Evaluation of agents for use in medium for selective isolation of lyme disease and relapsing feverBorrelia species

Barbour-Stoenner-Kelly (BSK) II medium containing fosfomycin, 5-fluorouracil, trimethoprim and sulfamethoxazole was evaluated for the selective isolation of theBorrelia species responsible for Lyme disease and relapsing fever. The maximum noninhibitory concentrations of fosfomycin, 5-fluorouracil, trimethoprim and sulfamethoxazole for six strains of borreliae were 500 to >1000 µg/ml, 250 to >500 µg/ml, 125 to 500 µg/ml and 125 to 500 µg/ml, respectively. The combination of four agents (fosfomycin 400 µg/ml, 5-fluorouracil 100 µg/ml, trimethoprim 10 µg/ml, sulfamethoxazole 50 µg/ml) did not inhibit the growth of borreliae, allowing growth in cultures inoculated with a few organisms (theoretically a single organism). In contrast, the fouragent combination completely inhibited the growth of 12 of 13 other bacterial strains tested as possible contaminants. This combination also allowed the selective growth of borreliae in experimentally contaminated specimens. The four-agent combination in BSK II medium may be useful for selective isolation ofBorrelia species responsible for Lyme disease and relapsing fever from clinical and environmental samples.     

Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage

The objective of this study was to assess the influence of specific factors on post-thaw development of mouse cryopreserved morulae. Thawed morulae (n = 206) were randomly distributed between 10 treatment groups: medium alone control (CT), Vero (VR) cells, leukaemia inhibitory factor (1 ng/ml), interleukin-6 (1 ng/ml), transforming growth factor (TGF) alpha (2 ng/ml), epidermal growth factor (EGF) (4 ng/ml), platelet-derived growth factor (1 ng/ml), insulin-like growth factor (IGF)-I (30 ng/ml), IGF-II (1 ng/ml) and TGFbeta (2 ng/ml). At 4, 8, 20, 30 and 48 h, a digitized image of each thawed embryo was captured and stored for later analysis. The following parameters were examined: blastocoel formation, blastocyst expansion, zona thickness and hatching. At termination of the experiment, cell number per embryo was determined by bisbenzimide staining. When contrasted to the medium alone control, co-culture consistently accelerated the development of frozen-thawed morulae to the hatched blastocyst stage, allowing embryos to recover rapidly from any damage sustained during the cryopreservation process. While no single growth factor/cytokine was able to completely mimic the results achieved with co-culture, all of the growth factors impacted positively on at least one of the morphological parameters studied. Cell proliferation was significantly stimulated by just 48 h exposure to growth factors, either through co-culture or by direct media supplementation. Co-culture again yielded the best results with a mean cell count of 217 +/- 76 cells per blastocyst as compared with 131 +/- 36 in control medium alone. Amongst the factors tested, IGF-I, IGF-II and EGF had the greatest impact, with mean cell counts of 172 +/- 50, 168 +/- 50 and 179 +/- 55 respectively. Whereas only 5% of CT embryos developed to blastocysts with > 200 cells, 51% of thawed embryos placed on co-culture monolayers and 25-32% of embryos cultured with IGF-I, IGF-II or EGF had > 200 cells. This study for the first time systematically describes the effect of culture regimen and growth factor additives on the post-thaw development of cryopreserved embryos.     

Persistent infection of BHK21/WI-2 cells with rubella virus and characterization of rubella variants

