Cycloguanil and its parent compound proguanil demonstrate distinct activities against Plasmodium falciparum malaria parasites transformed with human dihydrofolate reductase

The lack of suitable antimalarial agents to replace chloroquine and pyrimethamine/sulfadoxine threatens efforts to control the spread of drug-resistant strains of the malaria parasite Plasmodium falciparum. Here we describe a transformation system, involving WR99210 selection of parasites transformed with either wild-type or methotrexate-resistant human dihydrofolate reductase (DHFR), that has application for the screening of P. falciparum-specific DHFR inhibitors that are active against drug-resistant parasites. Using this system, we have found that the prophylactic drug cycloguanil has a mode of pharmacological action distinct from the activity of its parent compound proguanil. Complementation assays demonstrate that cycloguanil acts specifically on P. falciparum DHFR and has no other significant target. The target of proguanil itself is separate from DHFR. We propose a strategy of combination chemotherapy incorporating the use of multiple parasite-specific inhibitors that act at the same molecular target and thereby maintain, in combination, their effectiveness against alternative forms of resistance that arise from different sets of point mutations in the target. This approach could be combined with traditional forms of combination chemotherapy in which two or more compounds are used against separate targets.     

Synthesis and in vitro evaluation of 9-anilino-3,6-diaminoacridines active against a multidrug-resistant strain of the malaria parasite Plasmodium falciparum

A series of 9-anilinoacridines have been prepared and evaluated for their activity against a multidrug-resistant K1 strain of the malaria parasite Plasmodium falciparum in erythrocyte suspensions. 3,6-Diamino substitution on the acridine ring resulted in lower mammalian cell cytotoxicity and higher antiparasitic activity than other substitution patterns, providing compounds with the highest in vitro therapeutic indices. A new synthesis of 3,6-diamino-9-anilinoacridines, via reduction of the corresponding diazides, gives much higher yields than traditional methods. Within the subset of 3,6-diamino-9-anilinoacridines, there was considerable tolerance to substitution at the 1'-anilino position. In a sharp divergence with structure-activity relationships for high mammalian cell toxicity and anticancer effects, derivatives bearing electron-withdrawing 1'-substituents (e.g., SO2-NHR and CONHR) showed the most potent antimalarial activity (IC50 values of 10-20 nM). Representative compounds were shown to be potent inhibitors of the DNA strand-passing activity of human topoisomerase II and of the DNA decatenation activity of the corresponding parasite enzyme. The 1'-SO2NH2derivative 7n completely inhibited strand passage by Jurkat topoisomerase II at 20 microM, and an increase in linear DNA (indicative of inhibition of religation) was seen at or above 1 microM. It also inhibited the decatenating activity of the parasite topoisomerase II at 6 microM and above. In contrast, the analogous compound without the 3,6-diamino substituent was inactive in both assays up to 100 microM. Overall, there was a positive relationship between the ability of the drugs to inhibit parasite growth in culture and their ability to inhibit parasite topoisomerase II activity in an isolated enzyme assay. The 1'-SO2NH2 derivative 7n showed a high IVTI (1000) and was a potent inhibitor of both P. falciparum in vitro (IC50 20 nM) and P. falciparum-derived topoisomerase II. However, the compound was inactive against Plasmodium berghei in mice; reasons may include rapid metabolic inactivation (possibly by N-acetylation) and/or poor distribution.     

In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.     

Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes

BACKGROUND: Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. MATERIALS AND METHODS: To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. RESULTS: The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. CONCLUSIONS: In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.     

A novel class of monoglutamated antifolates exhibits tight-binding inhibition of human glycinamide ribonucleotide formyltransferase and potent activity against solid tumors

Tight-binding inhibition of recombinant human monofunctional glycinamide ribonucleotide formyltransferase by Lometrexol (6R-5,10-dideazatetrahydrofolate) requires polyglutamation. LY254155 and LY222306 differ from 5,10-dideazatetrahydrofolate in the replacement of the 1',4'- phenylene moiety by a 2',5'-thiophene and a 2',5'-furan, respectively. Compared to Lometrexol, the thiophene and furan analogues had 25- and 75-fold greater inhibitory potencies against human monofunctional glycinamides ribonucleotide formyltransferase (Ki = 2.1 and 0.77 nM, respectively). The binding affinities of the thiophene and furan analogues for membrane folate-binding protein from human KB cells were 6- and 350-fold weaker than Lometrexol, respectively. Both the thiophene analogue and 5,10-dideazatetrahydrofolate inhibited the in vivo growth of murine 6C3HED lymphosarcoma, murine C3H mammary carcinoma, and human xenograft HXGC3, HC1, and VRC5 colon carcinomas by 95-100%. The thiophene analogue was efficacious against human xenograft PANC-1, a pancreatic carcinoma which was completely resistant to 5,10- dideazatetrahydrofolate. These novel antifolates represent the first monoglutamated tight-binding inhibitors of glycinamide ribonucleotide formyltransferase. By eliminating the need for polyglutamation, this class of antifolates may have clinical activity in the treatment of solid tumors expressing low levels of folylpolyglutamate synthetase or tumors resistant to antifolate therapy due to increased gamma-glutamyl hydrolase activity.     

