Impact of hospital volume on risk-adjusted mortality following oesophagectomy in Japan.

BACKGROUND: Previous studies have reported that patients undergoing oesophagectomy in high-volume hospitals experience lower mortality rates. However, there has been ongoing discussion regarding the validity of evidence for this association. The purpose of this study was to investigate the relationship between hospital volume and risk-adjusted mortality following oesophagectomy in Japan, using a nationwide web-based database. METHODS: The study included patients registered in the database as having undergone oesophagectomy with reconstruction between 2011 and 2013. Outcome measures were 30-day and operative mortality rates. Logistic regression analysis was used to adjust for hospital volume, surgeon volume and risk factors for mortality after oesophagectomy. RESULTS: A total of 16 556 oesophagectomies at 988 hospitals were included; the overall unadjusted 30-day and operative mortality rates were 1·1 and 3·0 per cent respectively. The unadjusted operative mortality rate in hospitals performing fewer than ten procedures per year (5·1 per cent) was more than three times higher than that in hospitals conducting 30 or more procedures annually (1·5 per cent). Multivariable models indicated that hospital volume had a significant effect on 30-day (odds ratio 0·88 per 10-patient increase; P = 0·012) and operative (odds ratio 0·86 per 10-patient increase; P < 0·001) mortality. CONCLUSION: In Japan, high-volume hospitals had lower risk-adjusted 30-day and operative mortality rates following oesophagectomy compared with low-volume hospitals.

Hemodynamic assessment of celiaco-mesenteric anastomosis in patients with pancreaticoduodenal artery aneurysm concomitant with celiac artery occlusion using flow-sensitive four-dimensional magnetic resonance imaging.

OBJECTIVES: Many pancreaticoduodenal artery (PDA) aneurysms are associated with celiac artery (CA) stenosis. The pathogenesis of PDA aneurysm may be associated with hemodynamic changes due to CA stenosis/occlusion. The aim of this study was to assess the hemodynamic changes of celiaco-mesenteric anastomosis in patients with PDA aneurysms concomitant with CA occlusion using four-dimensional flow-sensitive magnetic resonance imaging (4D-Flow). METHODS: 4D-Flow was performed preoperatively on five patients. Seven age- and sex-matched individuals were used as controls. Hemodynamic parameters such as flow volume and maximum flow velocity in PDAs, gastroduodenal arteries, common hepatic arteries, and superior mesenteric arteries were compared between both groups. Wall shear stress (WSS) and oscillatory shear index (OSI) were mapped in both groups. RESULTS: In the patient group, 4D-Flow identified retrograde flow of both gastroduodenal arteries and common hepatic arteries. Heterogeneous distribution patterns of both WSS and OSI were identified across the entire PDA in the patient group. OSI mapping showed multiple regions with extremely high OSI values (OSI > 0.3) in all patients. All PDA aneurysms, which were surgically resected, were atherosclerotic. CONCLUSIONS: 4D-Flow identified hemodynamic changes in celiaco-mesenteric arteries in patients with PDA aneurysms with concomitant CA occlusion. These hemodynamic changes may be associated with PDA aneurysm formation.

Influence of age and gender on human lymphatic pumping pressure in the leg.

Lymph transportation is controlled, at least in part, by the intrinsic pumping of lymphatic vessels. The objectives of this study were to evaluate the influences of age and gender on leg lymphatic pumping pressure. A total of 399 subjects between the ages of 20 and 91 years (199 males and 200 females) volunteered to participate in this study. Lymphatic pumping was measured in 798 legs of the 399 participants. Indocyanine green (ICG) fluorescence lymphography was performed, and the real-time fluorescence images of lymph propulsion were obtained in a sitting position using an infrared-light camera system. A custom-made transparent sphygmomanometer cuff was wrapped around the lower leg and connected to a standard mercury sphygmomanometer. The cuff was inflated, and then gradually deflated until the fluorescent dye exceeded the upper border of the cuff. Lymph pumping pressure was defined as the value of the cuff pressure when the dye exceeded the upper border of the cuff. There was a significant correlation between the leg lymphatic pumping and age: r = -0.34 (p < 0.0001). Comparison of lymphatic pumping between males and females indicated that the age-related decrease in lymphatic pumping pressure was more marked in females of postmenopausal age.

Imaging mass spectrometry reveals unique lipid distribution in primary varicose veins.

