Characterization of the thrombin generation profile in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystemic inflammatory autoimmune disorder. Thrombotic events occur at a higher incidence among SLE patients. The investigation of thrombin generation (TG) with calibrated automated thrombogram (CAT) test as a global hemostasis assay is applicable for the overall functional assessment of the hemostasis. The aim of this study was to characterize the hemostatic alterations observed in SLE by CAT assay. In this study, CAT parameters and basic coagulation parameters of SLE patients (n = 22) and healthy control subjects (n = 34) were compared. CAT area under the curve (i.e., endogenous thrombin potential) was lower than normal in SLE (807 vs. 1,159 nM*min, respectively), whereas other CAT parameters (peak, lag time, time to peak, and velocity index) and the basic coagulation tests were within the normal range. The presence of anti-phospholipid antibodies and the applied therapy was not associated with hemostasis parameters in SLE. We concluded that the reported high risk of thrombosis is not related to TG potential.

A comparison of the lymphoproliferative capacity of pokeweed mitogen (PWM) and phytohaemagglutinin (PHA) in CCR-5 wild-type and heterozygous HIV-infected and uninfected subjects.

Effect of CCR-5 delta 32 heterozygosity in immunological protection was studied by a lymphocyte proliferation assay. Twenty of 86 HIV+ and eight of 32 healthy subjects showed heterozygous mutation (wt/mut) of the CCR-5 gene. Lymphocyte proliferation to pokeweed mitogen was found significantly higher (P < 0.005) in wt/mut versus wild type homozygous (wt/wt) HIV+ subjects in groups with CD4 > 500 and CD4 < 200 cell/ micro L. Phytohaemagglutinin induced stronger proliferation of cells from wt/mut HIV+ subjects with CD4 < 200 cell/ micro L (P = 0.03). Decline of lymphocyte response was more significant among wt/wt groups with different CD4+ cell counts than that between wt/mut groups to both mitogens. Reduced number of CCR-5 receptors on CD4+ cells may decrease the ability of HIV-1 envelope glycoproteins to transduce intracellular signals through CCR-5. Mutation in CCR-5 gene seems to have a benefit in preventing T-cells from HIV envelope-mediated immunopathogenic effects and maintain a relatively normal response to lectins.

Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals.

IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gamma mRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients.

Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals

IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gammamRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients

No change in impaired cellular immune response of HIV-negative homosexuals after 15 years of HIV epidemic in Eastern/Central European region.

Impaired cell-mediated immune reactivity to polyclonal mitogens was determined in HIV-negative homosexual men (HIV-MSM). Results were compared to those we reported in a complex clinical and immunological investigation in the same risk groups 15 years ago, before the onset of the AIDS epidemic in Hungary. Cellular immune reactivity to polyclonal mitogens was studied in 74 HIV-infected or HIV-uninfected homosexual men and heterosexual controls. Lymphocytes in whole-blood cultures were stimulated with various doses of phytohaemagglutinin (PHA), concanavalin-A (Con-A), and pokeweed mitogen (PWM) in a blast transformation assay. A significant difference (p = .0002) in lymphocyte proliferation between HIV-MSM vs. heterosexuals using PWM in both concentrations was found. Proliferative capacity was similar in HIV- MSM and HIV infected males with CD4+ > 500/microl. Con-A and PHA showed a less expressed proliferative response. Decreased lymphocyte reactivity to PWM, similar to the one in early HIV infection, could be observed in HIV-MSM. This HIV-independent mild immunodeficiency in MSM is a sign of an increased susceptibility and predisposes to subsequent HIV infection. It seems, however, that MSM's impaired immune response observed over a period of 15 years is an immunodeficiency not changed by the emerging HIV/AIDS epidemic. Our study provides an explanation why the incidence of new HIV cases in homo-/bisexual individuals is still high (> 70%), and it indicates that it remains high in Hungary.

