Characterisation and optimisation of foams for varicose vein sclerotherapy.

BACKGROUND: Foam sclerotherapy is the process of using an aqueous foam to deliver surfactant to a varicose vein to damage vein wall endothelial cells, causing the vein to spasm, collapse and ultimately be re-absorbed into the body. Aqueous foams are complex fluids that can exhibit a significant yield stress and high effective viscosity which depend on their composition, particularly the bubble size and liquid fraction. OBJECTIVE: To characterise the properties of foams used for varicose vein sclerotherapy and determine their effectiveness in the displacement of blood during sclerotherapy. METHODS: Foams are modelled as yield stress fluids and their flow profiles in a model vein are predicted. Values of the yield stress are determined from experimental data for three different foams using the Sauter mean of the bubble size distribution. Along with the measured liquid fraction of the foams, this information is collected into a Bingham number which entirely characterises the process of sclerotherapy. RESULTS: Polydispersity in bubble size has a strong effect on the yield stress of a foam and the Sauter mean of the size distribution better captures the effects of a few large bubbles. Reducing the polydispersity increases the yield stress, and a higher yield stress results in a larger plug region moving along the vein, which is more effective in displacing blood. The width of the plug region is proportional to the Bingham number, which also has a quadratic dependence on the liquid fraction of the foam. Assuming typical values for the rate of injection of a foam, we predict that for a vein of diameter 5 mm, the most effective foams have low liquid fraction, a narrow size distribution, and a Bingham number B ≈ 4.5. CONCLUSIONS: The Sauter mean radius provides the most appropriate measure of the bubble size for sclerotherapy and the Bingham number then provides a simple measure of the efficacy of foam sclerotherapy in a vein of a given size, and explains the ability of different foams to remove varicose veins. Foams containing small bubbles, with a narrow size distribution, and a low liquid fraction are beneficial for sclerotherapy.

Infection of human cells by dengue virus is modulated by different cell types and viral strains.

Although prior studies have investigated cellular infection by dengue virus (DV), many have used highly passaged strains. We have reassessed cellular infection by DV type 2 (DV2) using prototype and low-passage isolates representing genotypes from different geographic areas. We observed marked variation in the susceptibility to infection among cell types by different DV2 strains. HepG2 hepatoma cells were susceptible to infection by all DV2 strains assayed. Although the prototype strain generated higher titers of secreted virus than the low-passage isolates, this difference did not correspond to positive- or negative-strand viral RNA levels and thus may reflect variation in efficiency among DV2 isolates to translate viral proteins or package and/or secrete virus. In contrast, human foreskin fibroblasts were susceptible to the prototype and low-passage Thai isolates but not to five Nicaraguan strains tested, as reflected by the absence of accumulation of negative-strand viral RNA, viral antigen, and infectious virus. A similar pattern was observed with the antibody-dependent pathway of infection. U937 and THP-1 myeloid cells and peripheral blood monocytes were infected in the presence of enhancing antibodies by the prototype strain but not by low-passage Nicaraguan isolates. Again, the barrier appeared to be prior to negative-strand accumulation. Thus, depending on the cell type and viral isolate, blocks that limit the production of infectious virus in vitro may occur at distinct steps in the pathway of cellular infection.

Modulation of Dengue virus infection in human cells by alpha, beta, and gamma interferons.

A role for interferon (IFN) in modulating infection by dengue virus (DV) has been suggested by studies in DV-infected patients and IFN receptor-deficient mice. To address how IFN modulates DV type 2 infection, we have assayed IFN-alpha, -beta, and -gamma for the ability to enhance or diminish antibody-independent and antibody-dependent cell infection using a competitive, asymmetric reverse transcriptase-mediated PCR (RT-PCR) assay that quantitates positive and negative strands of viral RNA, a flow cytometric assay that measures viral antigen, and a plaque assay that analyzes virion production. Our data suggest that IFN-alpha and -beta protect cells against DV infection in vitro. Treatment of hepatoma cells with IFN-alpha or -beta decreases viral RNA levels greater than 1, 000-fold, the percentage of cells infected 90 to 95%, and the amount of infectious virus secreted 150- to 100,000-fold. These results have been reproduced with several cell types and viral strains, including low-passage isolates. In contrast, IFN-gamma has a more variable effect depending on the cell type and pathway of infection. Quantitative RT-PCR experiments indicate that IFN inhibits DV infection by preventing the accumulation of negative-strand viral RNA.