BACKGROUND: The lipid metabolism of varicose veins (VVs) remains unknown. To elucidate the pathogenesis of VV, we utilized the novel technique of imaging mass spectrometry (IMS). MATERIALS AND METHODS: We obtained VV tissues from 10 limbs of 10 VV patients who underwent great saphenous vein stripping. As control vein samples, we harvested segmental vein tissues from 6 limbs of 6 patients with peripheral artery occlusive disease who underwent infra-inguinal bypass with reversed saphenous vein grafting. To identify the localisation of lipid molecules in the VV tissues, we performed matrix-assisted laser desorption/ionization IMS (MALDI-IMS). We also performed MS/MS analyses to identify the structure of each molecule. RESULTS: We obtained mass spectra directly from control vein tissues and VV tissues and found a unique localisation of lipid molecules in the VV tissues. We localised lysophosphatidylcholine (LPC) (1-acyl 16:0), phosphatidylcholine (PC) (1-acyl 36:4) and sphingomyelin (SM) (d18:1/16:0) at the site of the VV valve. CONCLUSION: MALDI-IMS revealed the distribution of various lipid molecules in normal veins and VVs both. Accumulation of LPC (1-acyl 16:0), PC (1-acyl 36:4) and SM (d18:1/16:0) in the VV tissues suggested that inflammation associated with abnormal lipid metabolism may contribute to the development of VV.

Indocyanine green fluorescence angiography for intraoperative assessment of blood flow: a feasibility study.

OBJECTIVE: To introduce our preliminary experience with indocyanine green (ICG) fluorescence angiography for the assessment of lower leg bypasses. METHODS: 1ml of 0.5% indocyanine green was intravenously injected in 9 patients with PAD who underwent paramalleolar artery bypass using saphenous vein grafts. A newly developed near-infrared camera system (PDE; Hamamatsu Photonics K.K. Hamamatsu, Japan) was used for this study. RESULTS: ICG fluorescence angiography was performed without any adverse events. Fluorescence images of ICG angiography could be viewed as real-time images of the angiography in eight patients, while one patient underwent graft revision with the absence of fluorescence in ICG angiography. CONCLUSION: ICG fluorescence angiography is clinically feasible and may help surgeons assess the quality of lower leg bypasses.

Quantitative lymph imaging for assessment of lymph function using indocyanine green fluorescence lymphography.

OBJECTIVES: A new diagnostic imaging technique that can assess lymph function is needed as a screening test in daily practice. This study assessed the use of indocyanine green (ICG) fluorescence lymphography in subjects without leg oedema. METHODS: 0.3ml of ICG (0.5 %) was injected subcutaneously at the dorsum of the foot. Subsequently, the movement of ICG dye from the injection site to the groin was traced by visualizing its fluorescence signal with an infrared light camera. The time for the dye to reach the knee and groin were measured (Transit time to knee: TT(K), Transit time to groin: TT(G)). TT(G) was measured while standing, lying at a supine position, standing with massage, and sitting while using a cycle ergometer exercise at an intensity of 50W at 50rpm in ten healthy volunteers at intervals of 14 days. RESULTS: Mean TT(G) during standing was 357+/-289 and 653+/-564 seconds for the right and left legs respectively. Compared to TT(G) in the standing position, all other conditions shortened TT(G). In another seventeen subjects without leg oedema, we compared transit time obtained with ICG fluorescence lymphography to that with dynamic lymphoscintigraphy. A significant correlation between transit time measured with ICG lymphography and dynamic lymphoscintigraphy was identified (r(2)=0.64, p<0.01). CONCLUSIONS: ICG fluorescence lymphography has the potential to become an alternative lymphatic imaging technique to assess lymph function.

Loss of aquaporin 4 in lesions of neuromyelitis optica: distinction from multiple sclerosis.

Neuromyelitis optica (NMO) is an inflammatory and necrotizing disease clinically characterized by selective involvement of the optic nerves and spinal cord. There has been a long controversy as to whether NMO is a variant of multiple sclerosis (MS) or a distinct disease. Recently, an NMO-specific antibody (NMO-IgG) was found in the sera from patients with NMO, and its target antigen was identified as aquaporin 4 (AQP4) water channel protein, mainly expressed in astroglial foot processes. However, the pathogenetic role of the AQP4 in NMO remains unknown. We did an immunohistopathological study on the distribution of AQP4, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), activated complement C9neo and immunoglobulins in the spinal cord lesions and medulla oblongata of NMO (n = 12), MS (n = 6), brain and spinal infarction (n = 7) and normal control (n = 8). The most striking finding was that AQP4 immunoreactivity was lost in 60 out of a total of 67 acute and chronic NMO lesions (90%), but not in MS plaques. The extensive loss of AQP4 accompanied by decreased GFAP staining was evident, especially in the active perivascular lesions, where immunoglobulins and activated complements were deposited. Interestingly, in those NMO lesions, MBP-stained myelinated fibres were relatively preserved despite the loss of AQP4 and GFAP staining. The areas surrounding the lesions in NMO had enhanced expression of AQP4 and GFAP, which reflected reactive gliosis. In contrast, AQP4 immunoreactivity was well preserved and rather strongly stained in the demyelinating MS plaques, and infarcts were also stained for AQP4 from the very acute phase of necrosis to the chronic stage of astrogliosis. In normal controls, AQP4 was diffusely expressed in the entire tissue sections, but the staining in the spinal cord was stronger in the central grey matter than in the white matter. The present study demonstrated that the immunoreactivities of AQP4 and GFAP were consistently lost from the early stage of the lesions in NMO, notably in the perivascular regions with complement and immunoglobulin deposition. These features in NMO were distinct from those of MS and infarction as well as normal controls, and suggest that astrocytic impairment associated with the loss of AQP4 and humoral immunity may be important in the pathogenesis of NMO lesions.

