A Case of Cancer of Unknown Primary Presenting with Chylothorax.

A 56-year-old man presented to our hospital with dyspnea on exertion for two months. Bilateral pleural effusions were found, and a close examination revealed a chylothorax, including adenocarcinoma. The primary tumor could not be identified by systemic examination. Therefore, the patient was diagnosed with cancer of unknown primary origin (CUP) presenting with chylothorax. Chemotherapy was administered for CUP, and thoracentesis, pleurodesis, ascites puncture, and nutritional therapy were performed for chylothorax and chylous ascites. Although drainage frequency and tumor marker levels (CA19-9, DUPAN-2, and Span-1) temporarily decreased, disease control deteriorated, and the patient died 12 months after the initial diagnosis.

Brain-wide network analysis of resting-state neuromagnetic data.

The present study performed a brain-wide network analysis of resting-state magnetoencephalograms recorded from 53 healthy participants to visualize elaborate brain maps of phase- and amplitude-derived graph-theory metrics at different frequencies. To achieve this, we conducted a vertex-wise computation of threshold-independent graph metrics by combining proportional thresholding and a conjunction analysis and applied them to a correlation analysis of age and brain networks. Source power showed a frequency-dependent cortical distribution. Threshold-independent graph metrics derived from phase- and amplitude-based connectivity showed similar or different distributions depending on frequency. Vertex-wise age-brain correlation maps revealed that source power at the beta band and the amplitude-based degree at the alpha band changed with age in local regions. The present results indicate that a brain-wide analysis of neuromagnetic data has the potential to reveal neurophysiological network features in the human brain in a resting state.

Mediastinal Lymph Node Metastasis of Esophageal Cancer with Esophageal Stenosis Diagnosed via Transesophageal Endoscopic Ultrasound with Bronchoscope-guided Fine-needle Aspiration.

An 80-year-old man underwent follow-up examinations after endoscopic submucosal dissection (ESD) for esophageal cancer. Computed tomography showed enlarged lymph nodes of the right recurrent nerve. The patient had esophageal stenosis due to repeated ESD for multiple esophageal tumors. The stenosis made the passage of an endoscopic ultrasound (EUS) scope through the esophagus difficult. Thus, an endobronchial ultrasound bronchoscope, which had a thinner diameter than that of the EUS scope, was used for transesophageal endoscopic ultrasound with bronchoscope-guided fine-needle aspiration. This technique led to the diagnosis of mediastinal lymph node metastasis of esophageal cancer.

Pancreatic Exocrine Insufficiency in Intraductal Papillary Mucinous Carcinoma Presenting with Leg Edema Treated with Pancreatic Exocrine Replacement Therapy.

An 89-year-old woman underwent examinations for leg edema. Blood tests indicated low nutrition and low pancreatic enzymes, and a stool examination indicated fatty stool. Computed tomography showed pleural effusion, ascites, and cystic lesions in the pancreatic head and mural nodules within the cysts. Pancreatic juice cytology revealed adenocarcinoma. The diagnosis was pancreatic exocrine insufficiency caused by intraductal papillary mucinous carcinoma. The patient did not wish to undergo surgery. Therefore, diuretics, component nutrients, and pancreatic exocrine replacement therapy using pancrelipase were initiated. After starting treatment, her leg edema, pleural effusion, and ascites disappeared, and her activities of daily living improved markedly.

Metabolomic analysis and mass spectrometry imaging after neonatal stroke and cell therapies in mouse brains.

Ischemic brain injury provokes complex, time-dependent downstream pathways that ultimately lead to cell death. We aimed to demonstrate the levels of a wide range of metabolites in brain lysates and their on-tissue distribution following neonatal stroke and cell therapies. Postnatal day 12 mice underwent middle cerebral artery occlusion (MCAO) and were administered 1 × 105 cells after 48 h. Metabolomic analysis of the injured hemisphere demonstrated that a variety of amino acids were significantly increased and that tricarboxylic acid cycle intermediates and some related amino acids, such as glutamate, were decreased. With the exception of the changes in citric acid, neither mesenchymal stem/stromal cells nor CD34+ cells ameliorated these changes. On-tissue visualization with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging revealed that the signal intensity of glutamate was significantly decreased in the infarct area, consistent with the metabolomic analysis, while its intensity was significantly increased in the peri-infarct area after MCAO. Although cell therapies did not ameliorate the changes in metabolites in the infarct area, mesenchymal stem cells ameliorated the increased levels of glutamate and carnitine in the peri-infarct area. MALDI-MS imaging showed the location-specific effect of cell therapies even in this subacute setting after MCAO. These methodologies may be useful for further investigation of possible treatments for ischemic brain injury.

