Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase.

Monotherapy with (-)2',3'-dideoxy-3'-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 --> valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV-neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme.

Improvement of facial skin characteristics using copper oxide containing pillowcases: a double-blind, placebo-controlled, parallel, randomized study.

Copper plays a key role in several processes of skin formation and regeneration. Copper has been shown to be absorbed through intact skin. We hypothesized that sleeping on fabrics containing copper-impregnated fibres would have a positive cosmetic effect on the skin. The aim of this study was to confirm our hypothesis. A 4-week, double blind, parallel, randomized study was carried out in which 57 volunteers aged 40-60 years used either copper oxide containing pillowcases (0.4% weight/weight) or control pillowcases not containing copper. Photographs were taken by a professional photographer of each participant at the beginning of the study and at 2 and 4 weeks after the commencement of the study. Two expert graders (a dermatologist and a cosmetologist) evaluated the pictures for the effect on several cosmetic facial skin characteristics. The copper-containing pillowcases had a positive effect for the following facial characteristics: reduction of wrinkles (P < 0.001) and crow's feet/fine lines (P < 0.001) and improvement of general appearance (P < 0.001) at both 2 and 4 weeks. The differences were statistically significant (Wilcoxon scores and chi-squared tests). Consistent sleeping for 4 weeks on copper oxide containing pillowcases caused a significant reduction in the appearance of facial wrinkles and crow's feet/fine lines and significant improvement in the appearance of facial skin. In most trial participants, this effect was already noticeable within 2 weeks of using the copper oxide containing pillowcases.

Chronic immune activation associated with intestinal helminth infections results in impaired signal transduction and anergy.

Helminthic parasites cause widespread, persistent infections in humans. The immigration of Ethiopians to Israel (a group denoted here by "Eth."), many of them infested with helminths and in a chronic immune-activation state, enabled us to investigate the effects of such immune activation on immune responses. We studied the immune profile and immune functions of 190 Eth. and Israeli non-Eth. (Isr.) highly, partially, or non-immune-activated individuals. Immune cells from highly immune-activated individuals were defective in several signaling responses, all of which were restored gradually following anti-helminthic treatment. These cells showed poor transmembrane signaling, as seen by the phosphorylation of various tyrosine kinases and of the MAPK kinases, ERK1/2 and p38; deficient degradation of phosphorylated IkappaBalpha; increased expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), which appears to block proliferative responses in these cells; decreased beta-chemokine secretion by CD8(+) cells after stimulation; and reduced proliferation to recall antigen stimulation. Highly immune-activated individuals also showed decreased delayed-type skin hypersensitivity responses to recall antigen before deworming. These findings support the notion that chronic helminthic infections cause persistent immune activation that results in hyporesponsiveness and anergy. Such impaired immune functions may diminish the capacity of these individuals to cope with infections and to generate cellular protective immunity after vaccination.

A potent antihemorrhagin in the serum of the non-poisonous water snake Natrix tessellata: isolation, characterization and mechanism of neutralization.

The main natural antihemorrhagic factor (NtAH), which inhibits the hemorrhagic activity of Bothrops asper snake venom, was isolated from the serum of the non-poisonous water snake Natrix tessellata by ammonium sulfate precipitation at 35-55%, Sephadex G-75 gel filtration, ion exchange chromatography on DEAE-Sepharose and CM-Sepharose and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 4.5 and a molecular mass of about 880 kDa. The antihemorrhagic activity was stable between pH 5.5-11.7 and up to 50 degrees C, but lost activity after 20 min at 60 degrees C. It did not form a precipitin line with the main hemorrhagin of Bothrops asper snake venom (BaH1), nor with the whole venom, which suggests that the antihemorrhagic factor is not an immunoglobulin. The mechanism of neutralization by the isolated antihemorrhagic factor NtAH did not include digestion of the hemorrhagic toxin BaH1. Chromatography of NtAH with active 125I-labeled BaH1 toxin as well as ELISA experiments demonstrated that the mechanism of neutralization involves formation of an inactive soluble complex between the natural NtAH of the non-poisonous water snake and the main hemorrhagin of Bothrops asper venom.

Isolation, characterization and mode of neutralization of a potent antihemorrhagic factor from the serum of the snake Bothrops asper.