Multicenter phase II trial of combination chemotherapy with weekly paclitaxel and 5-fluorouracil for the treatment of advanced or recurrent gastric carcinoma.

The aim of this study was to investigate the efficacy and safety of combination chemotherapy with weekly paclitaxel and 5-fluorouracil (5-FU) as first-line treatment in patients with advanced or recurrent gastric carcinoma. A total of 65 patients were treated with the following regimen, administered every 28 days; 5-FU 600 mg/m2 by 24-hour continuous infusion from days 1 through 5, and weekly paclitaxel 80 mg/m2 by 3-hour intravenous infusion on days 8, 14, and 21. A total of 272 cycles were conducted with a median of 4 (2-13) cycles per case. Out of 57 patients with measurable disease by RECIST criteria, there were 2 complete responses (3.5%), 20 partial responses (35.1%) and 25 cases with stable disease (43.9%). The overall response rate was 38.6% (95%CI: 26.0-51.2%). The median survival time and 1-year survival rates were 329 days and 47.4%, respectively. Both hematologic and non-hematologic toxicities were well tolerated.

Prevention of hepatic metastasis of human colon cancer by angiogenesis inhibitor TNP-470.

The antimetastatic effect of a potent angiogenesis inhibitor, O-(chloroacetyl-carbamoyl)fumagillol (TNP-470), was investigated in nude mice implanted with human colon cancer. Small pieces of tumors from three established human colon cancer cell lines (TK-3, TK-4, and TK-9), which were maintained in nude mice, were implanted into the cecal wall of nude mice via a small incision in the serosa. TNP-470 (20 or 30 mg/kg) was given s.c. every other day from day 10 after implantation, and the mice were sacrificed after 6 weeks. There was no difference in the weight of the implanted tumors (control group: 0.45 +/- 0.29 g versus treated group: 0.49 +/- 0.27 g). An antimetastatic effect of TNP-470 was clearly demonstrated in a dose-dependent manner. In the mice given 20 mg/kg TNP-470, liver metastasis developed in 3 of 10 cases. In the 30-mg/kg group, metastasis developed in only 1 of 17 mice, while it developed in 22 of 32 mice of the control group. The number of metastatic foci was significantly less in the treated groups. TNP-470 effectively prevented liver metastasis, however, but had no effect on the growth of the primary tumor. These results indicate that the angiogenesis inhibitor TNP-470 has a strong inhibitory activity against in vivo hepatic metastasis of human colon cancer.

Antitumor effect of adriamycin entrapped in liposomes conjugated with anti-human alpha-fetoprotein monoclonal antibody.

The monoclonal antibody, 19-F-12 (IgG2b), against human alpha-fetoprotein was conjugated to liposomes containing Adriamycin, and the therapeutic effects of the conjugate were experimentally studied using the alpha-fetoprotein-producing human hepatoma strain, Li-7, maintained in BALB/c nu/nu male mice. Three i.v. injections of liposomes containing Adriamycin (7.5 mg/kg) into tumor-bearing mice significantly inhibited the tumor growth, and the therapeutic effect of the antibody-conjugated liposomes was greater than that of unconjugated liposomes, as judged from the tumor weights and histological findings. Furthermore, the experiments were repeated with Adriamycin (4-5 mg/kg) in free form, since administration of Adriamycin (7.5 mg/kg) in free form was highly toxic for the mice. The results still indicated that the therapeutic effect of Adriamycin in 19-F-12 conjugated liposomes was superior to that of free Adriamycin or Adriamycin in unconjugated liposomes. In contrast to the treatment for Li-7 in nude mice, the therapeutic effect of Adriamycin in 19-F-12 conjugated liposomes was not much different from that of Adriamycin in normal mouse IgG (IgG2b fraction) conjugated liposomes against alpha-fetoprotein-negative human breast cancer strain MX1. Tissue distribution studies after i.v. injection of Adriamycin in various forms into mice revealed that preferential delivery of Adriamycin to tumors occurred to some extent with antibody-conjugated liposomes as compared to the unconjugated liposomes. In addition, reduction of the distribution of Adriamycin to the heart was achieved by administering the drug in the liposome-entrapped form, and this enabled the use of a higher dose (7.5 mg/kg) of Adriamycin without toxic side effect.