Summary Persistently infected cell lines of BHK21/WI-2 cells have been established by infection with the wild type rubella virus strain M-33. These cell lines, BHK-MP1 and BHK-MP2, showed immunity-like resistance to superinfection with M-33 virus at both 34° and 39.5° C. They also showed intrinsic interference with the replication of Newcastle Disease Virus at 34° C but not at 39.5° C. They released a small number of infectious virus particles which were temperature sensitive variants, being able to form plaques at 34° C, but not at 39.5° C on BHK21/WI-2 and on its derivative, BSR.When BHK-MP1 cells were cultured at 34° C in growth medium containing 10–20 µg/ml of 5-bromodeoxyuridine (BudR) there was a 5- to 10-fold increase in infectious virus in the medium as compared with the untreated controls. Mitomycin C (0.5 µg/ml) treatment for 7 hours likewise stimulated the release of virus from these cells. The enhancement of viral release by BudR was completely blocked by pretreatment with actinomycin D (5 µg/ml) for 3 hours prior to BudR treatment. Since the variant can be induced by these prophage inducers and inhibited by actinomycin D it is suggested that the viral genome is converted to a DNA provirus which is analogous to the lysogenic state of bacteriophage.With 2 Figures     

Preparation of Chinese hamster bone marrow and spleen primary cell cultures for sister chromatid exchange and chromosomal aberration studies

Summary Procedures for preparing and culturing Chinese hamster bone marrow and spleen cells for cytogenetic studies are described. Animals are killed by cervical dislocation, then the bone marrow is flushed from femora and tibia with Ham's F12 medium into centrifuge tubes. Bone marrow cells are collected by low-speed centrifugation (285 ×g). Approximately one million cells are cultured in a 30-ml Falcon flask with 5 ml complete medium containing 3.95 ml Ham's F12 medium, 1.0 ml fetal bovine serum (FBS), and 0.05 ml penicillin-streptomycin (5000 U/ml and 5000 µg/ml stock). Spleens are obtained aseptically, transferred to centrifuge tubes containing 2 ml of RPMI 1640 and smashed with a sterile spatula. The debris is removed, cells are collected by low-speed centrifugation (285 ×g), and washed three times with phosphate buffered saline supplemented with 2% heat inactivated FBS. Approximately one million cells are suspended in a 30-ml Falcon flask with 5 ml complete medium containing 3.70 ml RPMI 1640. 1.0 ml heat inactivated FBS, 0.05 ml penicillin-streptomycin, 0.05 ml of 200 mM l-glutamine solution, 1 × 10^–5 M 2-mercaptoethanol, and 0.20 ml lipopolysaccharide. For sister chromatid exchange analysis, 20 µM of 5-bromo-2 -deoxyuridine is also added to the culture medium for 24 to 28 h for bone marrow cells and 36 to 40 h for spleen cells. These culture systems can also be used for chromosomal aberration studies.     

Effect of pH and Zn2+ on subcultured muscle cells from Macrobrachium nipponense

A cell culture system was devised for muscle cell of Macrobrachium nipponense in the study. The juvenile and adult shrimps were held in laboratory aquaria with penicillin 1000 IU/ml and streptomycin 1000 µg/ml for 12–24 hours. Cell cultures were established in medium 199 supplemented with 20% fetal bovine serum, 1 g/L glucose, 5.2 g/L NaCl, 1.43 g/L CaCl₂, 0.05 g/L MgCl₂, 100 IU/mL penicillin and 100 µg/ml streptomycin. Fibroblast-like cells were passaged up to three times and survived for 54 days. The results showed the optimum for subculture in vitro was in medium 199 with pH 7.6. Moreover, basal medium supplemented with Zn²⁺ 60 µg/L could enhance the growth of the muscle cells. It was found that better results for cell culture would be obtained more easily with juvenile shrimps caught in spring than adults in summer or autumn; and shrimps caught within 12 hours after ecdysis could grow much better than the intermoult shrimps.     