Efficacy of artemisinin and mefloquine combinations against Plasmodium falciparum. In vitro simulation of in vivo pharmacokinetics

The activity of artemisinin in combination with mefloquine was tested in vitro against a chloroquine-sensitive (F32) strain of Plasmodium falciparum. A method of repetitive dosing and extending the culture observation period to 28-30 days was used to mimic the in vivo pharmacokinetic situation. Plasmodium falciparum was exposed to artemisinin from 10(-8) to 10(-5) M, mefloquine from 3 x 10(-9) to 10(-5) M and their combinations. The exposure time for artemisinin was 3 hours twice daily and for mefloquine 24 hours. The drug-dosing duration was 3 days. Neither artemisinin nor mefloquine alone provided radical clearance of P. falciparum, even when maximum concentrations (10(-5) M) were applied. The antiparasitic activity of artemisinin and mefloquine were significantly higher when dosed alone. Effective concentrations for different degrees of inhibition (EC 50, 90 and 99) of both artemisinin and mefloquine respectively were significantly lower when used in combination. At concentrations normally reached in vivo, this effect was clearly synergistic (P = 0.016) Our in vitro model of intermittent dosing of artemisinin and mefloquine combinations for 3 days provides significant evidence of positive interaction between the two compounds. Lower combination concentrations around the MIC-values for the individual compounds showed synergistic effect, and high concentrations showed additive effect. This indicates that such drug combinations may provide radical clearance at concentrations lower than those required for single-drug treatment.     

The chemotherapy of rodent malaria, XXV. Antimalarial activity of WR 122,455 (a 9-phenanthrenemethanol) in vivo and in vitro

WR 122,455, 3,6-bis-(trifluoromethyl)-alpha-(2-piperidinyl)-9-phenanthrenemethanol HCl, suppresses infection with drug-sensitive Plasmodium berghei N strain in mice. It acts rapidly and affects all the stages of the asexual intraerythrocytic parasites, the effective dose levels being about three times those of chloroquine and one-twelfth to one-fifteenth those of quinine. Under the influence of WR 122,455 haemozoin seems to disappear from the affected parasites following an initial coarsening of the fine pigment granules. These changes are similar to those exerted by quinine. Large doses of WR 122,455 have a residual affect due in part, at least, to deposition of insoluble material in the tissues. The drug appears to exert an antagonistic action on chloroquine when both drugs are administered simultaneously. It has no causal prophylactic effect. In vitro WR 122,455 is a competitive antagonist of chloroquine in a similar manner to quinine, and appears to have a dissociation constant (Ki) of 2-26 x 10(-8) M, making it about 18 times as active as quinine. WR 122,455 interacts strongly with calf thymus DNA, but the mechanism of interaction has yet to be defined. Mice tolerate single doses of a saline/Tween 80 suspensions up to about 400 mg/kg but sc administration induces necrotic changes at the injection site. Up to 30 mg/kg daily po for seven consecutive days is well tolerated systemically but local tissue reaction may occur if the drug is given by the sc or ip routes. However, systemically up to 60 mg/kg is tolerated sc or ip. The relation of WR 122,455 to drug resistant malaria will be reported later.     

The effect of artemisinine, its derivatives and mefloquine against chloroquine-resistant strains of Plasmodium falciparum in vitro

A 48 h in vitro test of the efficacy of artemisinine, dihydroartemisinine, artemether, mefloquine and chloroquine was carried out against 3 chloroquine-resistant strains of Plasmodium falciparum, strains K1 and T996 from Thailand and LS21 from India. A sigmoid Emax model was fitted to all in vitro inhibition data for each combination of drug and strain. Strains K1 and LS21 were strongly resistant to chloroquine, whereas T996 was partially resistant. Artemisinine, dihydroartemisinine and artemether were active against all strains, with complete growth inhibition at 10(-7) M. Artemether and dihydroartemisinine were both more potent than artemisinine, with 50% effective (EC50) values of 0.57-1.6 nM and 0.36-3.1 nM respectively; the EC50 of artemisinine was 1.5-6.1 nM for the 3 strains. The EC50 values for mefloquine were 46-185 nM. At higher concentrations, strains K1 and LS21 were fully inhibited, while with strain T996 mefloquine did not fully inhibit even at the highest concentration, 1.28 x 10(-6) M. It is concluded that artemisinine and its derivatives were highly effective against the 3 chloroquine-resistant strains, one of which showed borderline resistance to mefloquine.     