Efficacy and Safety of the Modified EPOCH Regimen (Etoposide, Vincristine, Doxorubicin, Carboplatin, and Prednisolone) for Adult T-cell Leukemia/Lymphoma: A Multicenter Retrospective Study.

BACKGROUND: We retrospectively analyzed patients with untreated aggressive adult T-cell leukemia/lymphoma who received the modified EPOCH (mEPOCH) regimen. PATIENTS AND METHODS: Patients received up to 6 mEPOCH cycles. Etoposide (50 mg/m2/day), doxorubicin (10 mg/m2/day), and vincristine (0.4 mg/m2/day) were each given as a continuous 96-hour infusion on days 1 to 4. Prednisolone (40 mg/m2/day) was given intravenously or orally on days 1 to 4 and then tapered and stopped on day 7, and carboplatin (dose calculated for each patient individually using Calvert's formula according to a target under the curve of 3 mg/mL/min) was given as a 2-hour intravenous infusion on day 6. RESULTS: In 103 patients, overall response rate and complete response rate were 58% and 25%, respectively. With a median follow-up of 8.9 months, the median survival time was 9.8 months (95% confidence interval, 7.2-13.9 months). The median progression-free survival (PFS) was 4.2 months (95% confidence interval, 3.4-5.7 months). Patients who completed ≥ 4 cycles experienced significantly better overall survival and PFS compared with those who completed < 4 cycles. Twenty-eight patients underwent allogeneic hematopoietic stem cell transplantation after mEPOCH and demonstrated significantly prolonged overall survival and PFS compared with those who did not undergo transplantation. CONCLUSION: The mEPOCH regimen is effective with tolerable adverse effects and may be an alternative treatment option for adult T-cell leukemia/lymphoma.

Promising Gene Therapy Using an Adenovirus Vector Carrying REIC/Dkk-3 Gene for the Treatment of Biliary Cancer.

BACKGROUND: We previously demonstrated that the reduced expression in immortalized cells (REIC)/dikkopf-3 (Dkk-3) gene was downregulated in various malignant tumors, and that an adenovirus vector carrying the REIC/Dkk-3 gene, termed Ad-REIC induced cancer-selective apoptosis in pancreatic cancer and hepatocellular carcinoma. OBJECTIVE: In this study, we examined the therapeutic effects of Ad-REIC in biliary cancer using a second- generation Ad-REIC (Ad-SGE-REIC). METHODS: Human biliary cancer cell lines (G-415, TFK-1) were used in this study. The cell viability and apoptotic effect of Ad-SGE-REIC were assessed in vitro using an MTT assay and Hoechst staining. The anti-tumor effect in vivo was assessed in a mouse xenograft model. We also assessed the therapeutic effects of Ad-SGE-REIC therapy with cisplatin. Cell signaling was assessed by Western blotting. RESULTS: Ad-SGE-REIC reduced cell viability, and induced apoptosis in biliary cancer cell lines via the activation of the c-Jun N-terminal kinase pathway. Ad-SGE-REIC also inhibited tumor growth in a mouse xenograft model. This effect was further enhanced in combination with cisplatin. CONCLUSION: Ad-SGE-REIC induced apoptosis and inhibited tumor growth in biliary cancer cells. REIC/Dkk-3 gene therapy using Ad-SGE-REIC is an attractive therapeutic tool for biliary cancer.

Autologous cord blood cell therapy for neonatal hypoxic-ischaemic encephalopathy: a pilot study for feasibility and safety.

Neonatal hypoxic-ischaemic encephalopathy (HIE) is a serious condition; many survivors develop neurological impairments, including cerebral palsy and intellectual disability. Preclinical studies show that the systemic administration of umbilical cord blood cells (UCBCs) is beneficial for neonatal HIE. We conducted a single-arm clinical study to examine the feasibility and safety of intravenous infusion of autologous UCBCs for newborns with HIE. When a neonate was born with severe asphyxia, the UCB was collected, volume-reduced, and divided into three doses. The processed UCB was infused at 12-24, 36-48, and 60-72 hours after the birth. The designed enrolment was six newborns. All six newborns received UCBC therapy strictly adhering to the study protocol together with therapeutic hypothermia. The physiological parameters and peripheral blood parameters did not change much between pre- and postinfusion. There were no serious adverse events that might be related to cell therapy. At 30 days of age, the six infants survived without circulatory or respiratory support. At 18 months of age, neurofunctional development was normal without any impairment in four infants and delayed with cerebral palsy in two infants. This pilot study shows that autologous UCBC therapy is feasible and safe.