Endocrinology: Somatostatin action on rat ovarian steroidogenesis

To date, very few studies on the effect of somatostatin on femalereproductive function have been reported. In our study, we examinedthe effects of somatostatin on (i) androgen biosynthesis usingwhole ovarian dispersates, and (ii) aromatase activity and progesteroneproduction using granulosa cells. Whole ovarian dispersatesobtained from immature rats were cultured for 96 h in serum-freemedium with human chorionic gonadotrophin (HCG; 25 ng/ml) andinsulin (10 µg/ml) in the presence or absence of an increasingconcentration of somatostatin (0.03–3.00 ng/ml). HCG-and insulin-stimulated accumulation of androsterone by thesecells was inhibited significantly by somatostatin. Granulosacells from diethyl-stilbestrol-treated rats were cultured for48 h in serum-free medium with follicle-stimulating hormone(FSH; 20 ng/ml) and FSH plus insulin (1 µg/ml) with orwithout somatostatin (0.03–3.00 ng/ml). Both aromataseactivity and progesterone production stimulated by FSH and FSHplus insulin were significantly inhibited by somatostatin. Somatostatinby itself (1 ng/ml) did not have an effect on any of the evaluatedparameters. The action of somatostatin could be immunoneutralizedand did not influence the plated viable cell mass. These findingsindicate that somatostatin can regulate ovarian steroidogenesisby mediating gonadotrophin and growth factor action on differentovarian cell types.     

Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus in vitro

Summary Azathioprine (Aza) was found to have anti-human cytomegalovirus (HCMV) activity in vitro at concentrations used for immunosuppression therapy. The dose of Aza for 50% plaque reduction was 0.592µg/ml for HCMV in human embryonic lung (HEL) cells, but those of Aza for 50% plaque reduction for herpes simplex virus (HSV) and varicella-zoster virus were more than 20µg/ml. The dose of Aza for 50% reduction of the HCMV yield in infected cells was 0.25µg/ml, while that for 50% reduction of the HSV yield in infected cells was more than 50µg/ml. The dose of Aza for 50% growth inhibition of HEL cells was 30µg/ml, and 50.7 and 120 times greater than the doses for 50% reduction of the plaque formation and the yield of HCMV, respectively. Thus Aza was found to have a strong anti-HCMV activity at concentrations used for immunosuppression. When HCMV infected cells were treated with cyclosporine (CsA: 0.2µg/ml) and prednisolone (Pred: 0.3µg/ml) simultaneously with Aza, the doses of Aza for 50% reduction of plaque formation and the yield of HCMV were 0.73 and 0.32µg/ml, respectively. Thus an inhibitory effect of Aza was also observed in HCMV-infected cells treated with CsA and Pred at their concentrations used for immunosuppression. Maintenance of an anti-HCMV dose of Aza in combination with CsA and Pred might establish not only satisfactory immunosuppression but also suppression of HCMV infection in transplant recipients.     

Inhibitory effect of HIV-1 envelope glycoproteins gp120 and gp160 on the in vitro growth of enriched (CD34+) hematopoietic progenitor cells

Summary The effect of increasing concentrations (from 0.01 to 10 µg/ml) of HIV-1 envelope glycoproteins gp160, gp120, gp41 and core protein p24 was evaluated on the in vitro growth of enriched hematopoietic progenitors (CD34⁺ cells). Both gp120 and gp160, at concentrations from 0.01 to 10 µg/ml, caused a progressive and significant (p<0.05) decrease in viable CD34⁺ cell count in liquid cultures supplemented with 2 ng/ml of human recombinant (r) interleukin-3 (IL-3), evaluated by means of Trypan-blue exclusion and [³H]thymidine ([³H]TdR) incorporation. In the absence of rIL-3, no inhibitory effects were observed even at the highest gp160 and gp120 concentrations explored (10 µg/ml). On the contrary, gp41 and p24 did not affect the number of viable CD34⁺ cells, either in the presence or in the absence of rIL-3. Moreover, gp160 and gp120, but not gp41 and p24, significantly (p<0.05) inhibited the in vitro growth of granulomacrophage progenitors (CFU-GM) in a dose-dependent fashion. These data clearly demonstrate that HIV-1 envelope glycoproteins inhibit the growth of purified hematopoietic progenitors. We propose that HIV-1 can impair hematopoiesis through the interaction of gp120/gp160 with CD34⁺ cell surface, independently of an infectious process.     

