Inflammatory cells play important roles in progression of solid neoplasms including ovarian cancers. Tumor-associated macrophages (TAMs) contribute to angiogenesis and immune suppression by modulating microenvironment. Ovarian cancer develops occasionally on the bases of endometriosis, a chronic inflammatory disease. We have recently demonstrated differential expressions of CXCR3 variants in endometriosis and ovarian cancers. In this study, we showed impaired CXCL4 expression in TAMs of ovarian cancers arising in endometriosis. The expressions of CXCL4 and its variant CXCL4L1 were investigated among normal ovaries (n = 26), endometriosis (n = 18) and endometriosis-associated ovarian cancers (EAOCs) composed of clear cell (n = 13) and endometrioid (n = 11) types. In addition, four cases of EAOCs that contained both benign and cancer lesions contiguously in single cysts were investigated in the study. Western blot and quantitative RT-PCR analyses revealed significant downregulation of CXCL4 and CXCL4L1 in EAOCs compared with those in endometriosis. In all EAOCs coexisting with endometriosis in the single cyst, the expression levels of CXCL4 and CXCL4L1 were significantly lower in cancer lesions than in corresponding endometriosis. Histopathological study revealed that CXCL4 was strongly expressed in CD68 (+) infiltrating macrophages of endometriosis. In microscopically transitional zone between endometriosis and EAOC, CD68 (+) macrophages often demonstrated CXCL4 (-) pattern. The majority of CD68 (+) TAMs in overt cancer lesions were negative for CXCL4. Collective data indicate that that CXCL4 insufficiency may be involved in specific inflammatory microenvironment of ovarian cancers arising in endometriosis. Suppression of CXCL4 in cancer lesions is likely to be attributable to TAMs in part.

Angiotensin II receptor-like 1 (APJ), a G protein-coupled receptor that was identified as a homologue of angiotensin II type 1 (AT1) receptor, exerts antagonistic effects on AT1-mediated vasoconstriction. Studies on pregnancy-induced hypertension (PIH) revealed aberrant activation of AT1 downstream signaling. In contrast, little is known about APJ in the pathophysiology of human pregnancy. In this study, we investigated APJ expression in normal human and PIH placentas. mRNAs were extracted from 50 placental villous tissues of 18 cases with severe PIH (8 late-onset, 4 early-onset, and 6 superimposed PIH) and 32 control pregnancies (including 6 preterm cases). Histopathologic studies were conducted using paraffin-embedded placental tissues from 12 control placentas (from 23 to 39 wk) and 23 PIH placentas (from 24 to 41 wk). Reverse transcriptase-polymerase chain reaction showed that APJ was cooperatively expressed with its ligand apelin and AT1 in controls and in late-onset PIH placentas but was significantly downregulated in early-onset PIH placentas with poor fetal growth. Quantitative reverse transcriptase-polymerase chain reaction analysis revealed upregulated APJ in late-onset PIH placentas but significantly downregulated APJ in early-onset PIH. In immunohistochemical staining, APJ was detected strongly in villous capillary endothelial cells and trophoblasts of late-onset PIH placentas. In contrast, APJ was poorly stained in endothelial cells of hypoplastic villi of early-onset PIH placentas. Collective data indicate that the apelin-APJ system is involved in fetoplacental circulation during human pregnancy. Impaired APJ expression in early-onset PIH placentas may reflect an aggravated placental condition with poor fetal growth.

