Nucleosides. 146. 1-Methyl-5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil, the C-nucleoside isostere of the potent antiviral agent 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (FMAU). Studies directed toward the synthesis of 2'-deoxy-2'-substituted arabino nucleosides. 6.

The synthesis of 5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1-methyluracil (1, C-FMAU), an isostere of the potent antiviral and antitumor nucleoside 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (2'-fluoro-5-methyl-ara-U or FMAU), was achieved. Pseudouridine (2) was converted into 4,5'-anhydro-3'-O-acetyl-2'-O-triflylpseudouridine (4), which was treated with tris(dimethylamino)sulfur (1+) difluorotrimethylsilicate (TASF) to give 4,5'-anhydro-5-(3-O-acetyl-2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1- methyluracil (5b) in 40% yield. Acid hydrolysis of the 4,5'-anhydro linkage of 5b with Dowex 50 (H+) afforded C-FMAU. The inhibitory activity of C-FMAU against HSV-1 and HSV-2 was about 10-fold less than that of FMAU in tissue culture. This compound, however, did not show significant activity in mice inoculated with HSV-1 or HSV-2.

Nucleosides. 150. Synthesis and some biological properties of 5-monofluoromethyl, 5-difluoromethyl, and 5-trifluoromethyl derivatives of 2'-deoxyuridine and 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil.

A new synthesis of 5-(monofluoromethyl)- and 5-(difluoromethyl)-2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil (F-FMAU and F2-FMAU) is reported. 3',5'-Di-O-(tert-butyldiphenyl)silylated thymidine or FMAU was photochemically brominated with NBS to the corresponding alpha-monobromide, which was hydrolyzed to the 5-hydroxymethyl derivative. Further oxidation of the latter with MnO2 afforded the 5-formyluracil nucleoside. Treatment of these nucleosides with DAST in CH2Cl2 gave the protected alpha-fluorinated nucleosides. Desiylation with TBAF afforded the desired free nucleosides. Also, 5-(trifluoromethyl)-2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil (F3-FMAU) was synthesized by copper-catalyzed trifluoromethylation of 5-iodo-2'-fluoro-ara-U (FIAU). These new nucleosides were studied in comparison with the corresponding 2'-deoxy-erythro-pentofuranosyl derivatives, for their inhibitory activity against cellular thymidylate synthase (TS) and [3H]TdR incorporation into DNA, cytotoxicity against HL-60 cells, and antiviral activity against herpes simplex types 1 and 2 (HSV-1 and -2). F2-TDR and F3-TDR strongly inhibited TS and were also quite cytotoxic and antiherpetic, whereas FTDR was only active in the antiviral assay. In the 2'-fluoroarabino series, fluorine substitution at the alpha-methyl function did not alter significantly the antiherpetic activity. Although FMAU and F-FMAU did not inhibit TS to any significant extent, F2-FMAU and F3-FMAU were weakly inhibitory. The latter nucleosides did not inhibit [3H]TDR incorporation into DNA, while all the other alpha-fluorinated thymine nucleosides inhibited the incorporation of radioactivity of [3H]TDR into DNA to various extents. F2-FMAU and F3-FMAU were about 2 orders of magnitude less cytotoxic against HL-60 cells than were F2-TDR and F3-TDR. The results strongly suggest that in both the 2'-deoxy-2'-fluoroarabino and the 2'-deoxy-erythro-pentofurano series the cytotoxic action of the alpha,alpha-difluoro and alpha,alpha,alpha-trifluoro derivatives may involve the inhibition of TS. The synthesis of [2-14C]F2-FMAU, as an experimental imaging agent, is also described. Unfortunately, the highly selective uptake of the labeled compound within infected brain regions previously noted with [2-14C]FMAU was not detected with the derivative [2-14C]F2-FMAU.

Nucleic acid hybridization, a method to determine effects of antiviral compounds on herpes simplex virus type 1 DNA synthesis.