Peptidyl beta-homo-aspartals (3-amino-4-carboxybutyraldehydes): new specific inhibitors of caspases.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE, caspase-1) processes the IL-1 beta precursor to mature inflammatory cytokine IL-1 beta. ICE has been identified as a unique cysteine protease, which cleaves Asp-X bonds, shows resistance to E-64 (an inhibitor of most cysteine proteases) and has a primary structure that is homologous to CED-3, a protein required for apoptosis (programmed cell death) in the nematode Caenorhabditis elegans, and to mammalian cysteine proteases that initiate and execute apoptosis, e.g., apopain/CPP32/caspase-3. The inhibitors of the ICE/CED-3 family or caspases, as they are called recently, may constitute therapeutic agents for amelioration of inflammatory and apoptosis-associated diseases. The most efficient ICE inhibitors are peptide aldehydes and peptidyl chloro or (acyloxy)methanes. A recent study revealed that both D- and L-Asp are accepted by ICE at the P1 of such inhibitors, and the peptidyl (acyloxy)methane analogues having the beta-homo-aspartyl residue [-NH-CH(CH2COOH)-CH2CO-] are inactive. These findings we reexamined in terms of two issues. (a) ICE's resistance to E-64. Since it was thought to be caused by the enzyme's unique substrate specificity, we prepared substrate-based analogues, which were not inhibitory suggesting significant structural difference between the active centers of ICE and papain-like enzymes. (b) Tolerance for D-stereochemistry at the P1 of these inhibitors. In view of the mechanism of cysteine protease inhibition by peptidyl X-methanes, we thought that this phenomenon should be a general characteristic of cysteine proteases and the hAsp-containing analogues should behave as reversible inhibitors. Here, we analyzed the inhibition of ICE and apopain in comparison with that of papain, thrombin, and trypsin by peptide L/D-alpha-aldehydes and their L-beta-homo-aldehyde [-NH-CH(R)-CH2-CHO] analogues. The following results were found. (1) The peptidyl L-beta-homo-aspartals are potent inhibitors for caspases. (2) The L-beta-homo analogues of peptide aldehyde inhibitors designed for other proteases are not inhibitory. (3) Unlike trypsin and thrombin (serine proteases), papain (cysteine protease) shows tolerance for D-stereochemistry at the P1 site of peptide aldehydes in proportion to the lability of the alpha-hydrogen of the P1-D-residue. The complete tolerance of ICE for P1-D-Asp may arise from this residue's high tendency to epimerization. (4) Reaction of cysteine proteases with peptide aldehyde or peptidyl X-methane inhibitors containing P1-D-residues may include alpha-proton abstraction followed by asymmetric induction leading to P1-L-residue-containing products.

Studies on the antibodies to human herpesvirus type 6 among Hungarian patients with asymptomatic HIV infection.

The occurrence and the possible role in promoting HIV infection by human herpesvirus type 6 (HHV-6) have not yet been revealed in Hungary. In different groups of patients, serum titre of IgM and IgG antibodies, as well as avidity of IgG were quantitated by indirect immunofluorescence and an enzyme-linked immunosorbent assay, using isolate U1102 of HHV-6 variant A as antigen. In 60% of HIV-seronegative adult controls, high avidity IgG antibodies were found in low titre suggesting childhood infection. In HIV-seronegative persons with high risk behaviour for HIV-infection, both IgM and low avidity IgG were frequently found in higher titre, representing either primary or frequent reinfections, or reactivation of latent HHV-6. In asymptomatic HIV-seropositive patients, high titre of high avidity IgG antibodies was predominant, proving virus infection in the near past. These results indicate the contribution of HHV-6 to immunosuppression prior to AIDS, predisposing the organism to HIV infection.

Peptidyl beta-homo-aspartals: specific inhibitors of interleukin-1 beta converting enzyme and its homologues (caspases).

Inhibition of interleukin-1 beta converting enzyme (ICE), apopain, papain, thrombin and trypsin with substrate like peptidyl L- and D-alpha-aldehydes and their L-beta-homo-aldehyde analogues was investigated. The L-beta-homo-aspartals appear to be specific inhibitors for ICE and its homologues; the other enzymes were not inhibited with such L-beta-homo aldehydes. Papain shows tolerance for D-residues at P1 depending on their chiral stability.

The Dominant T-Helper Lymphocyte Function of HIV Infected Patients.