Dipeptide γ-secretase inhibitor treatment enhances the anti-tumor effects of cisplatin against gastric cancer by suppressing cancer stem cell properties.

The γ-secretase inhibitor blocks Notch activity by preventing its cleavage at the cell surface. In the present study, the effect of the γ-secretase inhibitor on the viability of gastric cancer cells when administered in combination with cisplatin was investigated, with particular focus on CD44highLgr-5high cancer cells. The four gastric cancer cell lines, MKN45, MKN74, SC-6-JCK and SH-10-TC, were used for the experiments. In the MTT assay, treatment with 25 µM dipeptide γ-secretase inhibitor (DAPT) alone did not affect cell proliferation in any of the four cell lines. Gastric cancer cells subjected to combination treatment with DAPT and cisplatin exhibited decreased viability when compared with those treated with cisplatin alone. Flow cytometry was performed to evaluate the expression of cluster of differentiation (CD)-44 and leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr-5), two cancer stem cell markers in gastric cancers. Treatment with cisplatin alone significantly increased the proportion of CD44highLgr-5high cells. However, the addition of DAPT to cisplatin reduced the CD44highLgr-5high fraction, suggesting that DAPT reduced the number of gastric cancer cells. In conclusion, the present study demonstrated the synergistic effects of DAPT in combination with cisplatin by decreasing the survival of gastric cancer cells. In addition, combination treatment with DAPT reduced the number of CD44highLgr-5high cells, which are thought to exhibit cancer stem cell properties. These results highlight the therapeutic potential of DAPT in gastric cancer treatment.

Bone Morphogenetic Protein (BMP)-3b Gene Depletion Causes High Mortality in a Mouse Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Bone morphogenetic proteins (BMPs) are a group of proteins that induce the formation of bone and the development of the nervous system. BMP-3b, also known as growth and differentiation factor 10, is a member of the BMPs that is highly expressed in the developing and adult brain. BMP-3b is therefore thought to play an important role in the brain even after physiological neurogenesis has completed. BMP-3b is induced in peri-infarct neurons in aged brains and is one of the most highly upregulated genes during the initiation of axonal sprouting. However, little is known about the role of BMP-3b in neonatal brain injury. In the present study, we aimed to describe the effects of BMP-3b gene depletion on neonatal hypoxic-ischemic encephalopathy, which frequently results in death or lifelong neurological disabilities, such as cerebral palsy and mental retardation. BMP-3b knockout and wild type mice were prepared at postnatal day 12. Mice of each genotype were divided into sham-surgery, mild hypoxia-ischemia (HI), and severe HI groups (n = 12-45). Mice in the HI groups were subjected to left common carotid artery ligation followed by 30 min (mild HI) or 50 min (severe HI) of systemic hypoxic insult. A battery of tests, including behavioral tests, was performed, and the brain was then removed and evaluated at 14 days after insult. Compared with wild type pups, BMP-3b knockout pups demonstrated the following characteristics. (1) The males exposed to severe HI had a strikingly higher mortality rate, and as many as 70% of the knockout pups but none of the wild type pups died; (2) significantly more hyperactive locomotion was observed in males exposed to severe HI; and (3) significantly more hyperactive rearing was observed in both males and females exposed to mild HI. However, BMP-3b gene depletion did not affect other parameters, such as cerebral blood flow, cylinder test and rotarod test performance, body weight gain, brain weight, spleen weight, and neuroanatomical injury. The results of this study suggest that BMP-3b may play a crucial role to survive in severe neonatal hypoxic-ischemic insult.

Adaptive flexibility of the within-hand attentional gradient in touch: An MEG study.

