Heparin-binding EGF-like growth factor (HB-EGF) antisense oligonucleotide protected against hyperlipidemia-associated atherosclerosis.

BACKGROUND AND AIMS: Heparin-binding EGF-like growth factor (HB-EGF) is a representative EGF family member that interacts with EGFR under diverse stress environment. Previously, we reported that the HB-EGF-targeting using antisense oligonucleotide (ASO) effectively suppressed an aortic aneurysm in the vessel wall and circulatory lipid levels. In this study, we further examined the effects of the HB-EGF ASO administration on the development of hyperlipidemia-associated atherosclerosis using an atherogenic mouse model. METHODS AND RESULTS: The male and female LDLR deficient mice under Western diet containing 21% fat and 0.2% cholesterol content were cotreated with control and HB-EGF ASOs for 12 weeks. We observed that the HB-EGF ASO administration effectively downregulated circulatory VLDL- and LDL-associated lipid levels in circulation; concordantly, the HB-EGF targeting effectively suppressed the development of atherosclerosis in the aorta. An EGFR blocker BIBX1382 administration suppressed the hepatic TG secretion rate, suggesting a positive role of the HB-EGF signaling for the hepatic VLDL production. We newly observed that there was a significant improvement of the insulin sensitivity by the HB-EGF ASO administration in a mouse model under the Western diet as demonstrated by the improvement of the glucose and insulin tolerances. CONCLUSION: The HB-EGF ASO administration effectively downregulated circulatory lipid levels by suppressing hepatic VLDL production rate, which leads to effective protection against atherosclerosis in the vascular wall.

Targeted delivery of antisense oligonucleotides to pancreatic ß-cells.

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting ß-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic ß-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

Referrals for movement disorder surgery: under-representation of females and reasons for refusal.

OBJECTIVE: Referral of movement disorder patients for deep brain stimulation surgery was examined to determine whether referred patients were representative of gender proportions in our population, and reasons why patients do not proceed to surgery. METHODS: Demographic information on referrals to the surgical program was retrospectively reviewed from our database and from a detailed chart review. RESULTS: Although almost equal numbers of movement disorder patients are male and female, of the 91 patients referred for surgery, only 31% were female. Sixty-one percent of referred patients did not undergo surgery. Of these, the majority were denied for medical reasons, including cognitive decline (21%), psychiatric concerns (5%) and neurological reasons (42%). CONCLUSIONS: Almost one-third of patients referred for movement disorder surgery were denied for medical reasons. This underscores the importance of evaluation of all potential patients by a multidisiplinary team to fully assess suitablity for stereotactic surgery. Interestingly, women were under-represented in those referred. In order that all appropriate patients have the opportunity to consider surgery, education of both physicians and patients, and different strategies to approach females regarding surgery may allow more patients to benefit from this treatment.

Polychlorinated naphthalenes in air and snow in the Norwegian Arctic: a local source or an Eastern Arctic phenomenon?

PCNs were measured in air and snow during separate field campaigns at Ny-Alesund (April 2001) and Tromsø (February/March 2003) in the Norwegian Arctic. Air concentrations ranged from 27 to 48 and 9 to 47 pg sigmaPCN m(-3) for Ny-Alesund (n=6) and Tromsø (n=10), respectively. These concentrations (including the tri-chlorinated naphthalenes) greatly exceeded concentrations previously measured in the Canadian Arctic, but did fall within the upper range of concentrations observed over the eastern Arctic Ocean and regional seas. Local sources appear to be affecting concentrations observed at both sites, with the presence of several hexa-chlorinated naphthalenes at Tromsø probably attributed to local/regional sources. Use of air mass back trajectories at Tromsø revealed that background air concentrations in the Norwegian Arctic are likely to range between <9 and 20 pg sigmaPCN m(-3) and that contemporary concentrations derived close to potential sources (i.e. arctic towns) may equal or exceed those of PCBs. The mean concentration in surface snow was 350 and 240 pg sigmaPCN L(-1) (meltwater) (or 0.014 and 0.01 pg g(-1) (snow)) at Ny-Alesund and Tromsø, respectively. The wide variation in concentrations observed between fresh snowfalls could be explained by different snow densities (as a surrogate of snow surface area), rather than attributed to varying air concentrations. A statistically significant inverse relationship was found between snow density and concentrations of tri- to penta-chlorinated homologues and compliments similar findings for the polychlorinated biphenyls (PCBs). This suggests that the vapour-sorbed quantity changes rapidly with snow ageing/compaction; with implications for the fate of these chemicals in the Arctic.

