Accumulation of a soluble form of human nectin-2 is required for exerting the resistance against herpes simplex virus type 2 infection in transfected cells.

Cell entry of herpes simplex virus type 2 (HSV-2) requires the interaction of viral glycoprotein D (gD) with the receptor nectin-1 and herpesvirus entry mediator (HVEM). In addition, it is known that nectin-2 is also functional as a receptor for HSV-2, although the binding to the gD is weak. To examine an antiviral potential of a soluble form of human nectin-2 (hNectin-2Ig), transfected Vero cells expressing the entire ectodomain of nectin-2 fused to the Fc portion of human IgG were established. Specific binding of hNectin-2Ig to HSV-2 gD was confirmed by ELISA. Competitive ELISA demonstrated that accumulation of hNectin-2Ig in transfected cells increased significantly in a cell culture time dependent manner. Viral growth of several HSV-2 strains was significantly inhibited in the transfected cells that were cultured for 72 hr compared with control Vero cells, but not in cells that were cultured for 24 hr. These results indicate that accumulation of a soluble form of nectin-2 is required for exerting the resistance against HSV-2 infection.

Acetyl-L-carnitine normalizes the impaired long-term potentiation and spine density in a rat model of global ischemia.

As a consequence of an ischemic episode, energy production is disturbed, leading to neuronal cell death. Despite intensive research, the quest for promising neuroprotective drugs has largely failed, not only because of ineffectiveness, but also because of serious side-effects and dosing difficulties. Acetyl-l-carnitine (ALC) is an essential nutrient which plays a key role in energy metabolism by transporting fatty acids into mitochondria for ß-oxidation. It is an endogenous compound and can be used at high dose without toxicity in research into ischemia. Its neuroprotective properties have been reported in many studies, but its potential action on long-term potentiation (LTP) and dendritic spine density has not been described to date. The aim of the present study was an evaluation of the possible protective effect of ALC after ischemic insults inflicted on hippocampal synaptic plasticity in a 2-vessel occlusion (2VO) model in rats. For electrophysiological measurements, LTP was tested on hippocampal slices. The Golgi-Cox staining technique was used to determine spine density. 2VO resulted in a decreased, unstable LTP and a significant loss of dendritic spines. ALC administered after 2VO was not protective, but as pretreatment prior to 2VO it restored LTP nearly to the control level. This finding paralleled the histological analysis: ALC pretreatment resulted in the reappearance of dendritic spines on the CA1 pyramidal cells. Our data demonstrate that ALC administration can restore hippocampal function and spine density. ALC probably acts by enhancing the aerobic metabolic pathway, which is inhibited during and following ischemic attacks.

Techniques to facilitate ERCP with a conventional endoscope in patients with previous pancreatoduodenectomy.

There is little guidance on the performance of endoscopic retrograde cholangiopancreatography (ERCP) in patients with previous pancreatoduodenectomy. We reviewed techniques for ERCP with a conventional endoscope and assessed its value in 10 patients with previous pancreatoduodenectomy (15 ERCPs). After exploration of the surgical reconstruction, we used a front-viewing endoscope, and we used a small firm pillow under the abdomen and hand compression for preventing loop formation. Successful insertion to the ductal anastomoses and biliary cannulation were achieved in 13 / 15 procedures (87 %). In 6 procedures where we attempted pancreatic cannulation, we could not identify the pancreatojejunostomy, but after spraying contrast around the suspected location of the ductal anastomosis we obtained a pancreatogram in 4 / 6 procedures (67 %). Endoscopic biliary interventions were successful in 6 / 7 procedures (86 %). No complications were encountered. Use of appropriate techniques makes ERCP with a conventional endoscope feasible, effective, and safe in patients with previous pancreatoduodenectomy. Endoscopic therapy can be performed successfully in the bile duct, but has limited value regarding the pancreatic duct.

Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice.

