Foscarnet Research Articles

New phosphonoformic acid derivatives of 3'-azido-3'-deoxythymidine

New 5'-alkyl ethoxy- and aminocarbonylphosphonates of 3'-azido-3'-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5'-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Synthesis and stability studies of phosphonoformate–amino acid conjugates: a new class of slowly releasing foscarnet prodrugs

Prodrugs of phosphonoformic acid (PFA), an anti-viral agent used clinically as the trisodium salt (foscarnet), are of interest due to the low bioavailability of the parent drug, which severely limits its utility. Neutral PFA triesters are known to be susceptible to P–C bond cleavage under hydrolytic de-esterification conditions, and it was previously found that P,C-dimethyl PFA P–N conjugates with amino acid ethyl esters did not release PFA at pH 7, and could not be fully deprotected under either acid or basic conditions, which led, respectively, to premature cleavage of the P–N linkage (with incomplete deprotection of the PFA ester moiety), or to P–C cleavage. Here we report that novel, fully deprotected PFA-amino acid P–N conjugates 4 can be prepared via coupling of C-methyl PFA dianion 2 with C-ethyl-protected amino acids using aqueous EDC, which gives a stable monoanionic intermediate 3 that resists P–C cleavage during subsequent alkaline deprotection of the two carboxylate ester groups. At 37 °C, the resulting new PFA-amino acid (Val, Leu, Phe) conjugates ( 4a– c) undergo P–N cleavage near neutral pH, cleanly releasing PFA. A kinetic investigation of 4a hydrolysis at pH values 6.7, 7.2, and 8.5 showed that PFA release was first-order in [ 4a] with respective t 1/2 values of 1.4, 3.8, and 10.6 h.

Phosphorylated derivatives that activate or inhibit mammalian adenosine kinase provide insights into the role of pentavalent ions in AK catalysis.

The enzyme adenosine kinase (AK) exhibits a nearly complete dependency on the presence of pentavalent ions (PVI) such as phosphate, arsenate, and vanadate. To understand its basis, the effect of a large number of phosphorylated compounds on AK activity was examined. Several compounds, such as phosphoribosyl pyrophosphate, phosphoenol pyruvate, creatine phosphate, phosphorous acid, phosphonoformic acid, and inorganic pyrophosphate, were found to substitute for PVI in stimulating AK activity. Similar to PVI, these compounds lowered the Km of AK for adenosine. In contrast, many other structurally related compounds (i.e., phosphonoacetic acid, 2-carboxyethyl phosphonic acid, N-phosphonomethyl glycine, N-phosphonomethyl iminodiacetic acid) inhibited AK activity. These compounds seemed to compete with the activators for binding to AK. Structural comparisons of different compounds indicate that all activating compounds contain a net positive charge on the pentavalent atom (e.g., phosphorous), which should enable it to act as an acceptor for a nucleophilic group. We suggest that a phosphate (or other activator) bound near the active site participates in AK catalysis by forming a transient pentavalent intermediate with a nonbridging oxygen of the beta-phosphate in ATP. This interaction likely facilitates the transfer of gamma-phosphate from ATP to adenosine, thus accounting for the stimulating role of PVI in AK catalysis. The insight provided by these studies concerning the structural features of activators and inhibitors should also prove helpful in the design of more potent inhibitors of AK.

Multiple 5′ ends of human cytomegalovirus UL57 transcripts identify a complex, cycloheximide-resistant promoter region that activates oriLyt

The human cytomegalovirus (HCMV) UL57 gene lies adjacent to HCMV oriLyt, from which it is separated by an organizationally conserved, mostly noncoding region that is thought to both regulate UL57 expression and activate oriLyt function. However, the UL57 promoter has not been studied. We determined the 5′ ends of UL57 transcripts toward an understanding of the potential relationship between UL57 expression and oriLyt activation. The results presented here identified three distinct 5′ ends spread over 800 bp, at nt 90302, 90530, and 91138; use of these sites exhibited differential sensitivity to phosphonoformic acid treatment. Interestingly, a 10-kb UL57 transcript accumulated in cycloheximide-treated infected cells, even though other early transcripts were not detectable. However, the 10-kb transcript did not accumulate in cells treated with the more stringent translation inhibitor anisomycin. Consistent with the notion that the identified 5′ ends arise from distinct transcription start sites, the sequences upstream of sites I and II functioned as promoters responsive to HCMV infection in transient assays. However, the origin-proximal promoter region III required downstream sequences for transcriptional activity. Mutation of candidate core promoter elements suggested that promoter III is regulated by an initiator region (Inr) and a downstream promoter element. Finally, a 42-bp sequence containing the candidate Inr activated a minimal oriLyt core construct in transient replication assays. Thus, these studies showed that a large, complex promoter region with novel features controls UL57 expression, and identified a sequence that regulates both UL57 transcription and oriLyt activation.