Atmospheric emissions of polybrominated diphenyl ethers and other persistent organic pollutants during a major anthropogenic combustion event.

Air samples were taken for the analysis of persistent organic pollutants before, during, and after the national U.K. "Bonfire Festival" in November 2000. As expected, ambient levels of polynuclear aromatic hydrocarbons (PAHs) increased sharply in response to the widespread diffusive combustion processes that occurred at the time. Polybrominated diphenyl ethers (PBDEs) also increased at the suburban sampling location, to a greater extent than the PAHs. The rise and fall in PBDE concentrations was rapid, coinciding closely with the PAH "combustion markers". These data provide evidence for a novel mechanism responsible for dissipation of PBDEs into the environment. It is hypothesized that products treated with the penta-BDE product--notably household furnishing foams and textiles--have been subject to (unsanctioned) burning on private bonfires; even if the majority of the PBDE burden of such products is debrominated/broken down in the fires, it is shown that only small amounts of the total "stock" of penta product need be emitted to generate the concentrations detected. The mixture of PBDEs in the air during the Bonfire Festival was enriched in higher brominated congeners (e.g., BDE-99, -153, and -154) compared to that in background air. Estimates are made of the masses of compound classes that may have been emitted to the atmosphere during the festival.

12-O-tetradecanoylphorbol-13-acetate induces apoptosis in renal epithelial cells through a growth signal conflict which is prevented by activated ras.

12-O-Tetradecanoylphorbol-13-acetate (TPA) induced apoptosis in the pig renal epithelial cell line LLC-PK1 after 24 h of treatment as assessed by caspase 3 activation. Cotreatment of the cells with bryostatin markedly reduced the apoptotic effects of TPA. Okadaic acid, another tumor promoter, also induced apoptosis. Expression of an activated ras gene prevented TPA-induced apoptosis, while a dominant negative ras retarded the process. Taken together, these results suggest that TPA-induced apoptosis in LLC-PK1 may be analogous to TPA-induced tumor promotion in the two-stage model of skin carcinogenesis. Mechanistically, TPA-induced apoptosis seemed to be the result of a conflict of the growth-promoting affects of serum and the growth-retarding effects of TPA. This was manifested by a pronounced hypophosphorylation of the retinoblastoma gene product, pRb, which was prevented by activated ras. Apoptosis and pRb hypophosphorylation were associated with a reduction in cyclin D1 levels, suggesting that the growth-retarding effects of TPA were produced by modulation of this cell cycle protein. Interestingly, the mechanism of protection by activated ras did not seem to result from downstream activation of phosphatidylinositol-3-kinase (PI3K) as has been implicated in other systems. Additional analysis revealed that TPA-induced apoptosis was associated with the downregulation of the anti-apoptotic proteins Bcl-x and Mcl-1 and dependent on the activity of the transcription factor Jun.

Acyl coenzyme A: cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis.

Two enzymes are responsible for cholesterol ester formation in tissues, acyl coenzyme A:cholesterol acyltransferase types 1 and 2 (ACAT1 and ACAT2). The available evidence suggests different cell locations, membrane orientations, and metabolic functions for each enzyme. ACAT1 and ACAT2 gene disruption experiments in mice have shown complementary results, with ACAT1 being responsible for cholesterol homeostasis in the brain, skin, adrenal, and macrophages. ACAT1 -/- mice have less atherosclerosis than their ACAT1 +/+ counterparts, presumably because of the decreased ACAT activity in the macrophages. By contrast, ACAT2 -/- mice have limited cholesterol absorption in the intestine, and decreased cholesterol ester content in the liver and plasma lipoproteins. Almost no cholesterol esterification was found when liver and intestinal microsomes from ACAT2 -/- mice were assayed. Studies in non-human primates have shown the presence of ACAT1 primarily in the Kupffer cells of the liver, in non-mucosal cell types in the intestine, and in kidney and adrenal cortical cells, whereas ACAT2 is present only in hepatocytes and in intestinal mucosal cells. The membrane topology for ACAT1 and ACAT2 is also apparently different, with ACAT1 having a serine essential for activity on the cytoplasmic side of the endoplasmic reticulum membrane, whereas the analogous serine is present on the lumenal side of the endoplasmic reticulum for ACAT2. Taken together, the data suggest that cholesterol ester formation by ACAT1 supports separate functions compared with cholesterol esterification by ACAT2. The latter enzyme appears to be responsible for cholesterol ester formation and secretion in lipoproteins, whereas ACAT1 appears to function to maintain appropriate cholesterol availability in cell membranes.

Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.

Two closely related enzymes with more than 50% sequence identity have been identified that catalyze the esterification of cholesterol using acyl-CoA substrates, namely acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2. Both are membrane-spanning proteins believed to reside in the endoplasmic reticulum of cells. ACAT2 has been hypothesized to be associated with lipoprotein particle secretion whereas ACAT1 is ubiquitous and may serve a more general role in cellular cholesterol homeostasis. We have prepared and affinity purified rabbit polyclonal antibodies unique to either ACAT enzyme to identify their cellular localization in liver and intestine, the two main lipoprotein-secreting tissues of the body, and for comparison, kidney and adrenal. In the liver, ACAT2 was identified in the rough endoplasmic reticulum of essentially all hepatocytes whereas ACAT1 was confined to cells lining the intercellular spaces among hepatocytes in a pattern typical of Kupffer cells. In the intestine, ACAT2 signal was strongly present in the apical third of the mucosal cells, whereas ACAT1 staining was diffuse throughout the mucosal cell, but with strong signal in goblet cells, Paneth cells, and villus macrophages. In the kidney, ACAT1 immunostaining was specific for the distal tubules and podocytes within the glomerulus. In the adrenal, ACAT1 signal was strongly present in the cells of the cortex, and absent from other adrenal cell types. No ACAT2 signal was identified in the kidney or adrenal. We conclude that only the cells of the liver and intestine that secrete apolipoprotein B-containing lipoproteins contain ACAT2, whereas ACAT1 is present in numerous other cell types. The data clearly suggest separate functions for these two closely related enzymes, with ACAT2 being most closely associated with plasma cholesterol levels.

ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane.

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser(269)) in the cytosol and the analogous residue in ACAT2 (Ser(249)) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.

Lateral leg raising in patients with Parkinson's disease: influence of equilibrium constraint.

Patients with Parkinson's disease often have difficulty maintaining postural stability. This impairment is attributed to postural adjustment deficits. We studied the postural adjustments associated with the performance of two complex tasks which differed only in the final equilibrium constraints. Ten patients with Parkinson's disease and six age-matched control subjects were asked to raise one leg laterally to an abduction angle of approximately 45 degrees as fast as possible to the right or left in random order. In the first series of tests, the subjects were instructed to maintain the leg at 45 degrees, whereas in the second series they were instructed to place their foot back on the ground. Recordings included ground reaction forces and kinematics. In the patients with Parkinson's disease the final posture for the first task was never maintained. The strategy used to shift the body weight was different for the two groups. In control subjects, it was initiated by a whole body rotation around the ankle followed by a trunk inclination around the hip. Conversely, in patients with Parkinson's disease, the shift of the body weight was initiated by a trunk inclination around the hip and then by a whole body rotation around the ankle. The amplitude of the trunk inclination toward the supporting side was smaller than in the control subjects. The second task with less severe equilibrium constraints was, on the whole, better performed by the patients even though the same postural adjustment deficits were present.

Home telecare system using cable television plants--an experimental field trial.

To solve the inconvenience of routine transportation of chronically ill and handicapped patients, this paper proposes a platform based on a hybrid fiber coaxial (HFC) network in Taiwan designed to make a home telecare system feasible. The aim of this home telecare system is to combine biomedical data, including three-channel electrocardiogram (ECG) and blood pressure (BP), video, and audio into a National Television Standard Committee (NTSC) channel for communication between the patient and healthcare provider. Digitized biomedical data and output from medical devices can be further modulated to a second audio program (SAP) subchannel which can be used for second-language audio in NTSC television signals. For long-distance transmission, we translate the digital biomedical data into the frequency domain using frequency shift key (FSK) technology and insert this signal into an SAP band. The whole system has been implemented and tested. The results obtained using this system clearly demonstrated that real-time video, audio, and biomedical data transmission are very clear with a carrier-to-noise ratio up to 43 dB.

Prognostic value of abdominal CT scanning and hepatic histopathology in patients with acute liver failure.