MDCK cell cultures supplemented with high concentrations of trypsin exhibit remarkable susceptibility to influenza C virus

Summary Multiplication of influenza C virus in MDCK cell cultures increased with increasing concentrations of trypsin up to 160 µg/ml, whereas maximum growth of influenza A virus in the same culture was observed at a concentration of 10 µg/ml. In the presence of 160 µg of trypsin per ml MDCK cells showed the same or even higher susceptibility to various strains of influenza C virus compared with HMV-II cells, a human melanoma cell line that has been reported to have high susceptibility to the virus. Complete cleavage of the HE precursor protein of MDCK-grown influenza C virus into HE₁ and HE₂ subunits was achieved by trypsin at a concentration of 160 µg/ml, whereas only partial cleavage was observed at 10 µg/ml. The present results thus demonstrate that MDCK cell cultures supplemented with trypsin at a concentration of 160 µg/ml become highly susceptible to influenza C virus.     

Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay

Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in α-MEM medium, supplemented with 10% of selected fetal calf serum, 1 μM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFκB ligand (RANKL, 50 ng/ml), and transforming growth factor-β1 (TGF-β1, 5 ng/ml). Thus, in addition to widely recognized osteoclast-generating factors M-CSF and RANKL, other medium supplements and lengthy culture times were necessary. This assay reliably detected inhibition of osteoclast formation (multinucleated cells positive for tartrate-resistant acid phosphatase) and activity (resorbed area and collagen fragments released from bone slices) in dose response curves with several classes of bone resorption inhibitors. Therefore, this assay can be applied for monitoring bone-resorbing activity of novel drugs and as an clinical test for determining the capacity of blood cells to generate bone-resorbing osteoclasts. Isolation of large quantities of active human osteoclast mRNA and protein is also made possible by this assay.     

Effects of histamine type-2 receptor antagonists on indomethacin and IL-2 immunotherapy of metastasis

Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 × 10⁵ C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 µg/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 µg/ml (Ran-lo) or 143 µg/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 µg/ml, or (c) cimetidine (Cim) at 107 µg/ml, all in the drinking water on days 5–24; (3) IL-2 (1.5 × 10³ Cetus U i.p. every 8 h on days 10–14 and 20–24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (⁵¹Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no differences were observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy.     

Beta-amyloid toxicity and reversal in embryonic rat septal neurons

Alzheimer's disease is characterized mainly by loss of neurons from the septal nucleus. In this study, neurons from the septal nucleus of the embryonic day 16 (E16) rat were grown in culture with a plane of astrocytes from the embryonic rat and in a defined medium in the absence of serum. Neurons were treated with beta-amyloid (Abeta: 0.1, 1 and 10 microM) on day in vitro (DIV) 1 and DIV 4 and fluorescent microscopy was used to measure survival and apoptosis following exposure of the treated cells on DIV 7. Reversal of neurotoxicity was studied using the potentially neuroprotective agents nerve growth factor (NGF, 100 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml), insulin-like growth factors (IGF1 and IGF2, 10 ng/ml) and estrogen (10 nM), administered on DIV 4 and DIV 5, that is, subsequent to the Abeta (10 microM)-induced neurotoxicity. Abeta caused a significant decrease in survival at 10 microM, and a significant increase in apoptosis at 0.1 and 10 microM. IGF1, IGF2 and bFGF all caused a reversal of the Abeta-induced neurotoxic effect on survival while NGF and estrogen did not under these experimental conditions.     

Epitope mapping and the detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated monoclonal antibodies in a fixed-cell ELISA