In vitro transcription of mutated Leishmania tarentolae spliced leader RNA genes approximates in vivo patterns.

To elucidate the process of transcription in the kinetoplastids and to aid in the purification of transcription factors, we have developed a transcriptionally-competent nuclear extract from Leishmania tarentolae for the study of the spliced leader (SL) RNA gene. The extract was competent to transcribe a tagged SL RNA gene. The in vitro SL RNA transcripts initiated accurately and their synthesis was dependent on the presence of the promoter defined in vivo. The nuclear extract was then challenged rigorously using an exhaustive set of mutated SL RNA gene templates previously tested for transcriptional activity in vivo. Mutation of four nucleotides (CCGG) at positions -34 to -31 had a detrimental effect on transcription in vitro: the CC dinucleotide overlaps one element necessary in vivo. Similarly. four nucleotides (TGAC; positions -67 to -64) important for transcription in vitro overlapped the other core promoter element defined in vivo, but were generally not effective as point mutations. The promoter-binding ability of the transcriptionally-competent extract for the -60 region mutations mirrored the in vitro transcription pattern. Although it does not reflect precisely the in vivo results, this in vitro system provides us with an important tool for monitoring the purification of potential transcription factors, as well as the basis for future reconstitution experiments.

Single nucleotide resolution of promoter activity and protein binding for the Leishmania tarentolae spliced leader RNA gene.

In Kinetoplastid protozoa, trans-splicing is a central step in the maturation of nuclear mRNAs. In Leishmania, a common 39 nt spliced-leader (SL) is transferred via trans-splicing from the precursor 96 nt SL RNA to the 5' terminus of all known protein-encoding RNAs. In this study, promoter elements of the L. tarentolae SL RNA gene have been identified with respect to transcriptional activity and putative transcription factor binding. We have mapped the essential regions in the SL RNA gene promoter at single nucleotide resolution using both in vivo transcription and in vitro protein/DNA binding approaches. Two regions located upstream of the SL RNA gene were identified: a GN3CCC element at -39 to -33 and a GACN5G element at -66 to -58 were essential for SL RNA gene transcription in stably transfected cells. Consistent with other known bipartite promoter elements, the spacing between the GN3CCC and GACN5G elements was found to be critical for proper promoter function and correct transcription start point selection, as was the distance between the two elements and the wild-type transcription start point. The GACN5G element interacts specifically and in a double-stranded form with a protein(s) in Leishmania nuclear extracts. The degree of this protein DNA interaction in vitro correlated with SL RNA gene transcription efficiency in vivo, consistent with a role of the protein as a transcription factor. The core nucleotides GACN5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.

Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR.

In recent years, dengue viruses (serotypes 1 to 4) have spread throughout tropical regions worldwide. In many places, multiple dengue virus serotypes are circulating concurrently, which may increase the risk for the more severe form of the disease, dengue hemorrhagic fever. For the control and prevention of dengue fever, it is important to rapidly detect and type the virus in clinical samples and mosquitoes. Assays based on reverse transcriptase (RT) PCR (RT-PCR) amplification of dengue viral RNA can offer a rapid, sensitive, and specific approach to the typing of dengue viruses. We have reduced a two-step nested RT-PCR protocol to a single-tube reaction with sensitivity equivalent to that of the two-step protocol (1 to 50 PFU) in order to maximize simplicity and minimize the risk of sample cross-contamination. This assay was also optimized for use with a thermostable RT-polymerase. We designed a plasmid-based internal control that produces a uniquely sized product and can be used to control for both reverse transcription or amplification steps without the risk of generating false-positive results. This single-tube RT-PCR procedure was used to type dengue viruses during the 1995 and 1997-1998 outbreaks in Nicaragua. In addition, an extraction procedure that permits the sensitive detection of viral RNA in pools of up to 50 mosquitoes without PCR inhibition or RNA degradation was developed. This assay should serve as a practical tool for use in countries where dengue fever is endemic, in conjunction with classical methods for surveillance and epidemiology of dengue viruses.