Acute liver failure has extremely high mortality without liver transplantation. We attempted to determine the value of abdominal CT scanning and liver biopsy in its management. A retrospective analysis of patients with acute liver failure was performed; demographic, clinical, radiologic and histopathologic features were noted. Over a period of 13 years, 177 patients were evaluated. The mean age was 39 years and 63% were females. The patients were divided into three groups. Fourteen percent survived with medical management (group I), 37% died (group II), and 49% had liver transplantation (group III). Most patients showed diffuse low density of the liver on CT scanning and the proportions were similar in the three groups. Moderate to large ascites was not present in group I but occurred in 31% of patients in group II and in 15% in group III. Mean hepatic volumes were similar in the three groups; however, 97% of the patients with a liver volume of less than 1000 ml either died or required liver transplantation. Liver biopsies among patients with spontaneous recovery (group I) were distinguished by the presence of regenerative changes and a hepatic parenchymal necrosis of less than 50%. These results suggest that in patients with acute liver failure a liver volume of less than 1000 ml and/or hepatic parenchymal necrosis of greater than 50% is indicative of a poor prognosis. This information may assist decision making in such patients, in particular, regarding the need for liver transplantation.

Cold preservation of the human colon and ileum with University of Wisconsin solution.

The inclusion of the colon in the intestinal graft resulted in worsening patient and graft outcome and increased the incidence of infection and rejection. In this study, we examine the role of ischemia on the barrier function of the epithelium during cold ischemia. Samples were collected from 15 harvested and transplanted human donor grafts (colon, 10; ileum, 6), which were immersed in University of Wisconsin (UW) solution. Ischemia (6, 12, 24, and 48 h) and reoxygenation were performed to evaluate the mucosal electrical status using the Ussing chamber technique. The functions of enterocytes and crypt cells were tested by glucose and theophylline challenge. Modified Park's classification was applied to evaluate the severity of mucosal damage under light microscopy. The colon had higher levels of baseline potential difference, short-circuit current, and resistance than the ileum during 6 48 h of ischemia. Colonic epithelial cells responded well to theophylline stimulation at 24 h of ischemia, while there was no ileal response. The colonic mucosa was histopathologically well preserved in UW solution for 48 h, and mucosal damage induced by reoxygenation was less than in the ileum. In conclusion, electrophysiologically and histopathologically, the colon is less susceptible to cold preservation damage than the ileum during storage with UW solution.

Recent advances in telemedicine.

With continuing advances in information technology, the applications of computers in medicine are increasing rapidly. Modern information technology not only affects the delivery of health care, but also significantly influences the doctor-patient relationship. Since the 1990s, technologic developments in high-bandwidth telecommunications systems and digitizing devices have led to a surge of interest in telemedicine. In recent years, the Internet, with its powerful penetration and scalability, has become an increasingly popular medical information resource and a new platform for telemedicine. The impact of modern technology on the advancement of telemedicine in Taiwan started with the 1995 National Information Infrastructure project, which uses networks of different bandwidths for teleconsultation and distance education programs. In 1998, National Taiwan University and Taipei Medical College in Taiwan, and the University of Pittsburgh and the University of Iowa in the USA, began cooperation on a new Cyber Medical Center (CMC) project that integrates the technologies of multimedia, database management, a multiple-site videoconferencing system, and the World Wide Web. The aim of the CMC is to create a multimedia network system for the management of electronic patient records, teleconsultation, online continuing medical education, and information services on the Web. In the future, telemedicine systems in Taiwan are expected to combine the Internet and cable television to connect clinics, hospitals, insurance organizations, and public health administrations; and, finally, to extend health services to every household.

Effects of test H-reflex size on reciprocal inhibition in forearm muscles.

We studied effects of test H-reflex size on reciprocal Ia inhibition in forearm muscles. In both healthy control subjects and hemiplegic patients, the amount of Ia inhibition decreased as the test H-reflex size increased. It is possible that forearm reciprocal Ia inhibition in hemiplegics reported previously might be underestimated due to larger test H-reflexes used in the hemiplegics than in the controls.

Quantitative kinematics of gait patterns during the recovery period after stroke.

BACKGROUND AND PURPOSE: The purpose of this study was to assess the potential of a new quantitative kinematic analysis for the documentation and evaluation of recovery of gait function after neurological injury. METHODS: We assessed the kinematics of gait function in 16 patients with hemiplegia at varying intervals over a 1-year period after a stroke, using a novel method for gait pattern assessment based on principal component analysis. Conventional measures such as gait speed and stride length were also evaluated. Testing started as soon as patients became ambulatory after stroke. RESULTS: Of the 16 patients assessed, 7 showed at least a 50% increase in self-selected gait speed from the first to the last test. The results of the pattern analysis closely mirrred self-selected gait speed at higher speeds, but relative rankings derived from gait speed and the pattern analysis did not match for 6 of the 16 patients. Kinematic pattern analysis suggested that different mechanisms were used to generate changes in gait speed at different speed levels. CONCLUSION: There is a sizable fraction of the stroke population for whom kinematic gait pattern analysis can provide information that is different from that provided by speed, stride length, and cadence. The kinematic analysis can potentially provide information about the mechanisms of pathological gait.