Previous studies have demonstrated the cortical mechanisms for the gradient of spatial attention in vision and audition, whereas those for touch have yet to be elucidated in detail. In order to examine the within-hand gradient of tactile spatial attention in the cerebral cortex, we used magnetoencephalography (MEG) to record cortical responses to an electrocutaneous stimulation presented randomly to any of the five fingers of the right hand at a random interstimulus interval (750-1250 ms). Participants attended to the index finger, ring finger, or both to detect a rare target stimulus in a sequence of frequent standard stimuli presented to the attended finger(s) by silent counting. Neuromagnetic responses around the contralateral primary and secondary somatosensory cortices (SIc and SIIc) at 50-70 ms and 80-100 ms, respectively, for the stimulation of the index or ring finger were stronger when that finger was attended than when the distant finger was attended or at rest. The amplitude of the SIIc response was also intermediate when the index and ring fingers were simultaneously attended. The SIIc response to the task-irrelevant stimulation of the thumb or little finger increased when the index or ring finger was attended, respectively, suggesting an across-finger gradient of tactile attention. Simultaneous attention to the index and ring fingers decreased the SIIc response to the task-irrelevant stimulation of the intervening middle finger more than that with attention to either one of the two fingers. The earliest attentional sign was observed for the SI M40c response, with the amplitude increasing with the stimulation of the unattended finger. Furthermore, late responses in the temporo-parietal junction (TPJ) and prefrontal cortex (PFC) were stronger with the stimulation of the unattended finger than with that of the attended finger. Thus, the present study provides cortical evidence for the adaptive control of the within-hand, across-finger gradient of tactile attention that depends on whether attention is focused on a single finger or divided into non-adjacent different fingers.

Regulation of expression and trafficking of perforin-2 by LPS and TNF-α.

Perforin-2 is constitutively expressed in macrophages that are required for bacterial control. In this study, we found that perforin-2 is expressed in human macrophages with two isoforms: full-length perforin-2a and a splice variant, perforin-2b. Two isoforms show different subcellular distributions. Perforin-2a was predominantly localized to the membrane of endosome-like vesicles by a C-terminal transmembrane domain. In contrast, the short isoform perforin-2b lacking the transmembrane domain failed to localize to the membrane of vesicles. Furthermore, we determined that the pro-inflammatory stimuli LPS and TNF-α induced perforin-2a expression via the NF-κB pathway and triggered perforin-2a vesicles fusion with lysosomes. On the other hand, we detected the secretion of perforin-2b in response to LPS stimulation. Taken together, our data provide the evidence that membrane-bound and secretory isoforms of perforin-2 are present in human macrophages and may play important roles in immune defense.

Achievement of disease control with donor-derived EB virus-specific cytotoxic T cells after allogeneic peripheral blood stem cell transplantation for aggressive NK-cell leukemia.

Aggressive NK-cell leukemia (ANKL) is characterized by systemic infiltration of Epstein-Barr virus (EBV)-associated natural killer cells and poor prognosis. We report a case of ANKL in which EBV-specific cytotoxic T lymphocytes (CTLs) were induced. A 41-year-old male suffered from fever, pancytopenia, and hepatosplenomegaly. The number of abnormal large granular lymphocytes in the bone marrow was increased and the cells were positive for CD56 and EBV-encoded small nuclear RNAs. The patient was diagnosed with ANKL and achieved a complete response following intensive chemotherapy. He then underwent allogeneic peripheral blood stem cell transplantation from his sister. Conditioning therapy consisted of total body irradiation and cyclophosphamide. Graft-versus-host disease prophylaxis consisted of cyclosporine and methotrexate. On day 31, complete donor chimerism was achieved and no acute graft-versus-host disease developed. The ANKL relapsed on day 80, and cyclosporine was rapidly tapered and chemotherapy was started. During hematopoietic recovery, the number of atypical lymphocytes increased, but they were donor-derived EBV-specific CTLs. The patient achieved a partial response and EBV viral load decreased to normal range. Unfortunately, ANKL worsen again when the CTLs disappeared from his blood. This is the first case report of ANKL in which induced EBV-specific CTLs may have contributed to disease control.

Assessment of testicular corticosterone biosynthesis in adult male rats.