A potent antihemorrhagic factor (BaSAH1) was isolated from the serum of the snake Bothrops asper by ammonium sulfate precipitation at 40-60%, Sephacryl S-200 and Sephadex G-50 gel filtration, DEAE-Sepharose, and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 5.2 and a molecular weight of 66 kDa. 4 micrograms of the purified factor BaSAH were needed to neutralize the hemorrhagic dose of B. asper whole venom compared to 60 micrograms of the clinically used horse polyvalent immunoglobulins. Moreover, 0.35 microgram of BaSAH were sufficient to achieve complete neutralization of the main hemorrhagic toxin (BaH1), with a molar ratio of 2:1. The antihemorrhagic activity was stable between pH 1.5-9 and up to 60 degrees C but lost activity completely after 30 min of heating at 70 degrees C. BaSAH did not digest the hemorrhagic toxin BaH1 or formed a precipitin line with it, nor with the whole venom. Both ELISA experiments and chromatography of BaSAH after incubation with the 125I-labeled hemorrhagic toxin BaH1 demonstrated that the mechanism of the neutralization involves a formation of an inactive soluble complex between the natural antihemorrhagin and the main hemorrhagin of B. asper venom.

Multi-targeting the entrance door to block HIV-1.

The multistep nature of HIV-1 entry provides multisite targeting at the entrance door of HIV-1 to cells. Blocking HIV-1 entry to its host cells has clear advantages over blocking subsequent stages in the life cycle of the virus. Indeed, potent cooperative and synergistic inhibition of HIV-1 proliferation has been observed in in vitro studies with several entry inhibitor combinations, interacting with different steps of the HIV-1-cell entry cascade. Targeting a compound to several steps of the viral-cell entry and also to subsequent steps in the viral life cycle promises an even more effective therapeutic, by reducing the probability of HIV-1 to develop resistance. Using one drug that can target multiple sites and/or steps in the viral life cycle will have obvious advantages in clinical use. In this article we review the multistep process of HIV-1 cell entry and the current repertoire of inhibitors of this critical stage in the viral life cycle, and introduce an example of multisite HIV-1 targeting of the cell entry and subsequent critical steps in the viral life cycle.

Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase.

OBJECTIVE: To measure the extent of HIV resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) within the context of monotherapy and to assess the presence of the M184V substitution in the case of 3TC-resistant viruses. Whether the success of 3TC in clinical trials could be due, in part, to an increase in the fidelity of HIV reverse transcriptase conferred by the M184V substitution was also considered. METHODS: Two separate monotherapy studies were evaluated, one involving adults with CD4 counts > or = 300 x 10(6)/l, and the second involving children, some of whom had received antiretroviral treatment previously, while others were drug naive. Peripheral blood and plasma samples were collected regularly, and HIV isolation and determinations of drug median inhibitory concentration values were performed using umbilical cord mononuclear cells as targets. Amplification of the 184 mutation was performed by the polymerase chain reaction, using specific primer pairs. Fidelity determinations using purified, recombinant HIV reverse transcriptase derived from either wild-type virus or viruses that contained the 184V substitution were performed. RESULTS: Phenotypic resistance was detected in almost all subjects at times ranging from 8-20 weeks after initiation of therapy. The 184V substitution was usually detected prior to the occurrence of phenotypic resistance to 3TC. Fidelity determinations revealed that the 184V substitution conferred an approximately 5- to 10-fold increase in HIV reverse transcriptase fidelity. In addition, titres of patient sera tested for their ability to neutralize autologous sequential viral isolates were stabilized in patients receiving 3TC therapy as opposed to other drugs. CONCLUSIONS: Resistance to 3TC developed in virtually all subjects treated with this drug, and was associated with the appearance of an M184V mutation in HIV reverse transcriptase. The clinical benefit of 3TC therapy may be attributable in part to selection of viruses that are less able to replicate and mutate than the wild types.

Inhibitory potency of R-region specific antisense oligonucleotides against in vitro DNA polymerization and template-switching reactions catalysed by HIV-1 reverse transcriptase.

