laminin-like substances, but a marked difference was found in expression of alpha-Gal, which was most strongly expressed by 74FL, followed by 74AD, and rarely by 74AD-f and 74FL-a; the staining intensity was positively correlated with their experimental lung-colonizing potential. Cell membrane laminin-like substances were 200, 97, 62, 56 and 46 kDa and among them 62 and 56 kDa molecules were glycosylated with alpha-Gal. The pretreatment of 74FL cells with antilaminin antibody or with human type A serum (containing natural antibody to alpha-Gal epitope) depressed remarkably the lung-colonizing potential of the cells. These results suggest that the expression of 62 and 56 kDa laminin-like substances with alpha-Gal residues on tumor cell surfaces is one of the determinants associated with lung-colonizing potential of these cells.     

Evolution of interleukin-18 binding proteins and interleukin-1 receptor, type II proteins

Interleukin-18 (IL-18) is one of the pivotal cytokines controlling the defense mechanism called inflammation. As a first step to develop proteins for controlling the IL-18 level, we initiated a study of IL-18-binding proteins (IL-18BPs). Twenty-four IL-18BP family members, 11 from vertebrates and 13 from chordopoxviruses, were picked from the NCBI database. Eight of these vertebrate IL-18BPs and two of the chordopoxvirus IL18-BPs were identified here and characterized as new members of the IL-18BP family. Their IL-18 binding domains were aligned and the distribution of highly conserved critical amino acid residues was analyzed and used to construct a phylogenetic tree. From this tree it was inferred that at least two independent events created two different ancestral viral IL-18BP genes by retroposition of IL-18BP genes from the vertebrate lineage. These two events are estimated to have occurred after an ancient mammalian IL-18BP gene diverged from birds, and before the mammalian IL-18BP gene diverged into human, ungulate and rodent IL-18BP genes. Moreover, our results suggest that IL-18BP and interleukin-1 receptor, type II (IL-1R2) had a common ancestral gene and diverged from the ancestral gene into IL-18BP and IL-1R2 genes in the fish period.     

Polymorphous low-grade adenocarcinoma of submandibular gland origin

A case of polymorphous low-grade adenocarcinoma (PLGA) in the submandibular gland is reported. A 72 year old woman presented with a 5 year history of a gradually expanding tumor in the submandibular region. The surgical specimen revealed a relatively well demarcated tumor, 35 × 35 × 20 mm in size. Macroscopically, necrosis and hemorrhage were not seen in the solid tumor. Histologically, the tumor growth pattern was variable, composed of tubular, papillary, solid, trabecular and cribriform structures. Immunohistochemically, some tumor cells were positive for epithelial membrane antigen (EMA), S-100 protein, keratin, and carcino-embryonic antigen (CEA). Electron microscopically, prominent microvilli projected into the luminal spaces, and basal lamina and hemidesmosomes were seen in the tumor cells adjacent to the connective tissues. The submandibular gland is an extremely rare location for PLGA. To the authors' knowledge, this is the first case of its kind reported in the English literature.     

Comparison of standard and hand-assisted laparoscopic radical nephrectomy for renal cell carcinoma.

A laparoscopic radical nephrectomy (LRN) for renal cancer can be performed using two methods, hand-assisted laparoscopic surgery (HALS) and standard laparoscopic surgery (SLS). This institute initially used HALS to perform all radical nephrectomy, but gradually shifted to SLS. This study compared the two methods of radical nephrectomy: HALS vs. SLS, which were performed at a single institute. From March 1999 to November 2006, a total 129 patients with pathologically confirmed renal cell carcinoma underwent LRN, including 73 patients with the HALS and 56 patients with SLS. The median operative time was 264 minutes, and median estimated blood loss was 200 ml in the HALS group, respectively. The median operative time and median estimated blood loss in the SLS were 215 minutes and 100 ml, respectively. There was no significant difference in either the operative time or estimated blood loss between HALS and SLS. The median time to both postoperative oral intake and ambulation in the SLS were 1 day. Neither of these events after SLS was significantly shorter than that after HALS. The 4-year disease-free and overall survival rates in the HALS patients were 97.5% and 98.2%, respectively. Both the 4-year disease-free and overall survival rates in the SLS patients were 100%. Since no significant differences were observed between the two operative methods (SLS and HALS) regarding the operative data, postoperative course and oncological outcome, the surgical method for LRN can be selected according to characteristics of each surgical method.