The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7-7.0. The enzyme had a Km of 0.53 mm for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond.

The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors.

The SLC20 family transport proteins were originally identified as retroviral receptors (called Glvr-1 and Ram-1). Since then, they have been shown to function as sodium-phosphate (Na/P(i)) cotransporters, and have subsequently been classified as type III Na/P(i) cotransporters (now called Pit-1 and Pit-2). The Pit cotransporters share approximately 60% sequence homology, they have a high affinity for P(i), they are electrogenic with a coupling stoichiometry of >1 Na(+) per P(i) ion cotransported, and are inhibited by alkaline pH and phosphonoformic acid (PFA). Pit-1 and Pit-2 expression and/or activity has also been shown to be regulated by P(i) deprivation in some, but not all cells and tissues examined. The Pit-1 and Pit-2 cotransporters are widely expressed, but cell-type specific expression has only been investigated in bone, kidney and intestine. Both proteins are likely expressed on the basolateral membranes of polarized epithelial cells, where they are likely involved in cellular P(i) homeostasis. The Pit-1 and Pit-2 gene promoters have been cloned and characterized. While the exact roles of the Pit cotransporters in different cell types has not been definitively determined, they may be involved in important physiological pathways in bone, aortic smooth muscle cells, parathyroid glands, kidney and intestine.

Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway—amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na + with various cations, were used to study MV Pi transport, which had been thought to be Na +-dependent. MVs induced rapid mineralization ∼3 h after addition to SCL; CPLX and HAP caused almost immediate induction; ACP required ∼1 h. Phosphonoformate (PFA), a Pi analog, potently delayed the onset and reduced the rate of mineral formation of MV and the intermediates with IC 50’s of 3–6 μM and ∼10 μM, respectively. PFA:Pi molar ratios required to reduce the rate of rapid mineralization by 50% were ∼1:30 for ACP, ∼1:20 for HAP, ∼1:3.3 for CPLX, and ∼1:2.0 for MVs. MV mineralization was not found to be strictly Na +-dependent: substitution of Li + or K + for Na + had minimal effect; while N-methyl d-glucamine (NMG +) was totally inhibitory, choline + was clearly stimulatory. Na + substitutions had minimal effect on HAP- and CPLX-seeded mineral formation. However with ACP, NMG + totally blocked and choline + stimulated, just as they did MV mineralization. Thus, kinetic analyses indicate that ACP is a key intermediate, nevertheless, formation of CPLX appears to be the rate-limiting factor in MV mineralization.

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence.

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.

Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes

The recently cloned NaPi-IIb cotransporter is an apical membrane protein that is involved in the absorption of phosphate in the intestine. To expedite functional and structural studies, the human intestinal NaPi-IIb cotransporter was stably expressed in hamster fibroblast (PS120) cells. The hNaPi-IIb cDNA stably transfected cells exhibited a 1.8-fold higher sodium-dependent phosphate uptake than vector DNA transfected cells, and had a K m for Pi of ∼106 μM and a K m for Na + of ∼34 mM. The hNaPi-IIb cotransporter was also expressed in Xenopus oocytes and it exhibited a K m for Pi of ∼113 μM and a K m for Na + of ∼65 mM. The hNaPi-IIb cotransporter expressed in both PS120 cells and oocytes was inhibited by high external pH. Furthermore, the phosphate uptake mediated by the hNaPi-IIb cotransporter was inhibited by 5 mM phosphonoformic acid (PFA), 1 mM arsenate and 100 nM phorbol myristate acetate (PMA). These results demonstrate that the human intestinal NaPi-IIb cotransporter is functional when expressed in hamster fibroblasts, and that this model system may be useful in the future to identify NaPi-IIb cotransporter-specific inhibitors.