Antisense oligonucleotides (AONs) targeted to the R-region near the 5'-LTR of HIV-1 genomic RNA inhibited both the synthesis of (-) strong stop DNA and the first template-switch reaction catalysed by HIV-1 reverse transcriptase (RT) in vitro. The 18 nucleotide (nt) AONs used were identical in sequence but differed in the sugar component of the 3'-terminal nucleotide, with either 2'-deoxy-D-ribose (DNA), 2'-deoxy-L-ribose (L), or arabinose (ARA) in this position. All three AONs hybridized to complementary 18 nt RNA (T(m) approximately 70 degrees C) and specifically interacted with the target RNA HIV-1 sequence at 37 degrees C. L was unable to serve as primer for RT-catalysed DNA polymerization, whereas priming from ARA was about 30% that noted with DNA. Each of the three AONs resulted in similar 85-95% decreases in the amount of full length (-) strong stop DNA and up to 75% decreases in the first template-switch reaction products formed by RT, implying that elongation of the AONs did not enhance the inhibitory activity in vitro. A concomitant increase in a truncated DNA product corresponding to polymerization termination at the 5'-end of the AON was noted, indicating that RT was unable to displace the AON. Interestingly, near maximal inhibition in vitro an AON:target RNA template ratio of 1:1 was noted. Our results confirm the validity of our in vitro system for the analysis of potential antisense oligonucleotide inhibitors, and suggest that antisense oligonucleotides directed to the R-region of HIV-1 RNA may be effective inhibitors of the initial stages of HIV-1 proviral DNA synthesis.

Echinhibin-1--an inhibitor of Sendai virus isolated from the venom of the snake Echis coloratus.

The snake venom of Echis coloratus was found to abolish the hemagglutinating activity, hemolytic activity and in vivo infectivity of Sendai virus. The active factor (Echinhibin-1) was purified by gel filtration on Sephadex G-50, followed by chromatography on DEAE-Sepharose and CM-Sepharose. Echinhibin-1 is a protease with a molecular weight of about 25 kDa, an isoelectric point of 7 and is stained by PAS, indicating that it is a glycoprotein. It showed a strong azocollase activity that was stable up to 68 degrees C and at pH values of 4.5-10.5. Ten micrograms/ml were sufficient to abolish the hemolytic effect of the virus on human erythrocytes when incubation was at 37 degrees C for 2 h, while 20 micrograms/ml abolished the hemagglutinating activity. Addition of Echinhibin-1 after the adsorption of Sendai virions onto washed erythrocytes at 4 degrees C did not inhibit the subsequently hemolytic activity at 37 degrees C, indicating that Echinhibin-1 interferes with virus adsorption to the cells. Of various protease inhibitors, only Na2 EDTA and o-phenanthroline inhibited the antiviral activity of the purified factor, indicating that it is a metalloproteinase. In vivo, mice inoculated intranasally with the virus pretreated with Echinhibin-1 developed well and gained weight, whereas untreated virus-infected mice lost weight and died within 1 week. Intravenous administrations of the purified factor up to 80 micrograms/mouse produced no signs of toxicity and subcutaneous injections caused no hemorrhagic activity, while the whole venom is very hemorrhagic with an LD50 of 250 micrograms/kg for mice.

Isolation and characterization of synergistic hemorrhagins from the venom of the snake Bothrops asper.

Three hemorrhagic factors (BaH1, BH2 and BH3) were isolated from the venom of Bothrops asper by gel filtration on Sephacryl S-200, DEAE-Sepharose chromatography, metal chelate affinity chromatography and hydrophobic interaction chromatography. They contain 55% of the total hemorrhagic activity of the whole venom when they are mixed, but lose almost half of the activity if they are separated, indicating a synergism between the three. The main hemorrhagin is BaH1 (Bothrops asper hemorrhagin 1); the other two are weak hemorrhagins but contribute to the synergism. They are acidic proteins with a pI of 4.5, 5.2 and 5; their mol. wt is 64,000, 26,000 and 55,000 respectively. The minimal hemorrhagic dose (MHD) of BaH1, BH2 and BH3 is 0.18, 2 and 1.6 micrograms, with a specific activity 55, 5 and 6.25 higher than that of the whole venom. The hemorrhagic activity of all three factors was inhibited by EDTA and ortho-phenathroline, indicating that the hemorrhagic activity is metal dependent. Phosphoramidon, soybean trypsin inhibitor, PMSF, pepstatin and aprotinin did not affect the hemorrhagic activity of the isolated factors.

Inhibition of the hemorrhagic activity of Bothrops asper venom by a novel neutralizing mixture.