In HIV infection, the decrease in the number and functional activity of lymphocytes is accompanied by atopia and an increased level of total IgE and some specific IgE antibodies. This could be explained by the Th2 dominance induced by HIV replication and so a Th1-Th2 switch could have prognostic value. We investigated the characteristic T-helper phenotype dominance and its relationship to cytokine expression and IgE immune response in the early stage of asymptomatic HIV infection. In the separated lymphocytes of i. asymptomatic HIV positive persons; ii. HIV negative homosexuals; iii. atopic patients; and iv. healthy controls, expression of mRNA for IFNg (Th1) and IL-10 (Th2) were determined by semiquantitative RT-PCR. The serum level of antibodies for HIV 1/2 and total/specific IgE were also determined. Transcription of mRNA of IFNg and IL-10 were more pronounced in HIV positive and atopic groups than in the healthy control, without lymphocyte phenotype dominance. In HIV negative persons, however, a significant Th2 dominance was detected. There was no significant difference in the IgE level between the 4 investigated groups. In the HIV positive cases, IL-10 expression and total serum IgE do not support a switch to Th2 dominance. In the atopic group, aside from the total IgE level, down regulation of IFNg was not observed. These results suggest a general activation of the immune system in the early stage of HIV infection.

The renin-angiotensin system and the effect of propranolol upon the cerebral cortical and hypothalamic circulation in hypoxia.

The regulatory mechanisms of the cerebral blood flow have preoccupied the physiology department of Cluj since the end of the 4th decade. These studies continued over the last years. The researches progressed from the studies of regulation by blood pressure changes to the nervous regulation and to the metabolic one. This paper's subject is the renin-angiotensin and adrenalin system influence on the changes of cerebral blood flow during the general hypoxic hypoxia and cephalic ischemia. Experiments were performed in 10 dogs anaesthetised with a mixture of chloralose, urethan and morphine. Hypoxic hypoxia was obtained by breathing a mixture of 11% oxygen in nitrogen, in a closed system and cerebral ischemic hypoxia by partial compression of the carotid arteries, after the ligation of the vertebral and thyroid arteries. The arterial blood pressure and the cerebral and hypothalamic blood flow, measured with the heated thermoelement, were registered. The plasma renin activity was tested radioimmunologically before, at 1.5 min, 5, 10 and 15 min, after the beginning of hypoxia. In ischemic hypoxia the experiment was repeated after venous perfusion with propranolol (0.6 mg/kg/h). The systemic blood pressure increased in both forms of hypoxia. The cortical and hypothalamic blood flow increased with the systemic arterial blood pressure. The hypothalamic blood flow remained stable or diminished a little. Propranolol increased the cerebral blood flow during ischemic hypoxia up to 300%. The i.v. administration of angiotensin (1-5 mg/kg) increased the cortical flow, while the hypothalamic flow remained self-regulated. Plasma renin activity increased more in general hypoxic hypoxia, than in cephalic ischemic hypoxia. After propranolol the increase was higher in this hypoxia. Propranolol produced a major activation of the renin-angiotensin system and of the cortical blood flow in ischemic cephalic hypoxia, the renin-angiotensin system being located in the cerebral structure. As well high doses of angiotensin produced cerebral vasodilatation in small cerebral vessels. This effect was found in our experiments in the cortical blood flow too. Our results indicate a beneficial propranolol effect on cortical circulation in ischemic hypoxia.

Active site-directed thrombin inhibitors: alpha-hydroxyacyl-prolyl-arginals, new orally active stable analogues of D-Phe-Pro-Arg-H.

D-alpha-Hydroxyacyl-prolyl-arginals, a new type of analogues of D-Phe-Pro-Arg-H (R1), have been prepared and evaluated. Unlike R1, whose terminal group is NH2, the new analogues with a terminal OH group are stable, as are the N-substituted derivatives of R1, that is, D-MePhe-Pro-Arg-H (R2), the highly potent and selective thrombin inhibitor, and Boc-D-Phe-Pro-Arg-H (R3), the much less favorable analogue. The most notable of the new analogues corresponds to the general formula D-Xaa-Pro-Arg-H, wherein Xaa means the acyl residue of mandelic acid (Man, 1), diphenyllactic acid (Dpl, 2), hexahydrophenyllactic acid (Hpl, 3), or hexahydromandelic acid (Hma, 4). In plasma clotting assays, 1 to 4 appeared to inhibit thrombin as well as some other clotting enzymes involved in thrombin generation, whereas R1 and R2 seemed to produce anticoagulation through inhibition of thrombin only. In the fibrin plate assay, 1 to 4 possessed even more moderate antifibrinolytic activities than R2. In in vivo evaluation in rats and rabbits, 2 to 4 proved to be potent anticoagulants/antithrombotics even on oral administration in a dose of 5 mg/kg. In view of these findings with the alpha-hydroxyacyl-prolyl-arginals, it is very likely that the less favorable biologic properties of Boc-D-Phe-Pro-Arg-H are due to the hydrophobicity and bulkiness of the terminal Boc-NH rather than its neutrality.