Summary A fixed-cell ELISA was developed using swine testicle (ST) cells infected with the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and purified biotinylated monoclonal antibodies (b-MAbs). Five of the b-MAbs were specific for the peplomer (E2), five reacted to the nucleocapsid (N), and one reacted to the E1 protein of the Miller strain of TGEV. Protein A-Sepharose purification of MAbs yielded protein concentrations ranging from 0.40 to 3 mg per ml of ascites. Separate pools of N-MAbs and E2-MAbs, and the E1-MAb were used to monitor synthesis of TGE viral antigen in ST cells from 0 to 16 h post-infection at various multiplicities of infection (MOI). Epitopes of N proteins appeared sooner and at a lower MOI than those for the E1 and E2 proteins. The fixed-cell ELISA was also used to examine relative binding affinities of TGEV MAbs. Concentrations of b-MAbs producing a half-maximal signal ranged from 0.11 to 3.8 µg/ml for E2-MAbs, from 0.05 to 0.82 µg/ml for N-MAbs, and 6 µg/ml for the E1-MAb. The assay was used to determine the 50% neutralization concentrations for four neutralizing E2-MAbs (0.1 µg/ml to 6.9 µg/ml) and one E1-MAb (1.2 µg/ml). Competition assays between b-MAbs and unlabeled competitors indicated that at least two major antigenic sites exist on the E2-protein and 2 to 3 antigenic sites are present on the N-protein of Miller TGEV.     

Anti-Candida albicans activity of essential oils including Lemongrass (Cymbopogon citratus) oil and its component, citral]

The effects of 12 essential oils, popularly used as antifungal treatments in aromatherapy, on growth of Candida albicans were investigated. Mycelial growth of C. albicans, which is known to give the fungus the capacity to invade mucosal tissues, was inhibited in the medium containing 100 micro g/ml of the oils: lemongrass (Cymbopogon citratus), thyme (Thymus vulgaris), patchouli (Pogostemon cablin) and cedarwood (Cedrus atlantica). Not only lemongrass oil but also citral, a major component of lemongrass oil (80%), in the range of 25 and 200 micro g/ml inhibited the mycelial growth but allowed yeast-form growth. More than 200 micro g/ml of citral clearly inhibited both mycelial and yeast-form growth of C. albicans. These results provide experimental evidence suggesting the potential value of lemongrass oil for the treatment of oral or vaginal candidiasis.     

Fungicidal activity of human peripheral blood leukocytes against Paracoccidioides brasiliensis yeast cells.

Although epidemiological findings suggest that normal humans are resistant to Paracoccidioides brasiliensis infection, the host defense mechanisms against this fungus have not been fully understood. Here we examined human leukocytes for antifungal activity against yeast cells of this fungus, using an improved mycological culture medium with high plating efficiency for the yeast cell. In an attempt to minimize the impairment of leukocyte activities during the isolation process, leukocytes removed by centrifugation from a buffy coat of peripheral blood were used in the antifungal assay without further fractionation. The leukocytes thus prepared effectively killed P. brasiliensis yeast cells within the first 4 h of co-culture. Adding interferon-gamma (37 ng/ml), granulocyte-macrophage colony-stimulating factor (5 ng/ml), interleukin (IL)-1beta (12 ng/ml), or IL-4 (12 ng/ml) to the assay system enhanced the leukocyte antifungal (growth inhibitory) activity by 48 h. By contrast, addition of IL-8 (50 ng/ml) impaired the leukocyte activity. Tumor-necrosis factor-alpha (50 ng/ml) or IL-10 (25 ng/ml) had no effect in this respect. Dexamethasone (1 micromol/l) reduced the antifungal activity of leukocytes. This is the first demonstration that human leukocytes are able to effectively kill yeast cells of P. brasiliensis.     

NCI-H292 as an alternative cell line for the isolation and propagation of the human paramyxoviruses

Summary Primary rhesus monkey kidney (MK) cells have long been the cells of choice for isolation and propagation of the human paramyxoviruses (parainfluenza 1, 2, 3, 4A, 4B, and mumps). However, problems with the supply and cost of MK cells and the presence of endogenous viruses, including herpes B virus and SV-5, necessitated a search for an alternative cell line. Continuous cell cultures of human origin (L132, A-549, HuT-292, HEK, G-293, G-401, A-498, A-704, CAKI-1, RD) and simian origin (LLC-MK2, BSC-1, MA-104, Vero) were evaluated for their capacity to support the growth of the human paramyxoviruses, as followed by cytopathic effect, hemadsorption, hemagglutination, and EIA. NCI-H292 (HuT-292) human lung mucoepidermoid carcinoma cells (ATCC # CRL-1848) proved to be the most sensitive line for cultivating all serotypes and strains of the paramyxoviruses. These cells were also shown to be a suitable substitute for MK in primary isolation of paramyxoviruses from clinical specimens. RPMI-1640 with 1.5µg/ml trypsin was the preferred maintenance medium; alternatively, Eagle's MEM supplemented with 1.5µg/ml trypsin and 0.1% ITS was satisfactory. NCI-H292 cells are a continuous line with excellent growth characteristics, although the genetic polyploidy of the cells may limit the number of passages of usable cells.     