This study screened 25 sera, 19 synthetic products and five antivenoms obtained after immunization for their ability to neutralize the hemorrhagic activity of venom from the snake Bothrops asper. Among the sera screened, the homologous serum of B. asper itself was found to possess the highest neutralizing capacity, abolishing the hemorrhagic effect of the venom at weight ratio of 3:1. It was more efficient than the antisera obtained by immunization. Among the synthetic compounds tested, only O-phenanthroline and EDTA salts inhibited the hemorrhagic activity at concentrations of 0.5-10 mM; however, only CaNa2EDTA was non-toxic at the concentrations studied. Intravenous injections and in situ administration of the non-toxic inhibitors revealed that a fraction of B. asper serum, the horse polyvalent antivenom and CaNa2EDTA were the most potent antihemorrhagic materials against B. asper venom, especially when administered in situ as a mixture. This work suggests that this neutralizing mixture could be highly useful in the neutralization of local and systemic hemorrhage developing after B. asper envenomation.

In vitro activity of BaH1, the main hemorrhagic toxin of Bothrops asper snake venom on bovine endothelial cells.

Incubation of BaH1, the main hemorrhagic toxin purified from the venom of Bothrops asper, with endothelial cells caused the appearance of spaces among the cells. This effect became more noticeable with increasing hemorrhagin concentration and longer incubation time. Later, the cells became rounded and detached from the substrate into the medium. Augmentation of Trypan blue did not stain the detached cells, indicating their viability. Moreover, after washing the floating cells from the toxin they could be recultivated: they again spread on the substrate and proliferated, demonstrating that BaHl is not directly cytotoxic to the endothelial cells.

Pathological changes induced by BaH1, a hemorrhagic proteinase isolated from Bothrops asper (Terciopelo) snake venom, on mouse capillary blood vessels.

The pathological changes induced in capillaries by BaH1, a hemorrhagic metalloproteinase isolated from the venom of Bothrops asper, were studied after i.m. injection in mouse gastrocnemius. Hemorrhage was observed macroscopically, and corroborated histologically, within the first 5 min. At the ultrastructural level, the earliest changes in endothelial cells, observed 1 min after toxin administration, consisted of a decrease in the number of pinocytotic vesicles, the presence of blebs and cytoplasmic projections pinching off to the vascular lumen and the detachment of endothelial cells from the surrounding basal lamina. These processes occurred concomitantly with a thinning of endothelial cells. In capillaries undergoing more advanced degenerative stages, there were gaps or breaks in endothelial cells through which erythrocytes were escaping to the extravascular space. In these cells, the basal lamina was usually absent. Throughout this process, intercellular junctions remained apparently intact and no evidence was found of extravasation through widened intercellular junctions. In addition to this morphological pattern of degeneration, some capillaries presented swollen endothelial cells with dilated endoplasmic reticulum and lacking pinocytotic vesicles. Many capillaries contained platelet plugs and fibrin. Thus, hemorrhage induced by BaH1 occurs per rhexis, as has been also described for other venoms and hemorrhagic toxins.

Effect of various Viperidae and Crotalidae snake venoms on endothelial cells in vitro.

The effect of various crotalid and viperid venoms at 10, 50 and 100 micrograms/ml was examined on bovine and murine endothelial cells in vitro. The venoms caused the cells to lose their processes, leading to the appearance of spaces which were gradually enlarged between clusters of cells. The cells became round and finally detached from the substrate. This effect was more pronounced on bovine normal cells than on murine transformed cells. Most of the venoms did not affect the viability of the cells even after 24 hr of incubation, as determined by the trypan blue dye exclusion procedure. Moreover, after the cells were washed from the venoms and transferred into fresh medium, they regained their original morphology after spreading on the substrate and they then proliferated normally. This reversible effect shows that most of the crotalid and viperid venoms examined were not directly cytotoxic to the endothelial cells at the concentrations tested.

Isolation and characterization of a metalloproteinase with weak hemorrhagic activity from the venom of the snake Bothrops asper (terciopelo).

A metalloproteinase, named BaP1, was purified to homogeneity from the venom of Bothrops asper (Pacific region) of Costa Rica by ion-exchange chromatography on CM-Sephadex and gel filtration on Sephacryl S-200. The enzyme has a mol. wt of 24,000 and contains few Cys and high numbers of Asp, Leu, Ser and Glu. BaP1 hydrolyzes casein, hide powder azure and fibrinogen, having an optimal pH of 8.0. It rapidly digests the A alpha-chain of fibrinogen and, later on, the B beta-chain, leaving the gamma-chain unaffected. Chelating agents (EDTA and 1,10-phenanthroline) inhibited proteolytic activity, whereas 2-mercaptoethanol and soybean trypsin inhibitor did not affect this activity. BaP1 has a weak hemorrhagic activity, with a minimum hemorrhagic dose of 20 micrograms; this activity was inhibited by EDTA and was abolished after incubation at 60 degrees C. In addition, BaP1 induces edema and a mild myotoxic effect, lacking coagulant, defibrinating and lethal effects.

