Fine needle aspiration cytology of thyroid nodules: how accurate is it and what are the causes of discrepant cases?

Fine-needle aspiration cytology (FNAC) is widely accepted as the most accurate, sensitive, specific and cost-effective diagnostic procedure in the assessment of thyroid nodules and helps to select people preoperatively for surgery. The purpose of this study was to evaluate the results of thyroid FNAC in our institution and to determine the reasons for discrepancies between the cytological and histological diagnosis. We evaluated the cytological and histological results of 254 FNACs obtained from 231 patients who underwent subsequent thyroid surgery. All of the material was blindly reviewed for quality control, by one experienced cytopathologist. All FNACs were carried out under ultrasound guidance. The cytological diagnosis was classified as benign, suspicious, malignant or unsatisfactory. The definitive histological study showed benign lesions in 195 of the 231 patients (84%). A benign diagnosis based on FNAC was correct in 105 of the 108 benign cases (97%). FNACs diagnosed as 'suspicious' resulted in a distribution of 49 benign (79%) and 13 malignant (21%) diagnoses. FNAC showed malignancy in 34 cases (13%) and in only one case did the final histology differ from cytology (correlation 97%). The percentage of FNACs that were inadequate for diagnosis was 20%. Review of cytological and histological slides did not lead to any change in the original diagnosis. Our study revealed a cytological-histological discrepancy (2%) in only 4 out of 231 cases over a period of 10 years, due to either a diagnostic or sampling error.

Oxidative inactivation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and subunit cross-linking involve different dithiol/disulfide centers.

Rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR) is extremely sensitive to oxidative inactivation by low concentrations (micromolar) of glutathione disulfide (GSSG) even in the presence of millimolar concentrations of glutathione (GSH). Inactivation involves the formation of an intramolecular protein-SS-protein disulfide in thiol/disulfide redox equilibrium with the reduced, active enzyme (Cappel, R.E., and Gilbert, H.F. (1988) J. Biol. Chem. 263, 12204-12212). In the absence of dithiothreitol, HMGR oxidation has been previously shown to cross-link the microsomal enzyme into a covalent dimer (Ness, G.C., McCreery, M.J., Sample, C.E., Smith, M., and Pendelton, L.C. (1985) J. Biol. Chem. 260, 12391-12393). Examination of the extent of HMGR cross-linking and residual HMGR activity in microsomes equilibrated with glutathione redox buffers establishes that inactivation and cross-linking result from oxidation of different dithiol pairs. The thiol/disulfide oxidation potential (K(ox)) for the oxidative inactivation of HMGR, E(SH)2,active + GSSG<-->E(S-S)inactive + 2 GSH, is 0.67 +/- 0.07 M. However, the equilibrium constant for HMGR cross-linking, E(SH)2,monomer + GSS<-->E(S-S)dimer + 2 GSH, is 0.19 +/- 0.02 M, significantly lower than that for inactivation (p < 0.001). Because of the significantly different oxidation potentials and the lack of a linear relationship between cross-linking and inactivation, the two processes must involve two different sets of vicinal dithiols. HMGR becomes 5-10-fold more difficult to oxidize in the presence of saturating levels of the substrate, HMG-CoA. Both inactivation and cross-linking exhibit significantly lower oxidation potentials in the presence of this substrate, 0.072 +/- 0.01 and 0.047 +/- 0.007 M, respectively. The decrease in oxidation potential caused by substrate binding is observed for both inactivation and cross-linking, showing that both processes are affected by the binding of substrate to the enzyme. The dithiols involved in HMGR subunit cross-linking are 2-3-fold more difficult to oxidize than the dithiols that affect the enzyme activity. Thus, the observation of partial cross-linking of HMGR in vivo would imply that conditions are sufficiently oxidizing to result in significant enzyme inactivation. The extreme thermodynamic sensitivity of HMGR to oxidative inactivation and cross-linking in glutathione redox buffers that span the physiological redox state implies that thiol/disulfide redox state changes could provide a mechanism for regulating the activity and/or stability of this enzyme.

Assessment of the antioxidant/prooxidant status of murine skin following topical treatment with 12-O-tetradecanoylphorbol-13-acetate and throughout the ontogeny of skin cancer. Part II: Quantitation of glutathione and glutathione disulfide.