Corticosterone is synthesized in the adrenal glands and is circulated throughout the body to perform regulatory functions in various tissues. The testis is known to synthesize and secrete testosterone and other androgens. We developed an accurate method to measure steroid content using liquid chromatography-mass spectrometry analysis. In the present study, significant levels of the precursor compounds of testosterone and corticosterone synthesis could be detected in rat testis using this method. After adrenalectomy, corticosterone remained in the blood and testicular tissue at approximately 1% of the amount present in the control testis. When the excised testicular tissue was washed and incubated with NADH, NADPH and progesterone, not only testosterone and its precursors but also 11-deoxycorticosterone and corticosterone were produced; the levels of 11-deoxycorticosterone and corticosterone increased with incubation time. The production rate of 11-deoxycorticosterone from progesterone was estimated to be approximately 1/20 that of 17-hydroxyprogesterone, and the corticosterone level was approximately 1/10 that of testosterone. These ratios coincided with those in the testicular tissue of the adrenalectomized rats, indicating that corticosterone was synthesized in the testis and not in the blood. A primary finding of this study was that corticosterone and testosterone were synthesized in a 1/10-20 ratio in the testis. It is concluded that corticosterone, which has various functions, such as the regulation of glycolysis and mediating spermatogenesis, is produced locally in the testis and that this the local production is convenient and functional to respond to local needs.

Downregulation of cytochrome P450scc as an initial adverse effect of adult exposure to diethylstilbestrol on testicular steroidogenesis.

Reproductive toxicities and endocrine disruptions caused by chemicals in adult males are still poorly understood. It is our objectives to understand further details of the initial adverse effects leading severe testicular toxicities of a pharmaceutical endocrine disruptor, diethylstilbestrol (DES). Downregulations of both testicular regulatory proteins, such as the steroidogenic acute regulatory protein (StAR) and the peripheral benzodiazepine receptor (PBR), which play important roles in the transport of cholesterol into the mitochondria, and cytochrome P450 mediating the cholesterol side chain cleavage reaction (P450scc), were observed in the rat orally administered DES (340 µg/kg/2 days) for 2 weeks. We found that after only 1 week treatment with DES, the blood and testicular testosterone (TS) levels were drastically decreased without abnormalities of the StAR and PBR; however, the protein and mRNA levels of P450scc were diminished. Decrease in the conversion rate of cholesterol to pregnenolone was delayed in the in vitro assay using the testicular mitochondrial fraction from the rat treated with DES for 1 week. When the precursors in TS biosynthesis containing the testis were identified and determined by liquid chromatography-mass spectrometry analysis, decreased levels of all precursors except cholesterol were observed. In conclusion, suppressed cytochrome P450scc expression in adult male rat was identified as an initial target of DES in testicular steroidogenesis disorder leading reproductive toxicities.

Siamycin attenuates fsr quorum sensing mediated by a gelatinase biosynthesis-activating pheromone in Enterococcus faecalis.

The expression of two Enterococcus faecalis virulence-related proteases, gelatinase (GelE) and serine protease (SprE), is positively regulated by a quorum-sensing system encoded by the fsr gene cluster. In this system, E. faecalis secretes an autoinducing peptide, gelatinase biosynthesis-activating pheromone (GBAP), which triggers the FsrC-FsrA two-component regulatory system controlling the expression of two transcripts, fsrBDC and gelE-sprE. In the present study, we screened actinomycete metabolites for inhibitors of fsr quorum sensing. E. faecalis was cultured with each actinomycete culture supernatant tested, and the production of gelatinase and the production of GBAP were examined using the first screening and the second screening, respectively. Culture supernatant of Streptomyces sp. strain Y33-1 had the most potent inhibitory effect on both gelatinase production and GBAP production without inhibiting E. faecalis cell growth. The inhibitor in the culture supernatant was identified as a known peptide antibiotic, siamycin I. Siamycin I inhibited both gelatinase production and GBAP production at submicromolar concentrations, and it inhibited E. faecalis cell growth at concentrations above micromolar concentrations. Quantitative analysis of fsrBDC and gelE-sprE transcripts revealed that siamycin I suppressed the expression of both transcripts at a sublethal concentration. Siamycin I attenuated gelatinase production even when an overdose of GBAP was exogenously added to the culture. These results suggested that siamycin I inhibited the GBAP signaling via the FsrC-FsrA two-component regulatory system in a noncompetitive manner. The sublethal concentrations of siamycin I also attenuated biofilm formation. Treatment with siamycin could be a novel means of treating enterococcal infections.

Revised model for Enterococcus faecalis fsr quorum-sensing system: the small open reading frame fsrD encodes the gelatinase biosynthesis-activating pheromone propeptide corresponding to staphylococcal agrd.

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.