Restriction landmark genomic scanning (RLGS-M)-based genome-wide scanning of mouse liver tumors for alterations in DNA methylation status.

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect, and subsequently clone, genomic regions with alterations in DNA methylation associated with tumorigenesis. Use of a methylation-sensitive enzyme for the landmark cleavage allows analysis of changes in methylation patterns. In this study, we used RLGS-M to analyze SV40 T antigen-induced mouse liver tumors derived from interspecific F1 hybrids between Mus spretus (S) and C57BL/6 (B6). Because 575 S- and B6-specific RLGS loci/spots have been mapped, tumor-related alterations in the RLGS profile could be immediately localized to specific chromosomal regions. We previously found that the loss of contiguous loci/spots could be attributed primarily to DNA loss, whereas loss of solitary loci/spots could be attributed primarily to DNA methylation. In this study, we examined 30 mouse liver tumor samples for loss of the 507 mapped loci/spots. Fourteen solitary loci/spots found to be absent or reduced in more than 75% of tumor samples were cloned and subjected to DNA sequence analyses. Two loci were identified as alpha4 integrin and p16/CDKN2, genes reported to be involved in tumorigenesis. Thus, RLGS-M can detect alterations in the methylation status of known tumor suppressor genes and provide a method for detecting and subsequently cloning novel genomic regions that undergo alterations in methylation during tumorigenesis.

Diverse roles of STING-dependent signaling on the development of cancer.

Stimulator of interferon genes (STING) is a cellular sensor that controls cytosolic DNA-activated innate immune signaling. We have previously demonstrated that STING-deficient mice are resistant to carcinogen-induced skin cancer, similar to myeloid differentiation primary response gene 88 (MyD88) deficient mice, since the production of STING-dependent DNA-damage-induced proinflammatory cytokines, that likely require MyD88 signaling to exert their growth-promoting activity, are prevented. In contrast, MyD88-deficient mice are sensitive to colitis-associated cancer (CAC), since selected cytokines generated following DNA-damage also activate repair pathways, which can help prevent tumor development. Here, we demonstrate that STING signaling facilitates wound repair processes and that analogous to MyD88-deficient mice, STING-deficient mice (SKO) are prone to CAC induced by DNA-damaging agents. SKO mice harboring tumors exhibited low levels of tumor-suppressive interleukin-22 binding protein (IL-22BP) compared to normal mice, a cytokine considered critical for preventing colon-related cancer. Our data indicate that STING constitutes a critical component of the host early response to intestinal damage and is essential for invigorating tissue repair pathways that may help prevent tumorigenesis.

On the replication and spread of rabies virus in the human central nervous system.

Ultrastructural and immunohistochemical studies on the brains of two autopsy cases of human rabies revealed: By the peroxidase-antiperoxidase method, viral antigens were present in all eosinophilic inclusions detected in formalin fixed paraffin sections. Numerous antigenic masses, which apparently corresponded to the matrices and cylindrical particles in neurites revealed by electron microscopy, were present in the neuropil remote from neuronal perikarya. There were virions in the intercellular spaces and virus-budding from the plasma membrane into the extracellular space in the absence of a matrix, strongly indicating that rabies virus in the human central nervous system could spread through the intercellular spaces and that the replication of the virus was not necessarily accompanied by the formation of inclusion bodies. The synapse was involved in rabies as indicated by virions in the synaptic terminals. The implications of these observations are discussed in conjunction with the results of previous in vitro and animal experiments.

Prediction of acute embolic stroke outcome after local intraarterial thrombolysis: value of pretreatment and posttreatment 99mTc-ethyl cysteinate dimer single photon emission computed tomography.