Immunological studies on BaH1 and BaP1, two hemorrhagic metalloproteinases from the venom of the snake Bothrops asper.

No immunological cross-reactivity was observed between BaH1 and BaP1, two hemorrhagic metalloproteinases isolated from B. asper venom, by gel immunodiffusion, Western blotting and neutralization studies. Cross-reactivity was detected with antisera against these toxins in several crotaline and viperine snake venoms by ELISA, whereas no reactivity was observed with either antiserum against the venoms of Bothrops nummifer, Crotalus durissus terrificus, Vipera russelli and several elapid venoms. Antiserum against native BaH1 neutralized hemorrhagic activity of the venoms of B. asper, B. atrox, B. jararaca, Crotalus atrox, C. durissus durissus, Echis carinatus and Trimeresurus flavoviridis, being ineffective against the venoms of Agkistrodon bilineatus and Lachesis muta.

Activity of hemorrhagic metalloproteinase BaH-1 and myotoxin II from Bothrops asper snake venom on capillary endothelial cells in vitro.

In vivo, hemorrhagic toxins isolated from snake venoms cause a disorganization of the basal lamina of capillaries, with a concomitant degenerative process of endothelial cells. In this study we investigated the effects of BaH-1, a hemorrhagic metalloproteinase purified from the venom of Bothrops asper, on a murine endothelial cell line of capillary origin. A quantitative cytotoxicity assay based on the release of lactic dehydrogenase was utilized. BaH-1, despite its potent hemorrhagic activity, did not exert direct cytolytic activity on the endothelial cells, even at concentrations as high as 65 micrograms/ml. The only visible effect of BaH-1 on the cultured cells was a relatively slow, moderate detachment of cells, interpreted as a consequence of proteolytic degradation of extracellular matrix components. In contrast, myotoxin II, a lysine-49 phospholipase A2 from the same venom, was clearly cytotoxic to this cell type, albeit being devoid of hemorrhagic activity. These findings suggest that the ability of venom metalloproteinases to induce hemorrhage is not related to a direct cytotoxic action on endothelial cells, and that the rapid degenerative changes of endothelium observed in vivo are probably the result of an indirect mechanism.

Inhibition of Sendai virus by various snake venom.

Viperid, elapid and crotalid snake venoms were screened in vitro for antiviral activity against Sendai virus. The hemolysis of 10(8) human erythrocytes in 1 ml, caused by 70 HAU of Sendai virus, was abolished when the virions were pretreated with 10 ug of the viperid venom of Echis coloratus, and was considerably diminished when pretreated with 10 ug of the venom of Echis carinatus sochureki, the cobra venoms of Naja atra and Naja nigricollis nigricollis. These venoms did not affect the erythrocytes but inhibited the virions themselves irreversibly. All other examined snake venoms had low or no antiviral activity. There was no correlation between the proteolytic and the antiviral activity of the venoms.

Reduction of healthcare-associated infections in a long-term care brain injury ward by replacing regular linens with biocidal copper oxide impregnated linens.

BACKGROUND: Contaminated textiles in hospitals contribute to endogenous, indirect-contact, and aerosol transmission of nosocomial related pathogens. Copper oxide impregnated linens have wide-spectrum antimicrobial, antifungal, and antiviral properties. Our aim was to determine if replacing non-biocidal linens with biocidal copper oxide impregnated linens would reduce the rates of healthcare-associated infections (HAI) in a long-term care ward. METHODS: We compared the rates of HAI in two analogous patient cohorts in a head injury care ward over two 6-month parallel periods before (period A) and after (period B) replacing all the regular non-biocidal linens and personnel uniforms with copper oxide impregnated biocidal products. RESULTS: During period B, in comparison to period A, there was a 24% reduction in the HAI per 1000 hospitalization-days (p<0.05), a 47% reduction in the number of fever days (>38.5°C) per 1000 hospitalization-days (p<0.01), and a 32.8% reduction in total number of days of antibiotic administration per 1000 hospitalization-days (p<0.0001). Accordingly there was saving of approximately 27% in costs of antibiotics, HAI-related treatments, X-rays, disposables, labor, and laundry, expenses during period B. CONCLUSIONS: The use of biocidal copper oxide impregnated textiles in a long-term care ward may significantly reduce HAI, fever, antibiotic consumption, and related treatment costs.