In vitro activity of lumefantrine (benflumetol) against clinical isolates of Plasmodium falciparum in Yaoundé, Cameroon

The in vitro antimalarial activity of the new Chinese synthetic drug, lumefantrine, also known as benflumetol (a fluorene derivative belonging to the aminoalcohol class), was determined by an isotopic microtest against 61 fresh clinical isolates of Plasmodium falciparum and compared with that of other established antimalarial agents. The geometric mean 50% inhibitory concentration of lumefantrine was 11.9 nmol/liter (95% confidence intervals, 10.4 to 13.6 nmol/liter; range, 3.3 to 25.6 nmol/liter). The in vitro activities of lumefantrine against the chloroquine-sensitive and the chloroquine-resistant isolates did not differ (P > 0.05). There was a significant positive correlation of responses between lumefantrine and two other aminoalcohols studied, mefloquine (r = 0.688) and halofantrine (r = 0.677), and between lumefantrine and artesunate (r = 0.420), suggesting a potential for in vitro cross-resistance. Our data suggest high in vitro activity of lumefantrine, comparable to that of mefloquine, and are in agreement with the promising results of preliminary clinical trials.     

Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur-based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine-resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (CH2OH-H2pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high-level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.     

Relationship between antimalarial drug activity, accumulation, and inhibition of heme polymerization in Plasmodium falciparum in vitro

We have investigated the contribution of drug accumulation and inhibition of heme polymerization to the in vitro activities of a series of antimalarial drugs. Only those compounds exhibiting structural relatedness to the quinolines inhibited heme polymerization. We could find no direct correlation between in vitro activity against chloroquine-susceptible or chloroquine-resistant isolates and either inhibition of heme polymerization or cellular drug accumulation for the drugs studied. However, in vitro activity against a chloroquine-susceptible isolate but not a chloroquine-resistant isolate showed a significant correlation with inhibition of heme polymerization when the activity was normalized for the extent of drug accumulation. The importance of these observations to the rational design of new quinoline-type drugs and the level of agreement of these conclusions with current views on quinoline drug action and resistance are discussed.     

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo

1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50 = 100 nM). NG-monomethyl-L-arginine (L-NMMA), N6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC50S 3-7 microM). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10 x selectivity for murine NOS II and 11 x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3 x selectivity for murine NOS II and 3.7 x selectivity for human recombinant NOS II. 3. Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50 = 1.5 microM) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 microM, 2-amino-4-methylpyridine did not inhibit the uptake of [3H]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies. 4. When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50 = 0.009 mg kg-1 min-1). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED50 = 0.060 mg kg-1 min-1) indicating 6.9 x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2 x and 8.6 x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo. 5. 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50 = 0.3 mg kg-1) or oral (ID50 = 20.8 mg kg-1) administration. 6. These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.     

Antagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB

The purpose of the present study was to examine the activity of the human Lactobacillus acidophilus strain LB, which secretes an antibacterial substance(s) against Helicobacter pylori in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of H. pylori in vitro independent of pH and lactic acid levels. Adhesion of H. pylori to the cultured human mucosecreting HT29-MTX cells decreased in parallel with the viability of H. pylori. In conventional mice, oral treatment with the LB-SCS protected against infection with Helicobacter felis. Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days postinfection), inhibition of stomach colonization by H. felis was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the H. pylori urease activity in vitro and in H. pylori that remained associated with the cultured human mucosecreting HT29-MTX cells. Moreover, a decrease in urease activity was detected in the stomach of the mice infected with H. felis and treated with LB-SCS.     