BACKGROUND: Although triple-negative breast cancer (TNBC) with epidermal growth factor receptor (EGFR) expression has been extensively studied, few studies have simultaneously examined EGFR expression and EGFR gene amplification. Here, we examined the correlations of EGFR expression with EGFR gene amplification, EGFR-activating mutations, and the expression of components of the Akt pathway. METHODS: Tumor tissues were obtained from 84 patients with TNBC. We analyzed the expression of EGFR, phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), and other relevant proteins using immunohistochemistry. We also analyzed EGFR gene and chromosome 7 copy numbers by dual-color in situ hybridization. DNA was extracted from formalin-fixed paraffin-embedded samples. Analysis of EGFR gene-activating mutations was performed using the smart amplification process version 2 assay. RESULTS: Most TNBCs expressing EGFR are non-specialized invasive ductal carcinomas, whereas others are likely to be rare specialized carcinomas, such as typical medullary carcinoma, apocrine carcinoma, metaplastic carcinoma, and adenoid cystic carcinoma. EGFR was expressed in samples from 28 of 84 (33.3 %) patients, but the EGFR gene was not amplified in any of the 84 samples. There were significant correlations between EGFR expression and the number of polysomic cells and the presence of high polysomy of chromosome 7. However, EGFR expression was not correlated with p-Akt or p-mTOR expression, nor with the other clinicopathological factors recorded in this study. We found no evidence of EGFR gene-activating mutations. CONCLUSIONS: EGFR gene amplification and EGFR-activating mutations might not be the mechanisms leading to the constitutive activation of EGFR in TNBC. Further investigation is needed to clarify the other molecular mechanisms for oncogenic activation of EGFR in TNBC.

In an attempt to evaluate the roles of the mismatch repair gene Msh2 in genome maintenance and in development during the fetal stage, spontaneous mutations and several developmental indices were studied in Msh2-deficient lacZ-transgenic mouse fetuses. Mutation levels in fetuses were elevated at 9.5dpc (days post coitum) when compared to wild-type mice, and the level of mutations continued to increase until the fetuses reached the newborn stage. The mutation levels in 4 different tissues of newborns showed similar magnitudes to those in the whole body. The levels remained similar after birth until 6 months of age. The molecular nature of the mutations examined in 12.5dpc fetuses of Msh2(+/+) and Msh2(-/-) revealed unique spectra which reflect errors produced during the DNA replication process, and those corrected by a mismatch repair system. Most base substitutions and simple deletions were reduced by the presence of the Msh2 gene, whereas G:C to A:T changes at CpG sequences were not affected, suggesting that the latter change was not influenced by mismatch repair. On the other hand, analysis of developmental indices revealed that there was very little effect, including the presence of malformations, resulting from Msh2-deficiencies. These results indicate that elevated mutation levels have little effect on the development of the fetus, even if a mutator phenotype appears at the organogenesis stage.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by fibrofolliculomas, renal tumors, and pulmonary cysts with recurrent pneumothorax. Multiple pulmonary cysts and pneumothorax are the key signs for diagnosing BHD syndrome. The pathologic features of BHD pulmonary cysts, however, are poorly understood. This disorder is caused by mutations in the gene that encodes folliculin (FLCN). FLCN is regarded as a tumor suppressor; it mediates cellular activities by interacting with the mammalian target of rapamycin (mTOR). In this study, we investigated the lungs of 11 patients from 9 BHD families. The majority of patients consulting doctors were women between 30 and 60 years of age who had pulmonary cysts and repeated pneumothoraces. Genomic DNA testing revealed 5 different mutation patterns. Histopathologic examination found that the inner surface of cysts was lined by epithelial cells, sometimes with a predominance of type II pneumocyte-like cuboidal cells. The cysts occasionally contained internal septa consisting of alveolar walls or showed an "alveoli within an alveolus" pattern. The cells constituting the cysts stained positive for phospho-S6 ribosomal protein expression, suggesting activation of the mTOR pathway. Although BHD pulmonary cysts are frequently misdiagnosed as nonspecific cystic diseases, they are distinctly different in histopathology from other bullous changes. Mechanical stress such as rupture and postrupture remodeling allows mesothelial invagination and fibrosis. Such modified BHD pulmonary cysts are virtually indistinguishable from nonspecific blebs and bullae. We propose a new insight, namely, that the BHD syndrome-associated pulmonary cyst may be considered a hamartoma-like cystic alveolar formation associated with deranged mTOR signaling.