Treatment of hilar cholangiocarcinoma (Klatskin tumors) with hepatic resection or transplantation.

BACKGROUND: Because of the rarity of hilar cholangiocarcinoma, its prognostic risk factors have not been sufficiently analyzed. This retrospective study was undertaken to evaluate various pathologic risk factors which influenced survival after curative hepatic resection or transplantation. METHODS: Between 1981 and 1996, 72 patients (43 males and 29 females) with hilar cholangiocarcinoma underwent hepatic resection (34 patients) or transplantation (38 patients) with curative intent. Medical records and pathologic specimens were reviewed to examine the various prognostic risk factors. Survival was calculated by the method of Kaplan-Meier using the log rank test with adjustment for the type of operation. Survival statistics were calculated first for each kind of treatment separately, and then combined for the calculation of the final significance value. RESULTS: Survival rates for 1, 3, and 5 years after hepatic resection were 74%, 34%, and 9%, respectively, and those after transplantation were 60%, 32%, and 25%, respectively. Univariate analysis revealed that T-3, positive lymph nodes, positive surgical margins, and pTNM stage III and IV were statistically significant poor prognostic factors. Multivariate analysis revealed that pTNM stage 0, I, and II, negative lymph node, and negative surgical margins were statistically significant good prognostic factors. For the patients in pTNM stage 0-II with negative surgical margins, 1-, 3-, and 5-year survivals were 80%, 73%, and 73%, respectively. For patients in pTNM stage IV-A with negative lymph nodes and surgical margins, 1-, 3-, and 5-year survivals were 66%, 37%, and 37%, respectively. CONCLUSIONS: Satisfactory longterm survivals can be obtained by curative surgery for hilar cholangiocarcinoma either with hepatic resection or liver transplantation. Redefining pTNM stage III and IV-A is proposed to better define prognosis.

Zinc deficiency increases hypothalamic neuropeptide Y and neuropeptide Y mRNA levels and does not block neuropeptide Y-induced feeding in rats.

Zinc deficiency reduces intake and produces an unusual approximately 3.5-d cycle of intake in rats. The mechanism underlying the anorexia and cycling has not yet been defined; current hypotheses suggest that alterations in amino acid metabolism and neurotransmitter concentrations may be a part of this anorexia. Recent reports indicate that appetite-stimulating neuropeptide Y (NPY) may be elevated during zinc deficiency. This suggests that a resistance to NPY may exist during zinc deficiency because NPY levels are high, yet appetite is low. The purpose of this study was to measure NPY peptide and mRNA concentrations during zinc deficiency in specific nuclei of the hypothalamus in which peptide and mRNA for NPY are known to be associated with appetite, and also to determine whether zinc-deficient rats are responsive to central infusions of NPY. Both NPY peptide levels in the paraventricular nucleus and NPY mRNA levels in the arcuate nucleus were higher (P < 0.05) in zinc-deficient rats than in zinc-adequate rats. When rats were administered exogenous NPY to the paraventricular nucleus, both zinc-deficient and zinc-adequate rats responded similarly by increasing food intake. These results suggest that NPY is elevated during zinc deficiency in an attempt to restore normal food intake levels, rather than being reduced and thereby contributing to the anorexia associated with zinc deficiency. During zinc deficiency, NPY receptors are able to bind NPY and initiate an orexigenic response.

Spatio-temporal and kinematic analysis of pointing movements performed by cerebellar patients with limb ataxia.

Three patients with cerebellar limb ataxia and three age-matched controls performed arm-pointing movements towards a visual stimulus during an experimental procedure using a double-step paradigm in a three-dimensional space. Four types of trajectories were defined: P1, single-step pointing movement towards the visual stimulus in the initial position S1; P2, double-step pointing movement towards S1; P3, double-step straight pointing movement towards the second position S2; and P4, double-step pointing movement towards S2 with an initial direction towards S1. We found that the cerebellar patients, as well as the controls, were able to modify their motor programs, but with impaired timing, severe anomalies in the direction and amplitude of the changed movement trajectories and alteration of the precision of the pointing movements.