Synergistic inhibition of herpesvirus replication by docosanol and antiviral nucleoside analogs

Interactions between docosanol ( n-docosanol, behenyl alcohol) and nucleoside or pyrophosphate analogs were investigated in vitro. The anti-HSV activity of acyclovir (ACV) was synergistically enhanced by treatment of cells with docosanol as judged by inhibition of progeny virus production and plaque formation. This drug interaction between ACV and docosanol was observed with laboratory strains of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), oral and genital clinical isolates of HSV, cytomegalovirus (CMV), and varicella zoster virus (VZV). Near optimal concentrations of docosanol plus ACV inhibited HSV replication >99% more than either drug alone, including emergence of ACV-resistant variants. The response was observed with African Green Monkey kidney cells, normal human foreskin cells, and normal human lung cells. Treatment of cells with docosanol also synergistically intensified the inhibition of HSV production by all tested nucleoside analogs, including trifluorothymidine (TFT), adenine arabinoside (Ara-A), and ribavirin. An additive anti-HSV effect was observed with docosanol and phosphonoformate (PFA). No evidence was found for either synergistic inhibition of cellular DNA synthesis or induction of overt cellular toxicity when docosanol was combined with ACV, TFT, Ara-A, ribavirin, PFA, 8-azaguanine, or 5-fluorouracil. The ability of docosanol treatment to increase the antiviral activities of nucleoside analog antiviral drugs, coupled with a lack of toxic interactions, translates to substantial improvements in drug selectivity ratios.

Cardiovascular manifestations of HIV infection.

Studies published over the past 3 years have tracked the incidence and course of human immunodeficiency virus (HIV) infection in relation to cardiac illness in both children and adults.1 These studies show that subclinical echocardiographic abnormalities independently predict adverse outcomes and identify high-risk groups to target for early intervention and therapy. The Joint United Nations Program on HIV/AIDS estimates that 36.1 million people were living with HIV infection at the end of the year 2000.2 If 8% to 10% of patients develop symptomatic heart failure over a 2- to 5-year period,3 then 3 million cases of HIV-related heart failure will present during that period.1 Cardiovascular manifestations of HIV have been altered by the introduction of highly active antiretroviral therapy (HAART) regimens. On one hand, HAART has significantly modified the course of HIV disease, lengthened survival, and improved the quality of life of HIV-infected patients. On the other hand, the early data have raised concerns that HAART is associated with an increase in both peripheral and coronary arterial diseases.1 The HAART-associated changes are relevant only to the minority of HIV-infected individuals worldwide who have access to HAART. Thus, studies conducted before HAART became available remain globally applicable. In this review article, the principal HIV-associated cardiovascular manifestations will be discussed, with an emphasis on new knowledge about prevalence, pathogenesis, and treatment. HIV disease is recognized as an important cause of dilated cardiomyopathy, with an estimated annual incidence of 15.9 in 1000 before the introduction of HAART3 (Table 1). The importance of cardiac dysfunction is demonstrated by its effect on survival in acquired immunodeficiency syndrome (AIDS). Median survival to AIDS-related death is 101 days in patients with left ventricular dysfunction and 472 days in patients with a normal heart as shown by echocardiography at a similar infection …

Management of viral infections in immunocompromised cancer patients.

Immunocompromised cancer patients are at elevated risk for serious viral disease. The introduction into clinical use of potent antiviral agents and of rapid diagnostic tests resulted in the development of efficient management strategies for infections due to herpes simplex virus, varicellazoster virus, and cytomegalovirus. The emergence of herpesvirus resistance to acyclovir, ganciclovir, cidofovir, or foscarnet represents an important challenge. The new neuraminidase inhibitors zanamivir and oseltamivir are efficacious in the treatment of influenza in otherwise healthy adults, and need to be investigated among immunodeficient patients with malignancy. Preliminary data on pleconaril, a first broad-spectrum anti-picornaviral compound, are promising.