Tissue glutathione (GSH) and glutathione disulfide (GSSG) contents were quantitated in the skins of female SENCAR mice following the topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), and in the skin tumors generated by an initiation-promotion protocol. Total epidermal GSHt (GSH + GSSG) and GSSG contents were not reproducibly and significantly altered 0.5, 4 or 24 h after one or four topical applications of 1 microgram TPA, relative to the values obtained in age-matched, solvent-treated mice. Similar findings held for dermal GSHt at all times of analyses, and for dermal GSSG contents 0.5 and 4 h after TPA application. However, dermal GSSG contents were slightly elevated 24 h after TPA application. The GSHt and GSSG contents of skins initiated with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and harvested 17, 29 and 37 days after the cessation of chronic treatment with acetone (14 weeks, twice a week) were comparable to the values measured in age-matched, non-treated skins. In contrast, GSHt contents of papillomas harvested 17, 29 and 37 days after the cessation of chronic treatment with 1 microgram TPA (14 weeks, twice a week) were 2- to 4-fold greater than the values measured in non-treated mice, and DMBA-initiated, acetone-promoted mice, and the non-tumorous tissue adjacent to the papillomas. Comparable changes did not occur in papilloma GSSG contents. GSHt contents in squamous cell carcinomas (SCC) were twice the values measured in papillomas and 5- to 8-fold greater than the values measured in non-treated skins, and the non-tumorous tissue adjacent to SCC. Similarly, GSSG contents in SCC were elevated multifold relative to papillomas, non-treated skin and the non-tumorous tissue adjacent to SCC. Epidermal cell suspensions prepared by the trypsin-flotation procedure retained less than 2% of their original GSHt content and had reduced GSHt/GSSG ratios. Collectively these studies suggest that (i) if promoting doses of TPA induce oxidative stress in murine epidermis, it cannot be detected by measurements of GSH/GSSG; (ii) the antioxidant capacity of epidermal cells prepared by the trypsin-flotation procedure is severely compromised; and (iii) GSHt contents progressively increase during skin tumor ontogeny.

Inhibition of glutathione disulfide reductase by glutathione.

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.

The effects of NADPH and 3-hydroxy-3-methylglutaryl-CoA on the thiol/disulfide redox behavior of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase.

Microsomal 3-hydroxy-3-methylglutaryl-CoA reductase isolated from the livers of rats fed a diet containing cholestyramine (HMGR-C) is oxidized to a protein-SS-protein disulfide via a thermodynamically favorable thiol/disulfide exchange in glutathione redox buffers which approach the normal in vivo redox poise. In the presence of either substrate (NADPH or 3-hydroxy-3-methylglutaryl-CoA), the equilibrium thiol/disulfide redox behavior of HMGR-C is substantially different than that observed in the absence of substrates or in the presence of both substrates. NADPH present during redox equilibrium in a glutathione redox buffer decreases the equilibrium constant for formation of the protein-SS-protein disulfide (Kox,i) from 0.55 +/- 0.07 M to 0.18 +/- 0.02 M and increases the Kox,m for formation of an inactive protein-SS-glutathione mixed disulfide from less than 1 to 6 +/- 1. The presence of 3-hydroxy-3-methylglutaryl-CoA during redox equilibrium has a similar effect, decreasing the Kox,i for protein-SS-protein disulfide formation to 0.10 +/- 0.02 M and increasing the Kox,m for protein-SS-glutathione mixed disulfide formation to 3.8 +/- 0.9. A three-state model is developed which describes the simultaneous accumulation of protein-SS-protein and protein-SS-glutathione mixed disulfides at redox equilibrium with glutathione redox buffers. Because of the different redox behavior of the free and substrate-liganded forms of the enzyme, addition of 3-hydroxy-3-methylglutaryl-CoA or NADPH to HMGR-C at redox equilibrium results in increased reduction and activation of the enzyme.

The effects of mevinolin on the thiol/disulfide exchange between 3-hydroxy-3-methyglutaryl-coenzyme A reductase and glutathione.