The present study demonstrates that a clonal derivative (HF10) of HSV-1 strain HF effectively treated disseminated peritoneal neoplasm in an immunocompetent animal model and that all of survived mice acquired resistance to rechallenge with tumor cells. The survival time of mice treated with HF10 was longer than that of mice treated with hrR3, indicating that the oncolytic effect of HF10 was more potent than that of hrR3 in this animal model. HF10 induces syncytia formation in vitro, whereas hrR3 forms rounded CPE. The sequential administration of HF10 gave a long term survival of more than 90 days after tumor injection, with no signs of disease, in 8 of the 9 treated mice. The results suggest that treatment of disseminated peritoneal tumor with HF10 induces a specific antitumor immune response. Genomic structure determination showed that HF10 has a deletion of 3.9-kilobase pair (kbp) in the right end of UL and UL/IRL junction, resulting in the loss of UL 56 expression. A 2.3 kbp deletion and extensive rearrangement were also observed in the left end of the genome.

Herpes simplex virus encodes a virion-associated protein which promotes long cellular processes in over-expressing cells.

BACKGROUND: Herpes simplex virus (HSV) possesses a number of accessory genes which are dispensable for replication in cell culture. A previous study showed that the UL21 gene product of HSV type 1 is a virion component that is not necessary for viral replication. The function of the gene product remains unknown. RESULTS: We found that the HSV-1 UL21 gene product, a capsid-associated tegument protein with an apparent molecular mass of 62 kDa, promotes the outgrowth of long cellular processes when it is over-expressed in non-neural cells. The UL21 protein co-localizes and physically associates with microtubules in the long processes. Analysis using mutant proteins implicates a proline-rich region in promotion of the processes. CONCLUSIONS: The results suggest that the UL21 protein, like tau and other MAPs, promotes the process by directly or indirectly interacting with microtubules and facilitates the intracellular transport of the virus.

The US11 gene product of herpes simplex virus has intercellular trafficking activity.

The US11 gene product of herpes simplex virus is an abundant virion structural protein with RNA-binding regulatory activity. Its carboxyl-terminal half consists of tandem tripeptide repeats of the sequence RXP. We demonstrate that the US11 protein has intercellular trafficking activity and accumulates in the nucleolus when singly expressed in cultured cells, and that the RXP repeats are responsible for this activity. These same properties were also observed in cells expressing a fusion protein linking US11 to the green fluorescent protein. Furthermore, exogenous US11 protein was internalized by cells at 4 degrees C, which suggests that US11 protein uptake occurs primarily through an energy-independent pathway.

Colonisation and transmission of Clostridium difficile in healthy individuals examined by PCR ribotyping and pulsed-field gel electrophoresis.

Healthy adults who had not been exposed to antimicrobial agents for the preceding 4 weeks were examined for intestinal carriage of Clostridium difficile. The 1234 individuals examined were composed of seven groups: three classes of university students, hospital workers at two hospitals, employees of a company and self-defence force personnel at a local station. Overall, 94 (7.6%) individuals were positive for C. difficile by faecal culture but carriage rates among the study groups ranged from 4.2% to 15.3%. Typing by PCR ribotyping and pulsed-field gel electrophoresis demonstrated clusters of carriers colonised by a single type in each of three groups, indicating that cross-transmission of C. difficile can occur in community settings. Follow-up culture was performed on 38 C. difficile-positive individuals and C. difficile was isolated again from 12 (32%) of them 5-7 months after the initial culture; six (50%) of these 12 individuals had a new strain on repeat culture. Two or more family members were C. difficile-positive in five of 22 families examined. C. difficile with an identical type was isolated from persons within a family in only one family. These results suggest that intestinal carriage by healthy adults may play a role as a reservoir for community-acquired C. difficile-associated diarrhoea, but that cross-transmission of C. difficile does not occur frequently among family members at home.

Identification and characterization of the UL24 gene product of herpes simplex virus type 2.