A novel retinoic acid receptor-selective retinoid, ALRT1550, has potent antitumor activity against human oral squamous carcinoma xenografts in nude mice

We have identified a novel retinoid, ALRT1550, that potently and selectively activates retinoic acid receptors (RARs). ALRT1550 binds RARs with Kd values of approximately equal to 1-4 nM, and retinoid X receptors with low affinities (Kd approximately equal to 270-556 nM). We studied the effects of ALRT1550 on cellular proliferation in squamous carcinoma cells. ALRT1550 inhibited in vitro proliferation of UMSCC-22B cells in a concentration-dependent manner with an IC50 value of 0.22 +/- 0.1 (SE) nM. 9-cis-Retinoic acid (ALRT1057), a pan agonist retinoid that activates RARs and retinoid X receptors, inhibited proliferation with an IC50 value of 81 +/- 29 nM. In vivo, as tumor xenografts in nude mice, UMSCC-22B formed well-differentiated squamous carcinomas, and oral administration (daily, 5 days/week) of ALRT1550, begun 3 days after implanting tumor cells, inhibited tumor growth by up to 89% in a dose-dependent manner over the range of 3-75 micrograms/kg. ALRT1550 (30 micrograms/kg) also inhibited growth of established tumors by 72 +/- 3% when tumors were allowed to grow to approximately equal to 100 mm3 before dosing began. In comparison, 9-cis retinoic acid at 30 mg/kg inhibited growth of established tumors by 73 +/- 5%. Interestingly, retinoids did not appear to alter tumor morphologies in UMSCC-22B tumors. Notably, ALRT1550 produced a therapeutic index of approximately equal to 17 in this model, indicating a separation between doses that inhibited tumor growth and that induced symptoms of hypervitaminosis A. In summary, ALRT1550 potently inhibits cellular proliferation in vitro and in vivo in this squamous cell carcinoma tumor model. These data support additional study of ALRT1550 for its potential for improving anticancer therapy in human clinical trials.     

Binding of malaria T cell epitopes to DR and DQ molecules in vitro correlates with immunogenicity in vivo: identification of a universal T cell epitope in the Plasmodium falciparum circumsporozoite protein

The efficacy of a malaria peptide vaccine would be enhanced by the inclusion of a parasite-derived universal T cell epitope to ensure that all vaccinees develop parasite-specific cellular and humoral immunity. Two circumsporozoite (CS) protein T cell epitopes, previously identified by CD4+ T cell clones derived from Plasmodium falciparum sporozoite-immunized volunteers, were studied to determine their HLA class II binding potential. One epitope, located in amino acid (aa) 326-345 of the P. falciparum (NF54 strain) CS protein, was "universal" in that it could bind to multiple DR and DQ molecules in vitro. In contrast, the second epitope, T1, which is located in the CS repeat region, was recognized by T cells in the context of DQ6 (DQB1*0603) and did not bind with high affinity to any of the class II molecules tested in the peptide binding assays. The in vitro patterns of peptide/HLA interactions correlated with immunogenicity in vivo. A multiple antigen peptide (MAP) containing the aa 326-345 epitope elicited responses in eight inbred strains (H-2(a,b,d,k,p,q,r,s)), while the T1 MAP was recognized by only a single haplotype, H-2b. The combination of the universal aa 326-345 T cell epitope and the T1 repeat in a di-epitope MAP overcame the genetic restriction to the P. falciparum CS repeat region and elicited antisporozoite Ab responses in all of the MAP-immunized mice. Synthetic peptide malaria vaccines containing the aa 326-345 universal T cell epitope would be expected to elicit parasite-specific immune responses in both sporozoite-primed and naive individuals of diverse genetic backgrounds.     

The effects of leukotrienes C4 and D4 on ciliary activity of human paranasal sinus mucosa in vitro

The effects of leukotrienes C4 and D4 on ciliary activity of human paranasal sinus mucosa were investigated in vitro. Normal mucosa was surgically obtained from human paranasal sinuses and incubated in the form of tissue culture. Ciliated cells were magnified under an inverted microscope, and ciliary activity was photoelectrically measured. LTD4 progressively inhibited ciliary activity, and showed a more potent effect on ciliary activity compared to LTC4. The concentrations of LTC4 and LTD4 in the incubation medium were determined by radioimmunoassay when the mucosa was incubated with 10(-8) M LTC4. The concentration of LTD4 gradually increased and after 90 min reached the maximum of 0.71 x 10(-8) M, while that of LTC4 was reduced to about 10% of its initial concentration within 60 min. These results suggested the possible conversion of LTC4 to LTD4 on the mucosa, and that LTC4 can inhibit ciliary activity by means of LTD4.     

The tryptase, mouse mast cell protease 7, exhibits anticoagulant activity in vivo and in vitro due to its ability to degrade fibrinogen in the presence of the diverse array of protease inhibitors in plasma

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34-55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the alpha-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the alpha-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.     