An application of the nucleic acid hybridization technique to screen effects of antiherpes compounds on herpes simplex virus type 1 (HSV-1) DNA synthesis is described. Whole cells are applied to nitrocellulose filters, their DNA is denatured and fixed to the filter. The resulting DNA spots are hybridized to cloned nick-translated HSV-1 DNA and the amount of hybridization is monitored by autoradiography or scintillation counting. Six antiherpes compounds: bromovinyldeoxyuridine, acyclovir, (R)- and (S)-enantiomers of 9-(3,4-dihydroxybutyl)guanine, 9-(4-hydroxybutyl)guanine and forscarnet, were evaluated for their effects on HSV-1 DNA synthesis. The most active compounds were bromovinyldeoxyuridine and acyclovir, with mean 50% inhibition values (IC50) for four different HSV-1 strains of 0.3 microM and 0.8 microM, respectively. The (R)-enantiomer of the new antiherpes compound 9-(3,4-dihydroxybutyl)guanine was found to be more active than the (S)-enantiomer, with mean IC50s of 6.5 and 14 microM, respectively, while mean IC50s of 2.5 and 68 microM were obtained for 9-(4-hydroxybutyl)guanine and foscarnet, respectively.

Nucleic acid hybridization for measurement of effects of antiviral compounds on human cytomegalovirus DNA replication.

A nucleic acid hybridization technique has been developed to study the effect of different antiviral compounds on the replication of human cytomegalovirus in vitro. One laboratory strain of human cytomegalovirus, Ad. 169, and six clinical isolates were studied. Doses needed for 50% inhibition of viral DNA replication were calculated for foscarnet, acyclovir, and arabinosyladenine. The mean 50% inhibition dose values obtained were 179 microM for foscarnet, 82 microM for acyclovir, and 44 microM for arabinosyladenine. This method yields values that agree with earlier reports, and it offers great advantages over usual methods to date for studying inhibition of viral DNA replication.

Increased serum alpha-fetoprotein levels in cytomegalovirus infections.

Among 54 patients with a clinically and serologically verified cytomegalovirus infection, 8 (15%) had raised alpha-fetoprotein (AFP) levels in sera taken after the onset of infection. No raised serum AFP was seen in acute or convalescent sera of 45 patients with other viral diseases, including infectious mononucleosis and acute herpes simplex virus infection. No definite correlation was found between raised AFP and raised S-ALAT values, indicating hepatic damage. The highest single AFP values were seen in two immunosuppressed patients and in a patient who received a blood transfusion. Two possible explanations for this phenomenon are advanced.

Distribution of tumor-associated antigen CEA and cross-reacting NCA in fetal organs.

The organ distribution of the tumor-associated carcinoembryonic antigen (CEA), and that of the normal tissue component NCA (non-specific cross-reacting antigen) have been investigated in the fetus. Organ extracts from five fetuses between 14 and 21 weeks of age were analysed by radioimmunoassay using specific antisera. CEA was detected in large amounts (800--1,650 ng/g) in fetal colon and in barely detectable amounts in lung and placental tissue. This differed from NCA, which could be detected in almost all organ extracts analysed. The highest concentration of NCA was measured in fetal colon and the content increased with the gestational age of the fetus. High amounts of NCA were also found in the liver, spleen and placental tissue. The gel elution profiles of CEA and NCA from an amniotic fluid pool and a pool of colonic extracts were also determined. CEA eluted similarly to the marker 125I--CEA purified from liver metastasis of colonic carcinoma. The NCA-reactive material was found in three distinct peaks.

Early interactions between human cytomegalovirus and cells.

Adsorption of cytomegalovirus (CMV) to human fibroblasts was not inhibited by preincubation with other herpes viruses (HSV-1, HSV-2, VZV). Transport of virus to the nucleus was studied using virus labelled with 3H-thymidine. Radioactivity was found in the nucleus 20 minutes after virus had been added to the cells. In order to assess the expression of the virus genome, synthesis of early (EA) and late (LA) antigens was studied. Assayed on permissive human lung fibroblasts, a plaque purified virus contained more EA inducing than both EA and LA inducing viral units. Since this was a consistent finding with virions of all densities, it seems to be an effect of viral dose rather than of virion density. Alternatively, LA-defectiveness is a virion property which does not vary with virion density.