Determination of HIV-1 Subtypes in Hungary by Synthetic Peptides Representing the V3 Loop of env.

For the determination of HIV-1 diversity and serotyping of HIV-1 subtypes, an enzyme immunoassay was developed based on synthetic peptides representing immunodominant epitopes of the V3 loop of HIV-1 subtypes A, B, C and E, respectively. Sera from 53 asymptomatic HIV-1 infected individuals were tested for their pattern of binding reactivity to the synthetic peptides. 45/52 (85%) of the sera reacted exclusively to V3 peptide representing HIV-1 B subtypes, 4/52 (7.6%) of the sera showed cross reacticity to A/B peptides and 1/52 (1.9%) of the sera reacted with both A and C peptides. No single reactivity with subtype A or E peptides have been observed. Results together with nucleotide sequence analysis of the V3 region of clinical isolates suggest that HIV-1 infection in Hungary has been induced predominantly by strains belonging to HIV-1 subtype B.

Active site-directed thrombin inhibitors: alpha-hydroxyacyl-prolyl-arginals. New orally active stable analogs of D-Phe-Pro-Arg-H.

D-alpha-Hydroxyacyl-prolyl-arginals have been designed and synthesized as orally active stable analogs of D-Phe-Pro-Arg-H, the active site-directed peptidyl thrombin inhibitor prototype. Many of the new analogs possess high in vitro anticoagulant activity while having little effect on fibrinolysis. Compounds GYKI-66104 (2), -66131 (3) and -66132 (5) effectively delay the clotting time in rabbits ex vivo and prevent thrombus formation in various thrombosis models in rabbits and rats when applied in a single oral dose of 5 mg kg-1.

Methodological aspects in the studies on the mechanism of action of synthetic direct thrombin antagonists.

Requirements reported for an ideal anticoagulant [12] and for an ideal antithrombotic [18] show the necessity of many-sided methodological approach in order to detect the molecular mechanism of action of a novel synthetic antagonist of thrombin. The lack of a protocol internationally accepted, on the one hand, and with regard to a general proposal accepted [16], on the other hand, authors applied a complex methodological system involving also the study on the possible interactions at molecular level of some novel thrombin antagonists with the main components of their site of action. The surprising contradiction found between in vitro and in vivo efficacy of several antagonists could be attributed and explained by the significant differences in Ki and IC50 values determined in complex clotting assays containing plasma proteins and/or blood cells versus those measured in reaction mixtures consisting of a synthetic chromogenic substrate, the target enzyme, thrombin, and the antagonist compound in buffer solution.

Screening for fibrinolysis inhibitory effect of synthetic thrombin inhibitors.

Fibrin plate assay (FPA) and thrombelastography (TEG) were used to assess the antifibrinolytic effects of D-Phe-Pro-Arg-H (1), the prototype of peptide aldehyde inhibitors of thrombin, and two of its more stable derivatives, D-MePhe-Pro-Arg-H (2) and Boc-D-Phe-Pro-Arg-H (3). Inhibition of plasmin generation by tissue plasminogen activator, urokinase and streptokinase were studied by both FPA and TEG while that of plasmin could only be examined by FPA. TEG was more sensitive than FPA in general and for the detection of streptokinase inhibition in particular. Derivative (3) was 2-50 times more inhibitory than (1) or (2) depending on the enzyme studied and the assay system used. The thrombin selectivities of (1)-(3) were defined as the thrombin to fibrinolytic enzyme potency ratios. Data obtained by the FPA and thrombin time assay indicated (1) and (2) to be 2-80 times more selective for thrombin than (3). On the other hand, the values determined by TEG and recalcification assay showed the thrombin selectivity of (2) to be two to three times higher than that of (1), and (3) to have no such selectivity. According to TEG studies, (1) and (2) assisted rather than inhibited fibrinolysis by reducing the elasticity of human plasma clots.

Inhibition by D-MePhe-Pro-Arg-H (GYKI-14766) of thrombus growth in experimental models of thrombosis.