Enterococcus faecium: in vitro activity of antimicrobial drugs, singly and combined, with and without defibrinated human blood, against multiple-antibiotic-resistant strains

The minimal inhibitory (MICs) and bactericidal concentrations of 14 antimicrobial drugs were determined against 17 clinical isolates of Enterococcus faecium, including 4 glycopeptide-resistant strains. Both teicoplanin and vancomycin lacked bactericidal activity against all 13 susceptible isolates. Time-kill experiments served to test various antibiotic combinations chiefly against glycopeptide-resistant strains in Mueller-Hinton broth (MHB) and in MHB supplemented with 65% (v/v) fresh defibrinated human blood. Co-trimoxazole, fusidic acid, and novobiocin yielded bacteriostatic effects. Rifampin was bactericidally active against rifampin-susceptible strains (MICs = 0.125 micrograms/ml), but less so against low-level-rifampin-resistant (MICs = 2-8 micrograms/ml) strains in MHB. However, in the presence of human blood, rifampin (2 micrograms/ml) combined with co-trimoxazole (0.25/4.75 micrograms/ml) killed rifampin-susceptible and low-level-rifampin-resistant, but not moderate-level-rifampin-resistant (MICs = 16-32 micrograms/ml) strains of E. faecium. Of two topical drugs examined, mupirocin merely inhibited strains of E. faecium; conversely, taurolidine at 2,000 micrograms/ml was efficacious against all strains examined, although the kinetics of bactericidal activity were retarded somewhat in the presence of 65 vol% human blood.     

Enhancement of in vitro and in vivo anti-tumor activity of anti-GD2 monoclonal antibody 220-51 against human neuroblastoma by granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Three new species of Eimeria are described from iguanid lizards of Central and South America. The oocysts of each species have no micropyles or residua and the sporocysts lack Stieda bodies, but all have a sporocyst residuum. Eimeria sanctaluciae n.sp. was found in the St. Lucia tree lizard, Anolis luciae, collected from the Maria Islands, Lesser Antilles. The oocysts are spherical to subspherical, averaging 17.3 x 16.5 microns, with a single layered colourless wall; about 60% contain polar granules. The sporocysts are ellipsoidal and average 7.7 x 5.5 microns. Eimeria liolaemi n.sp. was recovered from the blue-gold swift, Liolaemus taenius, from Chile. The oocysts are spherical to subspherical, measuring 21 x 20.1 microns with a single-layered colourless wall. The sporocysts are subspherical and average 7.4 x 6.8 microns. Eimeria caesicia n.sp. is described from the Brazilian collared iguanid, Tropidurus torquatus. The oocysts measure 27.4 x 23.7 microns, are spherical to subspherical, with a bilayered wall, the outer surface of which appears pale blue in colour, the thin, inner wall appearing brown, when viewed by direct light under the optical microscope. The sporocysts are subspherical and average 9.4 x 7.2 microns. Unnamed polysporocystid oocysts with dizoic sporocysts are reported from the faeces of the lesser St. Vincent tree lizard, Anolis trinitatis and the possibility of spurious parasitism briefly discussed. In addition, oocysts of an unnamed Isospora sp. with a smooth oocyst wall which closely resembles I. reui were recovered from A. trinitatis.     

The effect of gramicidin, a topical contraceptive and antimicrobial agent with anti-HIV activity, against herpes simplex viruses type 1 and 2 in vitro

The effect of an anti-HIV compound, gramicidin, previously used as a topical antibiotic and vaginal contraceptive, on the replication of herpes simplex viruses (HSV) type 1 and 2 has been examined. Human WI-38 fibroblasts were inoculated with either HSV type in the presence of serial dilutions of gramicidin and reduction in viral yield was measured by ELISA. The 50% inhibitory dose (IC50) of gramicidin against 3 HSV-1 and 4 HSV-2 isolates was equal to 0.3 microgram/ml and was comparable to the efficacy of the anti-HSV agent acyclovir (ACV). The IC50 of gramicidin required to protect WI-38 from cytolytic effect of HSV was 10 micrograms/ml at day 5 postinfection, indicating that at this time point the activity of gramicidin was inferior than that of ACV. Nevertheless, gramicidin suppressed the replication of ACV-resistant thymidine kinase and DNA polymerase HSV mutants at doses effective against ACV-sensitive strains. The results suggest that the antimicrobial and spermostatic agent, gramicidin, has potential against sexually transmitted diseases (STDs) and for prophylaxis of sex-borne HIV and HSV infections.