Adsorption of purified human cytomegalovirus and induction of early antigens in different cells.

Different human and nonhuman cells were assayed for their capacity to absorb human cytomegalovirus (CMV Ad.169) and to support CMV infection in vitro. The CMV adsorptive capacity was assayed by measuring cell-bound radioactivity after addition of purified 3H- or 125I-labeled CMV or by a bioassay for residual infectious virus in the supernatant fluid. Many of the human and nonhuman cell types adsorbed CMV. Induction of CMV early nuclear antigens in the same cells was assayed by anticomplement immunofluorescence staining of fixed cells 1-3 days after infection. CMV early antigens were induced in the human and nonhuman cells that showed a high degree of CMV adsorption.

Studies of cytomegalovirus infection in renal allograft recipients.

A prospective study of cytomegalovirus (CMV) infections has been carried out in 28 renal graft recipients. The protocol called for frequent blood and urine sampling during the first year after transplantation, but death or graft loss caused earlier termination in nearly half the patients. In this material 5/7 (71%) susceptible patients developed primary infections and 20/21 experienced a secondary infection (95%). Viruria was detected in 79% and viremia in 43%. The type of blood cell responsible for the viremic phase was studied by separating the blood cells on a density gradient. The polymorphonuclear cell fraction was the most common source of virus but virus could also be recovered from the mononuclear cell fraction. As some samples that were freeze-thawed repeatedly never yielded virus, it would appear that viable cells are needed for virus isolation. In both primary and secondary infections isolation of CMV from blood cells often preceded the isolation of CMV from urine. Among variables tested for a possible relationship to the occurrence of CMV viremia the only one to display such an association was the time at which rejection episodes occurred. In 19/28 such episodes recorded in 19 patients there was a temporal relationship to viremia (p less than 0.03). Seven of the patients experienced clinical symptoms suggestive of CMV infection as fever, cough, myalgia, arthralgia, chest pain and pneumonia. Laboratory signs included elevated amino acid transferase levels, leukopenia and thrombocytopenia and a specific anti-CMV antibody response.

Studies of cytomegalovirus infection in renal allograft recipients. II. Serological response to various viral antigens.

In a prospective study the antibody response to various cytomegalovirus (CMV) antigens was examined in 28 renal allograft recipients. Both primary and secondary infections were investigated. Antibodies against immediate early (IEA) and early antigens (EA) were studied by anti-complement immunofluorescence; IgM and IgG antibodies to nuclear late antigens were differentiated by enzyme-linked immunosorbent assay (ELISA). The results of the tests were compared with each other and with those of the complement fixation (CF) test. 5/7 susceptible patients (71%) contracted primary infections. Both IgM and IgG antibodies developed and antibodies to IEA and EA appeared somewhat later. The antibodies to IEA and EA remained detectable throughout the observation period. Secondary infections developed in 20/21 (95%) patients. All initially had CMV antibody levels in ELISA and CF. Rising CMV titers of IgG antibodies were taken as a measure of secondary infection. IgM antibodies developed in only 10/20 (50%) patients. The highest titers of CMV IgM antibody levels were lower in secondary than in primary infections. Antibodies to IEA and EA were present prior to transplantation in some patients, but did not develop in all with secondary infections. The antibody titers were lower just after than before the transplantation in some patients. but subsequently increased again. It thus seems as if the humoral immune response to these CMV antigens differs in primary and secondary infections.

Comparative methods for detection of thymidine kinase-deficient herpes simplex virus type 1 strains.

Four methods for analyzing viral susceptibility to antiviral substances were compared. In two methods viral products were measured: late viral proteins were measured by an enzyme-linked immunosorbent assay and viral DNA was measured by DNA hybridization. Infectious virus was quantified in the other two assays as the number of plaques and the yield of virus. The enzyme-linked immunosorbent assay procedure in our hands detected the smallest amounts (lowest proportions) of thymidine kinase-deficient herpes simplex virus type 1 mixed with wild-type virus. The thymidine kinase-deficient proportion of the herpes simplex virus type 1 isolate increased rapidly in the presence of acyclovir in cell culture.