Vascular calcification and inorganic phosphate

Vascular calcification is highly correlated with elevated serum phosphate levels in uremic patients. To shed light on this process, we examined the ability of extracellular inorganic phosphate (Pi) levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro. When cultured in media containing normal physiological levels of Pi (1.4 mmol/L Pi), HSMC grew in monolayers and did not mineralize. In contrast, HSMC cultured in media containing Pi levels comparable to those seen in hyperphosphatemic individuals (> 1.4 mmol/L), showed dose-dependent increases in cell culture calcium deposition. Mechanistic studies showed that elevated Pi treatment of HSMC also enhanced the expression of the osteogenic markers, osteocalcin and Cbfa-1. The effects of elevated Pi on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor, phosphonoformic acid (PFA), to dose-dependently inhibit Pi-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. Using polymerase chain reaction and Northern blot analyses, the NPC in HSMC was identified as Pit-1 (Glvr-1), a member of the type III NPCs. Interestingly, platelet-derived growth factor-BB (PDGF-BB), a potent atherogenic stimulus, increased the maximum velocity (Vmax) but not the affinity (Km) of phosphate uptake, enhanced the expression of Pit-1 mRNA, and induced HSMC culture calcification in a time- and dose-dependent manner. Importantly, in the presence of PDGF, HSMC culture calcification occurred under normophosphatemic conditions. These data suggest that elevated Pi may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and may help explain both the phenomena of human metastatic calcification under hyperphosphatemic conditions as well as increased calcification in PDGF-rich atherosclerotic lesions. © 2001 by the National Kidney Foundation, Inc.

Generation of hydroxyl radicals by the antiviral compound phosphonoformic acid (foscarnet).

Phosphonoformic acid (foscarnet) is an antiviral agent that is used to treat severe cytomegalovirus infections in AIDS patients. We demonstrate by using the ferrous iron indicator Ferrozine and ascorbic acid (vitamin C) that foscarnet can chelate ferric iron and form a redox-active iron complex. By using the hydroxyl radical indicator coumarin-3-carboxylic acid we found that the foscarnet-Fe3 complex formed can readily catalyze hydroxyl radical (.OH) generation by the Fenton reaction: (Fe2+ + H2O2-4Fe3+ + .OH + -OH) if hydrogen peroxide and ascorbic acid are present. Hydroxylation of coumarin-3-carboxylic acid could be blocked by addition of known hydroxyl radical scavengers such as mannitol, sucrose, glucose and dimethyl sulfoxide. Moreover, by using a DNA nicking assay, we found that foscarnet catalyzed hydroxyl radicals can induce single strand brakes in DNA. The potency of the hydroxyl radicals formed to induce damage could also be demonstrated in a phosphate-free buffer where the hydroxyl radicals formed attacked and liberated phosphate from the foscarnet molecule. Our results indicate that foscarnet catalyzed hydroxyl radical formation might take place during conditions where a peroxide generating system(s), vitamin C and transitions metals are present.

Generation of Hydroxyl Radicals by the Antiviral Compound Phosphonoformic Acid (Foscarnet)

Abstract: Phosphonoformic acid (foscarnet) is an antiviral agent that is used to treat severe cytomegalovirus infections in AIDS patients. We demonstrate by using the ferrous iron indicator Ferrozine and ascorbic acid (vitamin C) that foscarnet can chelate ferric iron and form a redox‐active iron complex. By using the hydroxyl radical indicator coumarin‐3‐carboxylic acid we found that the foscarnet‐Fe3 complex formed can readily catalyze hydroxyl radical (˙OH) generation by the Fenton reaction: (Fe2++ H2O2→Fe3++˙OH +−OH) if hydrogen peroxide and ascorbic acid are present. Hydroxylation of coumarin‐3‐carboxylic acid could be blocked by addition of known hydroxyl radical scavengers such as mannitol, sucrose, glucose and dimethyl sulfoxide. Moreover, by using a DNA nicking assay, we found that foscarnet catalyzed hydroxyl radicals can induce single strand brakes in DNA. The potency of the hydroxyl radicals formed to induce damage could also be demonstrated in a phosphate‐free buffer where the hydroxyl radicals formed attacked and liberated phosphate from the foscarnet molecule. Our results indicate that foscarnet catalyzed hydroxyl radical formation might take place during conditions where a peroxide generating system(s), vitamin C and transitions metals are present.