The aim of this study was to investigate the efficacy of pre- and posttreatment 99mTc-ethyl cysteinate dimer (99mTc-ECD) single photon emission computed tomography (SPECT) for predicting the ischemic outcome of embolic middle cerebral artery occlusion after treatment with local intraarterial thrombolysis. The authors examined 28 patients with a moderately ischemic area (ratio of affected regional activity to cerebellar activity (A/C ratio) of 0.4 to 0.7) determined using pretreatment SPECT, and with complete recanalization within 6 hours. Posttreatment dynamic and static SPECT studies were performed immediately after thrombolysis. The extent of the affected area outlined on pretreatment SPECT was used for the posttreatment SPECT images, and A/C ratios were calculated. The relative retention ratio of 99mTc-ECD in the affected area was also analyzed using posttreatment dynamic SPECT. Fourteen patients either without infarction or with small subcortical and basal ganglial infarction, 11 patients with medium or large cortical infarction, and 3 patients with hemorrhage were identified by follow-up computed tomography. Ischemic outcome correlated with the relative retention ratio of 99mTc-ECD more closely than either the pre- or posttreatment A/C ratios. In particular, a threshold value for the development of hemorrhage was distinct only in the relative retention ratio of 99mTc-ECD. Pretreatment 99mTc-ECD SPECT did not always predict the occurrence of hemorrhagic transformation, whereas dynamic 99mTc-ECD SPECT performed immediately after thrombolysis allowed clear identification of patients at risk for hemorrhagic transformation.

Comparative evaluation of 5'-end-sequence quality of clones in CAP trapper and other full-length-cDNA libraries.

To enhance the usefulness of the laboratory mouse and to facilitate the rapid assay of gene functions we have been collecting the entire set of mouse full-length cDNA by one-pass sequencing. To collect full-length cDNA clones efficiently, it is critical to construct high-quality cDNA libraries. In recent years, we have been developing a way to construct full-length cDNA libraries by using biotinylation of the cap structure (the 'CAP-trapper' method) coupled with treatment to increase reverse transcriptase efficiency at high temperature by the addition of trehalose. In this paper we report our evaluation of the quality of CAP trapper and a number of other full-length cDNA libraries, including the results of 5' end analysis of clones in CAP trapper and the other libraries. We used a procedure that compared the 5'-ends of cDNA clones with those of genes in the public databases. Our analysis showed that 63% of cDNA clones in CAP trapper libraries had sequences that were either the same length as those of equivalent genes in the public database or 5'-extended, and that 90% of these clones maintained their coding sequences. These results indicate that the CAP trapper library is a promising tool for collecting full-length cDNA in large-scale projects. Comparison of the quality of CAP trapper with that of other full-length-cDNA libraries confirmed the value of these libraries.

Correlation between sequence conservation of the 5' untranslated region and codon usage bias in Mus musculus genes.

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.

Comprehensive sequence analysis of translation termination sites in various eukaryotes.

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.

Prediction of human cDNA from its homologous mouse full-length cDNA and human shotgun database.

We propose a prediction method for human full-length cDNA by comparing sequence data between human genome shotgun sequence and mouse full-length cDNA. The human genome which is homologous to the mouse full-length cDNA is selected by a homology search program, and the predicted exons are connected at the exon-intron junction which gives the best homology score to the mouse full-length cDNA. The accuracy of the predicted human full-length coding region is 83.3%, and the false positive rate is 16.7%. Five human full-length proteins out of 20 proteins are correctly predicted.

Retrograde axoplasmic transport of adriamycin: an experimental form of motor neuron disease?

Adriamycin (ADM) is a DNA-directed RNA inhibitor. In attempts to produce an experimental form of motor neuron disease, we injected the agent into rat sciatic nerve. Retrograde axoplasmic flow conveyed ADM into soma of the spinal motor neurons, as confirmed by fluorescence microscopy. Motor neuron degeneration, which included nuclear heterochromatinization and diffuse chromatolysis, was observed after 6 to 8 days. After 2 weeks, many neurons that gave rise to sciatic nerve efferents underwent dissolution. Retrograde axoplasmic flow and DNA-injurious substances could affect survival of motor neurons.

Retrograde transport and differential accumulation of serum proteins in motor neurons: implications for motor neuron diseases.

We found immunocytochemical evidence of serum albumin and immunoglobulin G in motor neurons of rats under physiologic conditions, but not when axoplasmic flow was abolished. The amount of serum proteins was highest in somatic motor neurons of the spinal anterior horn, nucleus ambiguous, and trigeminal motor nucleus; less in hypoglossal and facial neurons; and sparse in preganglionic autonomic, oculomotor, and hypothalamic neurons. Toxic or trophic substances, bound to serum proteins, could also be incorporated into motor neurons. Quantitative differences in accumulation paralleled the selective vulnerability of somatic motor neurons in motor neuron diseases.