The feeding of mevinolin plus cholestyramine to rats results in the production of a form of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR-CM) having thiol/disulfide redox properties different from those of 3-hydroxy-3-methylglutaryl-CoA reductase isolated from animals which had been given only cholestyramine (HMGR-C). The second-order rate constant for the inactivation of HMGR-CM by GSSG is 7-fold slower than for HMGR-C, while the second-order rate constant for the reactivation of oxidized enzyme by GSH is 100-fold slower. However, in the presence of saturating concentrations of both substrates, the rate constants for thiol/disulfide exchange are similar for both forms of the enzyme. HMGR-CM behaves as if a protein-glutathione mixed disulfide having a Kox of 27 +/- 4 is formed at equilibrium. In contrast, HMGR-C has previously been shown to form a protein-protein disulfide (Cappel, R. E., and Gilbert, H. F. (1988) J. Biol. Chem. 263, 12204-12212). Both forms of the enzyme are more difficult to oxidize thermodynamically in the presence of saturating levels of both substrates. For HMGR-CM, NADPH alone has no effect on the equilibrium constant for oxidation, but hydroxymethylglutaryl-CoA alone makes the enzyme approximately twice as difficult to oxidize. Under physiological conditions, HMGR-CM is thermodynamically more difficult to oxidize than HMGR-C. HMGR-C can be converted to HMGR-CM by in vitro treatment with mevinolinate. A direct or indirect interaction of mevinolin with HMGR-C results in some persistent, as yet undefined, structural alteration which inhibits the formation of a protein-SS-protein disulfide upon oxidation by glutathione disulfide.

Thiol/disulfide exchange between 3-hydroxy-3-methylglutaryl-CoA reductase and glutathione. A thermodynamically facile dithiol oxidation.

In glutathione redox buffers, rat liver, microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase rapidly equilibrates between a reduced, active form and an oxidized, inactive form. At pH 7.0, 37 degrees C, the second order rate constant for inactivation of the reduced enzyme by GSSG is 1700 +/- 200 M-1 min-1, approximately 20-fold faster than the reaction of GSSG with a typical, unhindered thiol of pKa 7.7. High concentrations of GSH or lower concentrations of dithiothreitol restore the activity of the oxidized enzyme. The oxidation of the enzyme by GSSG is only 30-fold slower in the presence of saturating levels of both substrates. The incomplete inhibition of thiol/disulfide exchange by substrates can lead to significant changes in the activity of the enzyme during the assay when glutathione is present. At redox equilibrium, both in the absence and presence of substrates, the activity of the enzyme depends on the quantity [GSH]2/[GSSG], suggesting that the redox transition involves the formation of a protein-SS-protein disulfide. The equilibrium constant for the reaction HMGRred + GSSG in equilibrium HMGRox + 2 GSH is 0.55 +/- 0.07 M in the absence of substrates and 0.20 +/- 0.02 M in the presence of saturating levels of both substrates. Thus, HMG-CoA reductase is very sensitive to dithiol oxidation both kinetically and thermodynamically. Significant changes in the oxidation state and activity of this enzyme could be expected to result from normal changes in the thiol/disulfide oxidation state of the cellular glutathione redox buffer.

Thiol/disulfide redox equilibrium between glutathione and glycogen debranching enzyme (amylo-1,6-glucosidase/4-alpha-glucanotransferase) from rabbit muscle.

Rabbit skeletal muscle glycogen debranching enzyme is inactivated in a kinetically biphasic manner by GSSG at pH 8.0. The rapid phase results in the loss of 30% activity, while the slower phase leads to total enzyme inactivation. Both the glucosidase and the transferase activities of the enzyme are inhibited by GSSG. The inactivation by disulfides is fully and rapidly reversed in a biphasic manner by reduction with excess reduced dithiothreitol or GSH. After a fast initial recovery of 70% of the initial activity, the remaining 30% of the activity is recovered more slowly. Equilibration of the enzyme with a redox buffer of GSH and GSSG shows a monophasic equilibration of the activity. The ratio of GSH/GSSG where the enzyme is 50% active (R0.5) is 0.06 +/- 0.03. The R0.5 does not vary significantly with the total concentration of glutathione species suggesting formation of protein-SSG mixed disulfides. The ratios of the observed second-order rate constants for GSSG inactivation and GSH reactivation do not lead to a correct value of the observed thiol/disulfide oxidation equilibrium constant. Although the enzyme has sulfhydryl groups, the oxidation of which leads to activity changes, the kinetic and thermodynamic resistance to oxidation suggests that the enzyme is not likely to be subject to regulation by thiol/disulfide exchange in vivo.

