Evaluation of an infectivity standard for real-time quality control of human immunodeficiency virus type 1 quantitative micrococulture assays. Participating Laboratories of The AIDS Clinical Trials Group

Quantitative microculture assays of cryopreserved human immunodeficiency virus type 1-infected cell suspensions and culture supernatants were compared among seven assays sites. There was no significant change in titer during 1 year of storage. The overall standard deviation for infected cell suspensions was approximately 0.8 log10 virus titer. A method for detecting deviant assay results was developed and was used to identify two donor cell preparations (n = 54) that gave consistently low titers.     

Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome

Swine infectious agents, especially viruses, are potential public health risks associated with the use of pig organs for xenotransplantation in humans. Therefore, there is a need for better characterization of swine viruses and for the development of diagnostic tests for their detection. We report here isolation of a novel strain of porcine circovirus (PCV) from pigs with postweaning multisystemic wasting syndrome (PMWS). Affected pigs exhibited severe interstitial pneumonia and lymphoid depletion. The complete nucleotide sequence (1,768 nucleotides) of the genome of the PCV isolate was determined and compared with the sequence of the PCV strain isolated from PK-15 cells. Sequence comparison revealed significant differences between the two PCV strains, with an overall DNA homology of 76%. Two major open reading frames (ORFs) were identified. ORF1 was more conserved between the two strains, with 83% nucleotide homology and 86% amino acid homology. ORF2 was more variable, with nucleotide homology of 67% and amino acid homology of 65%. PCR and in situ hybridization demonstrated abundant viral DNA in various organs of pigs with PMWS. In situ hybridization demonstrated that this strain of PCV targets multiple organs and infects macrophages, lymphocytes, endothelial cells, and epithelial cells.     

Influence of tidal volume on the distribution of gas between the lungs and stomach in the nonintubated patient receiving positive-pressure ventilation

The technique involving filtration of diluted blood enables the separate analysis of the flow properties of different cell subpopulations. This study was designed to assess the changes occurring in the flow properties and function of blood cells in stored bank blood and salvaged blood compared to patient blood in a given clinical situation. We measured hydrogen peroxide production by neutrophils and the filterability, through 5 microm Nucleopore filters, of isolated red blood cells and of diluted blood. Samples were obtained from patients undergoing aortic surgery and blood intended for transfusion: either salvaged during surgery or stored bank blood. Both salvaged and bank blood were much less filterable than patient blood, with reduced deformability of both red and white blood cells. However, salvaged blood contained highly activated neutrophils with a prolonged transit time of the 'fast-flowing' cells in the analysis compared to bank blood. Bank blood contained significantly more particles which acted as pore-blockers. Cells in bank and salvaged blood therefore have markedly abnormal flow and biochemical properties compared to patient blood.     

Role of spontaneous and interleukin-2-induced natural killer cell activity in the cytotoxicity and rejection of Fas+ and Fas- tumor cells

In the current study, we investigated whether the naive, poly I:C or interleukin-2 (IL-2)-induced natural killer (NK)/lymphokine-activated killer (LAK) cells use perforin and/or Fas ligand (FasL) to mediated cytotoxicity. We correlated these findings with the ability of mice to reject syngeneic Fas+ and Fas- tumor cells either spontaneously or after IL-2 treatment. The spontaneous NK-cell-mediated cytotoxicity was primarily perforin based, whereas the poly I:C and IL-2-induced NK/LAK activity was both FasL and perforin dependent. L1210 Fas+ tumor targets were more sensitive than L1210 Fas- targets to poly I:C and IL-2-induced cytotoxicity in wild-type, gld/gld, and perforin knockout mice. When L1210 Fas+ and Fas- tumor cells were injected subcutaneously (sc) or intraperitoneally into syngeneic mice, Fas- tumor cells caused mortality earlier than Fas+ tumor cells. Also, approximately 20% of the mice injected sc with L1210 Fas+ tumor cells survived the challenge(>60 days), whereas all mice injected similarly with L1210 Fas- tumor cells died. When immunotherapy using IL-2 (10,000 U, three times/d for a week, followed by once/d for an additional week) was attempted in mice injected sc with tumor cells, IL-2 treatment was very effective against mice bearing L1210 Fas+ (40% survival) but not L1210 Fas- (0% survival) tumors. These data correlated with the finding that the LAK cells from IL-2-injected mice caused increased cytotoxicity against L1210 Fas+ when compared with L1210 Fas- targets. Also, L1210 Fas+ tumor-bearing mice showed increased tumor-specific cytotoxic T lymphocyte (CTL) activity when compared with those bearing L1210 Fas- tumor cells. Together our studies show for the first time that expression of Fas on tumor targets makes them more immunogenic as well as susceptible to CTL- and IL-2-induced LAK activity. The Fas+ tumor cells are also more responsive to immunotherapy with IL-2.     