Glucagon-like peptide-1 (GLP-1) and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important to elucidate a mechanism to maintain energy balance. In the present study, coadministration of subthreshold GLP-1 and leptin dramatically reduced feeding in rats. Although coadministration of GLP-1 with leptin did not enhance leptin signal transduction in the hypothalamus, it significantly decreased phosphorylation of AMP-activated protein kinase (AMPK). In addition, coadministration of GLP-1 with leptin significantly increased proopiomelanocortin (POMC) mRNA levels. Considering that α-melanocortin stimulating hormone (α-MSH) is derived from POMC and functions through the melanocortin-4-receptor (MC4-R) as a key molecule involved in feeding reduction, the interaction of GLP-1 and leptin on feeding reduction may be mediated through the α-MSH/MC4-R system. As expected, the interaction of GLP-1 and leptin was abolished by intracerebroventricular preadministration of the MC4-R antagonists agouti-related peptide and SHU9119. Taken together, GLP-1 and leptin cooperatively reduce feeding at least in part via inhibition of AMPK following binding of α-MSH to MC4-R.

We report a case of glomerular cystic kidney disease (GCKD) associated with tuberous sclerosis complex (TSC) in a neonate. The patient displayed progressive abdominal enlargement attributed to GCKD associated with TSC. After birth, the right kidney was resected because it compressed his liver and right lung, and possible malignancy could not be excluded. Macroscopically, the resected kidney was markedly enlarged, and histologically the kidney had numerous glomerular cysts accompanied by papillary epithelial growth. Notably, a small area of normal parenchyma was observed at the lower pole. The epithelial cells of the cysts displaying a papillary growth pattern were positive for mTOR, phosphorylated mTOR, and phosphorylated S6 ribosomal protein (p-S6). The morphologically noncystic, normal-looking tubular epithelium was also positive for p-S6. These results imply that one more molecular event might be necessary for cyst formation in GCKD associated with TSC, in addition to the activation of mTOR signaling.

Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1-12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1-12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids ((15)RKESKK(20)) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.

Birt-Hogg-Dubé syndrome is a familial genodermatosis, of which patients frequently develop renal neoplasms, fibrofolliculomas and pneumatocele. Here, we report a mother and daughter with renal neoplasms surgically resected (69 and 46 years-of-age at surgery, respectively). The mother's tumor was diagnosed as an unclassified type renal cell carcinoma associated with microscopic tumorous nodules, whereas the daughter's tumor was a hybrid oncocytic/chromophobe tumor. The germline mutation analysis of the responsible gene, FCLN (the folliculin gene), showed a deletion of 18 bp in exon 5 (c.332_349del/p.H111_Q116del), predicting an alteration of the amino acid sequence of "HPSHPQ" replaced by a single amino acid,"L". This is a novel germline mutation of the FCLN gene that has not been previously reported.

Mirror therapy is an effective technique for pain relief and motor function recovery. It has been demonstrated that magnetic 20-Hz activity is induced in the primary motor cortex (M1) after median nerve stimulation and that the amount of the stimulus-induced 20-Hz activity is decreased when the M1 is activated. In the present study, we investigated how the image or the mirror reflection of a hand holding a pencil modulates the stimulus-induced 20-Hz activity in the M1. Neuromagnetic brain activity was recorded from 13 healthy right-handed subjects while they were either viewing directly their hand holding a pencil or viewing a mirror reflection of their hand holding a pencil. The 20-Hz activity in the left or the right M1 was examined after the right or the left median nerve stimulation, respectively, and the suppression of the stimulus-induced 20-Hz in the M1 by viewing directly one hand holding a pencil or by viewing the mirror image of the hand holding a pencil was assumed to indicate the activation of the M1. The results indicated that the M1 innervating the dominant hand was suppressed either by viewing directly the dominant hand holding a pencil or by viewing the mirror image of the non-dominant hand holding a pencil. On the other hand, the M1 innervating the non-dominant hand was activated by viewing the mirror image of the dominant hand holding a pencil, but was not activated by viewing directly the non-dominant hand holding a pencil. The M1 innervating either the dominant or the non-dominant hand, however, was not activated by viewing the hand on the side ipsilateral to the M1 examined or the mirror image of the hand on the side contralateral to the M1 exaimined. Such activation of the M1 might induce some therapeutic effects of mirror therapy.