Cooperative behavior in the thiol oxidation of rabbit muscle glycogen phosphorylase in cysteamine/cystamine redox buffers.

Glycogen phosphorylase a and b are irreversibly inactivated by oxidation with the disulfide cystamine. The mechanism is complex and involves oxidation of at least two classes of sulfhydryl groups. The oxidation of one or more of the first class of 4 +/- 1 sulfhydryl groups is reversible, but the equilibrium constant for the oxidation is so unfavorable (1 X 10(-4)) that the micromolar concentrations of cysteamine released stoichiometrically with enzyme oxidation are sufficient to prevent complete oxidation even in the presence of 100 mM cystamine. The rapid phase of inactivation of phosphorylase b, which is first order in cystamine (k = 2.9 +/- 0.3 M-1 min-1), is followed by the oxidation of 5 +/- 1 groups in an irreversible process that is second order in cystamine concentration (k = 3.9 +/- M-2 min-1). Similar behavior is observed for phosphorylase a, although the behavior is complicated by association/dissociation equilibrium. The second-order dependence of the rate of irreversible inactivation on cystamine concentration is interpreted in terms of a "cooperative" model in which a rapidly reversible thermodynamically unfavorable equilibrium oxidation of one or more sulfhydryl groups must precede the irreversible oxidation of one or more additional sulfhydryl groups. The thiol/disulfide oxidation equilibrium constant for the initial reversible reaction is estimated to be at least 10(4) less favorable than that for the reversible oxidation of phosphofructokinase.

Clinical efficacy of a herpes simplex subunit vaccine.

A DNA-free herpes simplex type 2 subunit vaccine was administered to 18 volunteers without past evidence of herpes simplex type 1 (HSV 1) or herpes simplex type 2 (HSV 2) infection, to 44 patients with severe recurrent genital HSV 2 infection, and to 15 patients with severe oral type 1 HSV recurrences. The vaccine elicited both humoral and cell-mediated immunity in 97% of the subjects without past HSV infections and boosted significantly the cell-mediated immunity and antibody titers in almost all the patients with recurrent HSV 1 or HSV 2. The vaccine elicited particularly the production of complement-dependent cytotoxic antibodies in 96% of the patients with recurrent HSV 2 infections. This might, at least partly, explain the clinical efficacy of the vaccine. Indeed, we observed a significant decrease (t test, p less than 0.01) in the attack rate of the recurrences and also a significant shortening of the time needed to complete healing of the lesions (t test, p less than 0.01).

Effect of thymopentin on the mortality and immune response after an experimental herpes simplex infection in mice.

The effect of thymopentin on the mortality rate of mice treated with lethal doses (LD90) of herpes virus 2 and on the cytotoxic T cell activity after sublethal doses (LD10) of herpes virus was investigated in two series of experiments. Doses of 1, 0.1 or 0.01 ng of thymopentin per g/mouse were administered i.p. in each experiment, either 3 days before, 3 days (66 h) after, or 3 and 6 days after the herpes virus infection. The cumulative mortality rate was evaluated 10 days after the infection. Cytotoxic T cell activity was measured 3, 7 and 14 days after the infection. The 0.1-ng dose of thymopentin reduced the mortality rate to less than 50% (p = 0.0000) if it was administered 3 days before the infection. A single injection of any dose after infection did not reduce the mortality at all, while two injections of 0.1 ng reduced it by about 25% (p = 0.0038). A 1-ng dose showed a mild but significant reduction (p = 0.0313) if it was applied 3 days before the infection. The cytotoxic T cell activity was either not influenced or significantly modified (p less than 0.05), i.e. increased or decreased as compared to the control, depending on the dose and timing of thymopentin. A correlation between increased cytotoxic T cell activity and protection against mortality can be demonstrated, while no protection was observed in dose regimens where the cytotoxic T cell activity became reduced. The results are discussed in connection with earlier clinical studies in which the beneficial effect of thymopentin has been demonstrated in frequently relapsing herpes labialis and herpes genitalis patients.

Preliminary results of hepatitis B vaccination (HEVAC B) in healthy subjects and haemodialysis patients.