Three small nucleolar RNAs identified from the spliced leader-associated RNA locus in kinetoplastid protozoans.

First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and approximately 450 or approximately 350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2'-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.

Evaluation of fluorine-18-BPA-fructose for boron neutron capture treatment planning.

UNLABELLED: Boron neutron capture therapy (BNCT) using 4-[10B]boronophenylalanine-fructose (BPA-Fr) is in Phase II clinical trials to validate BNCT as a treatment for glioblastoma multiforme and melanoma. Successful BNCT depends on knowledge of the distribution of boron-containing agents in both tumor and normal tissue as currently determined by chemical confirmation of boron deposition in surgically removed malignant tissue before BNCT. METHODS: We used PET to noninvasively obtain in vivo information on the pharmacokinetics of the 18F-labeled analog of BPA-Fr in two patients with glioblastoma multiforme. Time-activity curves generated from the bolus injection of 18F-BPA-Fr were coinvolved to simulate a continuous infusion used for BNCT therapy. RESULTS: Distribution of 18F-BPA-Fr by PET was found to be consistent with tumor as identified by MR imaging. The 18F-BPA-Fr tumor-to-normal brain uptake ratio was 1.9 in Patient 1 and 3.1 in Patient 2 at 52 min after injection. The 18F-BPA-Fr uptake ratio in glioblastoma paralleled that of nonlabeled BPA-Fr seen in patients as previously determined by boron analysis of human glioblastoma tissue obtained from pre-BNCT surgical biopsy. CONCLUSION: Knowledge of the biodistribution of BPA-Fr enables pre-BNCT calculation of expected tissue dosimetry for a selected dose of BPA-Fr at a specific neutron exposure. Fluorine-18-BPA-Fr PET is capable of providing in vivo BPA-Fr biodistribution data that may prove valuable for patient selection and pre-BNCT treatment planning.

The SLA RNA gene of Trypanosoma brucei is organized in a tandem array which encodes several small RNAs.

We have recently identified the Spliced Leader Associated RNA (SLA RNA) which is implicated in pre-messenger RNA splicing in Trypanosoma brucei by virtue of its interaction with the 5' splice site of the trans spliced spliced leader RNA (SL RNA) in vivo. Southern analyses reveal that the SLA RNA gene is found in a tandem array of 10-11 copies per haploid genome in T. brucei. Each repeat unit in the array encodes three additional small RNAs of unknown function. RNA polymerase inhibition studies are consistent with transcription of all four genes by the same polymerase, but do not clearly differentiate between RNA polymerases II and III. The SLA RNA has homologs in other kinetoplastid protozoa and we have determined the sequence from two additional species. Trypanosoma cruzi and Crithidia fasciculata. Features of both secondary structure and sequence are conserved in these organisms. One conserved element, 5'-UGUAGUG-3', has the potential to base-pair to the SL RNA upstream of the 5' splice site. This potential interaction is consistent with the sites of SL RNA to SLA RNA psoralen cross-linking in vivo [1].

Hepatic free radical production after cold storage: Kupffer cell-dependent and -independent mechanisms in rats.

BACKGROUND/AIMS: Free radicals are important mediators of reperfusion injury; however, the mechanism(s) of oxyradical production after liver reimplantation are not well understood. A model of cold storage and reperfusion using low-level chemiluminescence to directly measure oxyradical production during reperfusion was developed. METHODS: Rat livers were harvested and stored at 4 degrees C in University of Wisconsin cold-storage solution or Euro-Collins solution for 0-48 hours and then flushed and reperfused with warm oxygenated (37 degrees C) Krebs-Henseleit buffer. Liver chemiluminescence was measured using a sensitive photomultiplier tube, and hepatocellular injury was assessed by measuring aspartate aminotransferase release into the perfusate. RESULTS: Chemiluminescence reached a maximum within 5 minutes of reperfusion and then decreased to a baseline within 30 minutes. There was a marked increase in chemiluminescence after only a short period of storage in University of Wisconsin cold-storage solution. Chemiluminescence decreased with longer periods of storage but steadily increased again after 16 hours of storage. Chemiluminescence after 22 hours of storage, but not after 3 hours of storage, was decreased by pretreatment with the Kupffer cell inactivator gadolinium chloride. CONCLUSIONS: The data suggest two mechanisms of oxyradical production during cold storage and reperfusion of the rat liver. The later phase seems to be Kupffer cell dependent.

Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 RNAs of Trypanosoma brucei.

Messenger RNA maturation in trypanosomes involves an RNA trans-splicing reaction in which a 39 nucleotide 5'-spliced leader (SL), derived from an independently transcribed 139 nucleotide SL RNA, is joined to pre-mRNAs. Trans-splicing intermediates are structurally consistent with a mechanism of SL addition which is similar to that of cis-splicing of nuclear pre-mRNAs; homologous components (e.g. the U small nuclear RNAs) exist in both cis- and trans-splicing systems, suggesting that these also participate in the two types of splicing reactions. In this study, ribonucleoprotein (RNP) complexes containing the trypanosome SL and U2 RNAs were purified and characterized. Although present at low levels in cellular extracts, the SL and U2 RNPs are the two most abundant of the several non-ribosomal small RNP complexes in these cells. The purification scheme utilizes ion-exchange chromatography, equilibrium density centrifugation, and gel filtration chromatography and reveals that the SL RNP shares biophysical properties with U RNPs of trypanosomes and other eukaryotes; its sedimentation coefficient in sucrose gradients is approximately 10 S, and it is resistant to dissociation during Cs2SO4 equilibrium density centrifugation. Complete separation of the SL and U2 RNPs was achieved by non-denaturing polyacrylamide gel electrophoresis. Proteins purifying with the SL and U2 RNPs were identified by 125I-labeling of tyrosine residues. Four SL RNP proteins with approximate molecular masses of 36, 32, 30, and 27 kDa and one U2 RNP protein of 31 kDa were identified, suggesting that different polypeptides are associated with these two RNAs. These particles are not immunoprecipitated by anti-Sm sera which recognizes U snRNP proteins of other eukaryotes including humans plants and yeast.

Laser self-focusing in the atmosphere.

The theory of self-focusing has been applied to beams propagating in the atmosphere. The effects of density variation, absorption, and air breakdown which limit the self-focusing through power driven instabilities are estimated. It is found that laser pulses whose pulse lengths are between 10 ns and 10 micros with powers from 10(2)P(c) to 10(3)P(c) have a chance to self-focus. Here P(c) is the critical power for self-focusing in an ideal nonattenuating medium with a nonlinear index of refraction.

Prevention of platelet deposition and thrombus formation on hemodialysis membranes: a double-blind randomized trial of aspirin and dipyridamole.

A double-blind crossover study comparing low-dose aspirin (ASA) and dipyridamole (DPM) (100 mg ASA + 75 mg DPM, t.d.s.), high-dose ASA and DPM (300 mg ASA + 75 mg DPM, t.d.s.), and placebo on platelet deposition and thrombus formation on hemodialysis membranes was undertaken in 17 long-term dialysis patients. The high-dose combination significantly reduced the fall in platelet count during dialysis and also significantly increased postdialysis heparin concentrations. Scanning electron microscopy of the Cuprophan membranes showed a reduction in platelet deposition and fibrin formation during both treatment schedules, but this was most marked with the high-dose combination. The results of this study indicate that there is a graded response to combined ASA-DPM treatment and that this can significantly reduce platelet consumption and contact activation of fibrin during hemodialysis with Cuprophan membranes.

Haemofiltration for profound dialysis-induced hypotension: removal of sodium and water without blood-pressure change.

Two patients with profound dialysis-induced hypotension were seen, in both of whom sequential ultrafiltration and haemodialysis failed to alleviate their symptoms; in one bicarbonate dialysis similarly produced no improvement. The hypotension was frequently severe enough to necessitate premature termination of the dialysis. Haemofiltration was associated with almost total resolution of symptoms, adequate biochemical control of uraemia, and satisfactory removal of weight gain between dialysis sessions. Profound hypotension during dialysis may become more common as older patients enter dialysis programmes; haemofiltration is a valuable technique in such cases.