A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis

von Willebrand factor (vWf) deficiency causes severe von Willebrand disease in humans. We generated a mouse model for this disease by using gene targeting. vWf-deficient mice appeared normal at birth; they were viable and fertile. Neither vWf nor vWf propolypeptide (von Willebrand antigen II) were detectable in plasma, platelets, or endothelial cells of the homozygous mutant mice. The mutant mice exhibited defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events in approximately 10% of neonates. As in the human disease, the factor VIII level in these mice was reduced strongly as a result of the lack of protection provided by vWf. Defective thrombosis in mutant mice was also evident in an in vivo model of vascular injury. In this model, the exteriorized mesentery was superfused with ferric chloride and the accumulation of fluorescently labeled platelets was observed by intravital microscopy. We conclude that these mice very closely mimic severe human von Willebrand disease and will be very useful for investigating the role of vWf in normal physiology and in disease models.     

Transjugular intrahepatic portosystemic shunts (TIPS)

Transjugular intrahepatic portosystemic shunt (TIPS) is a procedure recently introduced for the management of complications of portal hypertension. TIPS can be placed in the liver with relative ease by a skilled radiologist with a low risk of mortality. The major complications following the procedure are infection, especially in patients undergoing emergency TIPS, intra-abdominal haemorrhage from capsular punctures, and long-term problems related to encephalopathy and stenosis of the shunt. Encephalopathy is more of a problem in older patients with wide diameter shunts. Stenosis of the shunt is related to pseudo-intimal hyperplasia, probably related to transection of bile ductules during placement of the shunt. In view of the high rate of encephalopathy and stenosis following the shunt, a careful follow-up of all patients, including ultrasonographic and angiographic examination of the shunt, is mandatory. TIPS is used predominantly for the control of acute variceal haemorrhage, prevention of recurrent variceal bleeding, and refractory ascites when conventional treatment has failed. However, the role of TIPS in the management of complications of portal hypertension still awaits the outcome of clinical trials.     

The use of hormone replacement therapy in women with acute myocardial infarction: an audit of current practice

We undertook criterion-based audit of the current practice of prescribing hormone replacement therapy for women with acute myocardial infarction; the audit included 181 consecutive women admitted to one hospital with this diagnosis in one calendar year. The set standard was that, barring any contraindication, all postmenopausal women with acute myocardial infarction should be prescribed hormone replacement therapy before discharge from hospital. The evidence base of this standard derives from more than 30 epidemiological and clinical studies and a large body of biological data. Only 4.7% of the women were current users of hormone replacement therapy and the set standard was met in only 3% of eligible nonusers. Professionals caring for women who have had a myocardial infarction need to consider hormone replacement therapy as a secondary prophylaxis of myocardial infarction. Gynaecologists should liaise with colleagues in other specialties and general practice to ensure that information on the nongynaecological benefits of hormone replacement therapy is widely disseminated.     