Hepatitis B vaccine (Hevac B, Pasteur) was assessed in 52 healthy and 25 haemodialysis individuals. The percentage of hepatitis B surface antibody seroconversion was 100% in the first group but only 57.7% in the other one. The mean levels of hepatitis B surface antibody, 3 months after the first injection were respectively 222 and 42 milli International Units per ml. Five health-care workers, who experienced an accidental exposure were protected by a combined passive-active immunization.

Creatine kinase activity in normal and Duchenne muscular dystrophy fibroblasts.

Cultured human skin fibroblasts from 9 patients with Duchenne muscular dystrophy (DMD) and 8 normal age- and sex-matched controls were examined for creatine kinase (CK) activity. Both the normal and the DMD fibroblasts were found to have significant levels of CK activity (approximately 10 x 10(-3) IU per milligram of fibroblast protein). The control cells had slightly higher CK activity than the DMD lines, but this difference was not significant (0.2 less than P less than 0.1). The MM (muscle) isozyme, the BB (brain) isozyme, and the MB (hybrid) isozyme, of CK were found to be present in fibroblasts. The isozymes were separated by electrophoresis and the relative amount of each was determined for both normal and DMD cells. In normal fibroblasts, approximately 48% of the total CK activity was of the MM type, 40% was of the BB type, and 12% was of the MB type with no significant differences apparent between normal and DMD groups. The presence in human fibroblasts of significant levels of CK activity with a characteristic isozyme profile is an important consideration for studies of this "marker" enzyme in the pseudohypertrophic muscle of DMD.

Immune responses to DNA free herpes simplex proteins in man.

The data presented confirm in human volunteers our previous observations in animal models. The DNA free HSV 2 subunit vaccine used elicited an antibody and a cell-mediated immune response in 15 subjects without past evidence of HSV 1 or HSV 2 infections and increased the immunity level in 28 subjects suffering from HSV 1 or HSV 2 infections. Although we did not follow a double-blind, placebo controlled protocol our results suggest that the vaccine may reduce the frequency and severity of HSV infections. The time between the recurrences, the pain and the time to complete healing decreased significantly after the vaccination.

Immune response to a DNA free herpes simplex vaccine in man.

A DNA-free subunit herpes simplex virus (HSV) vaccine was administered to 15 volunteers without past evidence of HSV infection and to 25 patients with severe recurrent HSV infection. The immune response to the vaccine in these patients was compared to the immunological status of 20 non-vaccinated control patients with recurrent HSV infection. The vaccine elicited antibody and cell-mediated immunity (CMI) in the 15 subjects without past evidence of HSV infection and this response was similar to that observed after a natural infection. Among the 25 patients who were suffering from recurrent HSV infection the vaccine elicited complement dependent cytotoxic antibodies in 13 of these patients who did not possess these antibodies and increased significantly the titers of these antibodies in the 12 other patients. The vaccine gave a significant increase of the titers of the other specific antibodies as well as the level of cell-mediated immunity. The increase of the immunity level in these latter patient was not due to normal variations since in the non-vaccinated control group the antibody titers and CMI remained stable during the same period of time.

Expression of retrovirus-related antigen in pregnancy. II. Cytotoxic and blocking specificities of immunoglobulins eluted from the placenta.

Immunoglobulins, mostly of the IgG class, were detected in eluates of the placenta of 75% of 50 healthy women in their first or second pregnancy, 92% of 30 women with more than two pregnancies, and 87% of 23 pre-eclamptic patients. The immunoglobulins were assayed for complement-dependent cytotoxicity on human and monkey cell-lines, as well as on the same cells chronically infected with either Mason-Pfizer Virus (M-P V) or Baboon Endogenous Virus (BeV). The frequency of cytotoxic reactions was very low, except with immunoglobulins from the pre-eclamptic placentae, where one third of the samples lysed virus-infected cells with occasional killing of virus-free cells. All placental immunoglobulins which were not cytotoxic were then assayed for blocking activity by testing whether they could compete with the action of anticellular sera of virus-free cells, or with the toxic effect of antiviral sera on virus producing cells. 64% of the immunoglobulins from normal placentae competed with antiviral antibodies while only 17% blocked the action of anticellular sera. The frequency of blocking immunoglobulins was no greater in eluates from pre-eclamptic placentae. The data indicate that the placenta possesses retrovirus antigen sites which bind blocking antibodies in normal pregnancy and complement-dependent cytotoxic antibodies in pre-eclampsia.