The UL24 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 281 amino acid protein with a molecular mass of 30.5 kDa. In this study, the HSV-2 UL24 gene product has been identified by using a rabbit polyclonal antiserum produced against a recombinant protein containing the full-length UL24 gene product of HSV-2 fused to glutathione-S-transferase. The antiserum reacted specifically with a 32 kDa protein in HSV-2 186-infected Vero cells and with 31 and 32 kDa proteins in UL24-expressing Cos-7 cells. Accumulation of UL24 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. UL24 protein was found to be associated with purified HSV-2 virions and C capsids. Indirect immunofluorescence analysis demonstrated that the UL24-specific fluorescence was detected in perinuclear regions of the cytoplasm and/or in the nucleus as small discrete granules from 9h post infection (hpi). Furthermore, the UL24 protein expressed singly was detected predominantly in the nucleus and slightly in the cytoplasm at 24 h after transfection, with branch-like cytoplasmic protruding structures. Strong nucleolus staining was visible in partial cells.

A chimeric CYP11B1/CYP11B2 gene in glucocorticoid-insuppressible familial hyperaldosteronism.

Although a chimeric gene combining the 11beta-hydroxylase gene (CYP11B1) and the aldosterone synthase gene (CYP11B2) explains the pathophysiology of familial hyperaldosteronism (FH) type I, the contribution of this abnormality to FH type II has not been tested. We screened genomic DNA from a Japanese family with FH type II for the CYP11B1/CYP11B2 gene. The index patient was a 27-year-old woman with hypertension. Hypokalaemia, elevated plasma aldosterone and suppressed plasma renin activity suggested primary aldosteronism. Though computed tomography failed to reveal an adrenal tumour, left adrenalectomy was indicated due to a high aldosterone concentration in left adrenal venous blood. The resected adrenal gland contained an adenoma. As her mother had also been diagnosed with primary aldosteronism due to an adenoma, we administered oral dexamethasone to our patient before the operation and observed the response of the blood pressure and plasma aldosterone concentration for 2 weeks. Both parameters remained elevated during the treatment period, confirming the diagnosis of FH type II. Total DNA was isolated from blood cells of the index patient, her mother, and an unaffected brother. Samples were amplified by polymerase chain reaction using specific primers from CYP11B1 and CYP11B2. Unique DNA fragments of 1.4 kb were obtained from the index patient and her mother, but not from the healthy subject. The CYP11B1/CYP11B2 chimeric gene was found in a Japanese family with FH type II.

Diurnal variation of hemodynamic indices in non-dipper hypertensive patients.

The purpose of this study was to elucidate the underlying mechanisms of blunted nocturnal blood pressure reduction in non-dipper hypertensive patients. We studied the diurnal variations in systemic hemodynamic indices and baroreflex sensitivity. In 45 subjects with essential hypertension (24 men; mean age, 49+/-1 years), intra-arterial pressure was monitored telemetrically. Non-dippers were defined as those with a nocturnal reduction of systolic blood pressure of less than 10% of daytime systolic blood pressure. Stroke volume was determined using Wesseling's pulse contour method, calibrated with indocyanine green dilution. Baroreflex sensitivity was calculated as deltapulse interval/deltasystolic blood pressure on spontaneous variations. The mean values of the hemodynamic parameters were calculated every 30 min. Twenty-six subjects were classified as non-dippers. Daytime blood pressure was not significantly different between dippers (149+/-4/87+/-3 mmHg) and non-dippers (147+/-3/82+/-2 mmHg), while the nighttime blood pressure was significantly reduced in dippers (131+/-3/77+/-2 mmHg) but not in non-dippers (145+/-3/80+/-2 mmHg). Nocturnal decreases in both cardiac index and stroke index were smaller in non-dippers (-12.0+/-1.2% and 1.5+/-1.0%) than in dippers (-17.5+/-1.4% and -2.2+/-1.1%). Baroreflex sensitivity significantly increased at nighttime both in dippers (6.5+/-0.6 to 8.0+/-0.7 ms/mmHg) and in non-dippers (5.1+/-0.3 to 6.4+/-0.4 ms/mmHg). Neither daytime nor nighttime baroreflex sensitivity was significantly different between the groups. We conclude that the hemodynamics of non-dipper essential hypertension are characterized by an inadequate nocturnal decrease in cardiac index and stroke index, suggestive of relative volume expansion or malsuppressed sympathetic activity.