Specific perforation of muscle cell membranes with preserved SR functions by saponin treatment

Summary The effects of saponin onXenopus and frog skeletal muscle fibres were examined. The twitch ofXenopus single fibres was first potentiated slightly and then irreversibly abolished by 5–10 µg/ml of saponin. Treatment with saponin at 5–10 µg/ml or higher concentrations for 30 min resulted in perforation of the muscle cell membrane, indicated by the following evidence. (i) Fibres became responsive to contractile activating solutions with a pCa-tension relationship similar to that of mechanically skinned fibres. (ii) Removal and re-introduction of MgATP became effective in bringing fibres into rigor and the relaxed state, respectively. (iii) After the saponin treatment large contractions due to Ca release from the SR could be elicited by substitution of Cl for methanesulphonate in the medium. (iv) The treatment decreased the optical path length across the fibre, indicating loss of soluble proteins. (v) The lattice spacing of myofilaments was wider after the treatment as in mechanically skinned fibres. Contractile response of mechanically skinned fibres and their SR responses such as Ca uptake, Ca-induced Ca release and Cl-induced Ca release were not affected by treatment with 50 µg/ml saponin for 30 min, while 150 µg/ml or higher concentrations severely impaired the SR functions. It is possible, therefore, to make chemically skinned skeletal muscle fibres in which the functions of the SR are preserved by applying 10–50 µg/ml saponin.     

Comparative activity of various compounds against clinical strains of herpes simplex virus

The following compounds were evaluated for their inhibitory activity against clinical strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in both primary rabbit kidney (PRK) and HeLa cell cultures: (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), 9-(2-phosphonylmethoxyethyl)adenine (PMEA), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), (RS)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine (HPMPDAP), 5-(5-bromothien-2-yl)-2-deoxyuridine (BTDU), 5-(5-chlorothien-2-yl)-2-deoxyuridine (CTDU), 9-(2-deoxy-2-hydroxymethyl--D-erythro-oxetanosyl)guanine (OXT-G), pentosan polysulfate, heparin, dextran sulfate (MW 10,000), acyclovir, 9-(2-hydroxyethoxymethyl)guanine (ACV), (E)-5-(2-bromovinyl)-2-deoxyuridine (BVDU), 1--D-arabinofuranosyl-(E)-5-(2-bromovinyl)-uracil (BVaraU), vidarabine (9--D-arabinofuranosyladenine) (ara-A) and phosphonoformate (PFA). The most potent inhibitors of HSV-1 were (in order of decreasing activity in PRK cells) BVDU, ACV, BVaraU and OXT-G, their mean 50 % inhibitory concentration (IC₅₀) ranging from 0.02 µg/ml to 0.9 µg/ml. Then followed BTDU and CTDU (IC₅₀ 1–2 µg/ml), the sulfated polysaccharides (IC₅₀ 1.3–5.8 µg/ml), the phosphonylmethoxyalkyl derivatives (IC₅₀ 5.6–25 µg/ml), ara-A (IC₅₀ 11 µg/ml) and PFA (IC₅₀ 38.5 µg/ml). Except for BVDU, BVaraU, BTDU and CTDU, the compounds did not discriminate between HSV-2 and HSV-1. All the compounds studied could be considered specific anti-HSV agents. Their selectivity indexes varied from 3 (PFA) to 6400 (BVDU).