A rapid assay for evaluation of antiviral activity against coxsackie virus B3, influenza virus A, and herpes simplex virus type 1

In order to identify new potential antiviral drugs, small amounts of extracts or compounds have to be examined for cytotoxicity and antiviral activity in primary screening using a rapid, easy, inexpensive, and highly standardised test system. In this study, high-throughput cytopathic effect (CPE) inhibitory assays were established for coxsackie virus B3 on HeLa Ohio cells, influenza virus A on Madin–Darby canine kidney cells, and herpes simplex virus type 1 (HSV-1) on green monkey kidney cells that meet these requirements. The cytotoxic and the antiviral effects were quantified using a crystal violet uptake assay allowing automated handling of large numbers of candidate agents. To ensure comparable results with plaque reduction assays, the 50 and 90% plaque inhibitory concentrations of guanidine, amantadine, and phosphonoformic acid were used to standardise the anti-coxsackie virus B3, anti-influenza virus A, and anti-HSV-1 tests, respectively. The strong correlation between the antiviral activity determined by CPE-inhibitory assays and plaque reduction assay was further proved for other antivirals. In summary, low amounts of large numbers of compounds may be tested inexpensively and standardised within 24 h (coxsackie virus B3 and influenza virus A) or 48 h (herpes simplex virus type 1) post-infection using CPE inhibitory assays.

The Valine-to-Threonine 75 Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Relation with Stavudine Resistance

The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5'-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site.

Flow Cytometric Evaluation of Antiviral Agents against Human Herpesvirus 6

Antiviral activities of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV), penciclovir (9-[4-hydroxy-3-(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9-(1,3-dihydroxy-2-propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV-6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV-6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV-6 followed by PFA, PCV and ACV. For HHV-6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 microM and 34.7 microM respectively, whereas the IC50 of ACV and PCV were found to be 53.7 microM and 37.9 microM respectively. For HHV-6B, the IC50 of GCV and PFA were found to be 5.7 microM and 71.4 microM respectively, whereas the IC50 of ACV and PCV were found to be 119.0 microM and 77.8 microM respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV-6.

Sensibilidad del citomegalovirus frente a fármacos antivíricos en pacientes infectados por el virus de la inmunodeficiencia humana con coriorretinitis en España

To determine the prevalence of the in vitro resistance rate of cytomegalovirus (CMV) to gancyclovir (GCV) and foscarnet (FOS) among patients with AIDS and chorioretinitis. Furthermore, in vitro sensitivity results were compared with the clinical response to therapy. Thirty-six patients with the diagnosis of CMV retinitis and AIDS were included in the study. Antiviral sensitivity testing was performed to 51 clinical CMV strains from these patients. The in vitro sensitivity was compared with the clinical response to therapy. The resistance criteria were the inhibitory dose50 (ID50) GCV > 5 M and ID50 FOS > 400 M. None of the CMV tested strains was resistant to GCV or FOS; however, six patients who had relapses of chorioretinitis while on maintenance therapy and isolation during the condition had mean ID50 of strains (n=8) of 1,95 M for GCV (standard deviation [SD] of 0.71) and 115.2 for FOS (SD, 34.7). These patients responded well when drugs were used at induction doses. The in vitro sensitivity testing is not a good predictor of infection control when the drug is used at maintenance doses. This suggests that these strains should be classified in the intermediate resistant category.

Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism

In a previous investigation we showed that phosphate ions (Pi) induced apoptosis of terminally differentiated hypertrophic chondrocytes. To explore the mechanism by which Pi induces cell death, we asked the following two questions. First, can we prevent Pi-induced apoptosis by inhibiting plasma membrane Na-Pi cotransport? Second, which specific Na-Pi transporters are expressed in chondrocytes and are they developmentally regulated? Terminally differentiated hypertrophic chondrocytes were isolated from chick tibial cartilage and cell death was measured in the presence of 3–7 mmol/L Pi. To ascertain whether apoptosis was linked to a rise in cellular Pi loading, we examined the effect of phosphonoformic acid (PFA), a competitive inhibitor of Na-Pi cotransport on Pi-induced apoptosis in chondrocytes. We found that 1 mmol/L PFA blocked anion-induced cell death and prevented an increase in the cell Pi content. In a parallel study, we determined that the bisphosphonate, alendronate, also protected chondrocytes from death, albeit at a lower concentration than PFA. Using a DNA end-labeling procedure, we showed that the Pi-treated cells were apoptotic and, as might be predicted, the presence of PFA blocked induction of the death sequence. Next, we examined the expression of two Pi transporters in relation to chondrocyte maturation and anion treatment. We noted that there was expression of the constitutive transporter, Glvr-1, and a type II cotransporter in chick growth plate cells. Although these transport systems are active in terminally differentiated cells, it is probable that the initiation of apoptosis may require the induction of other Pi-transport systems. It is concluded that, at the mineralization front, cell death is linked directly to the elevation in environmental anion concentration and the concomitant rise in intracellular Pi levels.