Utility of magnetic resonance arteriography for distal lower extremity revascularization

PURPOSE: Magnetic resonance arteriography (MRA) of the lower extremities affords several possible advantages over conventional contrast arteriography (CA). We hypothesized that MRA of the infrageniculate vessels was sufficiently accurate to replace CA before revascularization procedures in patients with limb-threatening ischemia. METHODS: Fifty-three extremities in 49 patients were prospectively evaluated before attempted infrageniculate revascularization procedures with preoperative infrageniculate time-of-flight MRA (cost, $170/study) and standard contrast arteriography (cost, $1310/study) of the aortoiliac and runoff vessels. Independent operative plans were formulated based on the MRA and CA results before the revascularization procedure. Intraoperative, prebypass arteriograms (IOA; cost, $46/study) were obtained in all patients to confirm the adequacy of the distal runoff. The preoperative plans formulated by the results of MRA and CA were compared with the actual procedure performed based on the IOA. All arteriograms (CA, MRA, IOA) were reviewed after the operation by two independent reviewers, and the number of patent vessel segments and those with < 50% stenosis was determined. RESULTS: Revascularization procedures were performed in 44 of 53 extremities (83%), and amputation was performed in nine extremities (17%) because of an absence of a suitable bypass target. The CA and MRA were equally effective in predicting the optimal operative plans as determined from IOA (CA, 42 of 53 [77%] vs MRA, 40 of 53 [75%]; p = 0.79). More patent vessel segments were seen on CA than MRA (reviewer A, 229 vs 174, kappa = 0.32; reviewer B, 321 vs 314, kappa = 0.46); however, a comparable number of segments were seen if the vessels of the foot were excluded. The accuracy (reviewer A, 78% vs 68%, p = 0.003; reviewer B, 75% vs 67%, p = 0.003) and sensitivity (reviewer A, 69% vs 51%, p = 0.001; reviewer B, 68% vs 46%, p = 0.0001) of CA relative to IOA were superior to those of MRA, although the specificity was comparable (reviewer A, 86% vs 90%, p = 0.31; reviewer B, 82% vs 87%, p = 0.52). The combination of MRA and IOA would have resulted in the optimal operative plan in 51 of the 53 cases (96%) and was comparable with CA and IOA (53 of 53; 100%; p = 0.50). Substitution of MRA and IOA for CA and IOA could potentially have saved an estimated $60,420. CONCLUSIONS: The combination of MRA and IOA provides an accurate, cost-efficient strategy for visualization of the infrageniculate vessels before revascularization procedures.     

A multicenter study of bispectral electroencephalogram analysis for monitoring anesthetic effect

Occludin is the only known integral membrane protein localized at the points of membrane- membrane interaction of the tight junction. We have used the Xenopus embryo as an assay system to examine: (a) whether the expression of mutant occludin in embryos will disrupt the barrier function of tight junctions, and (b) whether there are signals within the occludin structure that are required for targeting to the sites of junctional interaction. mRNAs transcribed from a series of COOH-terminally truncated occludin mutants were microinjected into the antero-dorsal blastomere of eight-cell embryos. 8 h after injection, the full-length and the five COOH-terminally truncated proteins were all detected at tight junctions as defined by colocalization with both endogenous occludin and zonula occludens-1 demonstrating that exogenous occludin correctly targeted to the tight junction. Importantly, our data show that tight junctions containing four of the COOH-terminally truncated occludin proteins were leaky; the intercellular spaces between the apical cells were penetrated by sulfosuccinimidyl-6-(biotinamido) Hexanoate (NHS-LC-biotin). In contrast, embryos injected with mRNAs coding for the full-length, the least truncated, or the soluble COOH terminus remained impermeable to the NHS-LC-biotin tracer. The leakage induced by the mutant occludins could be rescued by coinjection with full-length occludin mRNA. Immunoprecipitation analysis of detergent-solubilized embryo membranes revealed that the exogenous occludin was bound to endogenous Xenopus occludin in vivo, indicating that occludin oligomerized during tight junction assembly. Our data demonstrate that the COOH terminus of occludin is required for the correct assembly of tight junction barrier function. We also provide evidence for the first time that occludin forms oligomers during the normal process of tight junction assembly. Our data suggest that mutant occludins target to the tight junction by virtue of their ability to oligomerize with full-length endogenous molecules.