Carcinoembryonic antigen in amniotic fluid.

Carcinoembryonic antigen (CEA), a substance which is known to occur in high amounts in the fetal gut and also in certain tumors of the gastrointestinal tract, has been demonstrated in amniotic fluids from different stages of pregnancy. Radioimmunoassays of CEA in amniotic fluids of 91 normal pregnancies showed a decrease from a mean of 53 ng/ml at 19 weeks to 25 ng/ml at the end of gestation. The CEA activity in amniotic fluid was eluted in the same volume as a standard 125I-CEA on a Sephadex G200 column. Amniotic fluid therefore contains CEA similar in molecular weight to the CEA purified from liver metastases of colonic cancer. Among 17 cases of abnormal pregnancies, CEA elevations were observed in five with anomalous fetuses.

Activity of the cytomegalovirus genome in the presence of PPi analogs.

PPi analogs and esters of these were studied for their effect on cytomegalovirus (CMV) multiplication. Five aromatic monoesters of phosphonoformate esterified either in the phosphono or the carboxylic group and two diesters were demonstrated to inhibit CMV DNA synthesis and late viral protein synthesis. In a direct assay, the monoesters but not the diesters inhibited CMV DNA polymerase activity. The production of early CMV antigens was not inhibited by any of the compounds. After incubation with either drug for periods up to 7 days, renewed viral production occurred on withdrawal of the compound. All inhibitory esters as well as PPi analogs showed a CMV multiplicity dependence. This was demonstrated both for CMV strain Ad.169 and for all tested CMV isolates. Evidence was found that the esters are hydrolyzed to phosphonoformate and, therefore, may be of importance as useful prodrugs in the specific therapy of CMV infections. The general phenomenon of reversibility to the productive state and the multiplicity dependence of CMV are important factors in any treatment schedule.

5-substituted 2'-fluoro-arabinonucleosides as potential antiviral agents.

In order to study the electronic effects of 5-substituents of 2'-fluoro-ara-U on antiviral activity, eight nucleosides were synthesized and screened for their activity. Preliminary in vitro studies revealed that 5-thiocyano-, 5-hydroxy- and 5-formyl-ara-U are moderately active against HSV-1, HSV-2 and VZV, but they are also cytotoxic. None of these showed significant activity against CMV.

Secretion of gamma-interferon at the cellular level. Induction by Epstein-Barr virus.

Using a haemolytic plaque assay for gamma-interferon (IFN-gamma) secretion we found that in vitro Epstein-Barr virus (EBV) exposure of peripheral blood mononuclear cells from EBV immune individuals led to IFN-gamma secretion, which was apparent within 6 h after virus contact and peaked 12-24 h after induction. Live, ultraviolet-light-irradiated and heat-inactivated virions all caused IFN-gamma secretion. In contrast, blood mononuclear cells from EBV non-immune adults or neonates could not be activated to IFN-gamma production by EBV.

AIDS-related vacuolar myelopathy is not associated with coinfection by human T-lymphotropic virus type I.

Assessment of antibodies against human T-lymphotropic virus type I (HTLV-I) by enzyme-linked immunoassay, immunofluorescence, and Western blot was undertaken in patients with pathologically or clinically diagnosed acquired immunodeficiency syndrome-related vacuolar myelopathy to determine whether this retrovirus could be etiologically implicated in this disorder. No serological evidence for HTLV-I was found in the patients with vacuolar myelopathy, though 1 patient with an atypical myelopathy did have antibodies against HTLV-I.

Reflexiones pedagógicas, psicológicas y filosóficas acerca del quehacer odontológico en el área clínica / Pedagogic, psychologic and phylosophic reflections on dental duty in the clinical area

Las reflexiones pedagógicas, filosóficas y psicológicas contenidas en este artículo, se fundamentan en los supuestos teóricos del texto Educación y Plenitud Humana, escrito por Juan Mantovani en 1972. Uno de los principales objetivos del seminario, fue que a partir de la concreción clínica, el docente concientizara la trascendencia del hecho educativo, científico y humano en función de la salud del paciente