The antithrombotic action of the highly effective synthetic thrombin inhibitor D-MePhe-Pro-Arg-H (GYKI-14766) was studied in various models of experimental thrombosis. The compound administered to rats and rabbits by i.v. bolus injections, continuous i.v. infusions, subcutaneously and orally, respectively, induced significant decrease in thrombus weight (i) in a quantitative venous thrombosis model with stasis based on vascular lesion in rats, (ii) in an extracorporeal arterio-venous shunt model in rabbits, and (iii) prevented the occlusion of the vessel in arterial thrombosis induced by mechanical damage in rats. By using the arterio-venous shunt model in rabbits the inhibitory effect on thrombus growth could be demonstrated as a function of dose and time in self-controlled experiments. Blood level of the inhibitor determined by a bioassay varied between 0.09-0.67 microgram/ml whole blood when doses of 15 and 20 mg/kg were administered orally. A correlation was found between thrombin time, platelet aggregation induced by thrombin ex vivo and the weight of thrombi formed.

Comparative studies in vitro and ex vivo on the anticoagulant effect of a reversible and an irreversible tripeptide inhibitor of thrombin.

Comparative studies on the anticoagulant effect of D-Phe-Pro-Arg-H (ALD) and D-Phe-Pro-Arg-CH2Cl (CMK) were carried out in order to estimate whether the reversible or the irreversible tripeptide inhibitor of thrombin would be more suitable to develop as a novel anticoagulant. Conventional screening assay methods in vitro were focused on the functional stability of the compounds in whole blood and blood components while ex vivo the changes in whole blood clotting time under parenteral application of the inhibitors were investigated. The efficacy of ALD relative to that of CMK was found to depend on the complexity of the test systems. Thus CMK was the more inhibitory in citrated plasma, but ALD showed the higher potency in whole blood. When incubated in various systems such as human whole blood, serum, solutions of isolated plasma proteins, digestive juices and tissue homogenates, respectively, the inhibitory activity of ALD showed only slight decreases for several hours while marked or substantial loss of activity was observed with CMK under identical conditions. ALD administered parenterally to rabbits proved to be powerful anticoagulant; CMK exhibited only a weak and transient anticoagulant effect presumably due to its ability to bind irreversibly to various plasma and tissue proteins. Accordingly, the reversible inhibitor ALD should be more suitable to develop as an anti-coagulant than CMK, its irreversibly acting analogue.

In vitro inhibition of blood coagulation by tripeptide aldehydes--a retrospective screening study focused on the stable D-MePhe-Pro-Arg-H.H2SO4.

A series of peptide aldehydes synthetized in our institute during the last 15 years were screened to detect their inhibitory effect on blood coagulation. Simple conventional clotting assays, platelet function tests and fibrinolytic methods were used to evaluate the inhibitory potency of the compounds in complex clotting systems as well as their supposed antifibrinolytic effect in vitro. Special attention was paid to the possible interactions with blood cells and plasma proteins, and to the functional stability of the inhibitors in several tissue homogenates. D-Phe-Pro-Arg-H (GYKI-14166, RGH-2958), Boc-D-Phe-Pro-Arg-H (GYKI-14451) and D-MePhe-Pro-Arg-H (GYKI-14766) were found to be the most potent inhibitors. The peptide aldehydes via formation of reversible complexes with thrombin impede the enzyme to react with the coagulation factors, platelet membrane and vessel wall. The compounds inhibit platelet aggregation induced by thrombin specifically without changing the sensitivity of platelets to other inducers. D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H showed no antifibrinolytic effect. D-MePhe-Pro-Arg-H and Boc-D-Phe-Pro-Arg-H proved to be stable in dry state for years and in solution at room temperature for several days. The anticoagulant activity of the compounds was declared in NIH antithrombin units.

In vivo anticoagulant and antiplatelet effect of D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H synthetized in our institute were administered to mice, rats, rabbits and beagle dogs. The kinetics of the anticoagulant and antiplatelet effect was recorded by measuring various clotting parameters, platelet count and aggregation, and evaluated as proposed by Verstraete and Verwilghen. The minimum effective doses were found to be 0.25-0.5 mg kg-1h-1 by intravenous continuous infusions and 0.5-1.0 mg/kg by single injections. The dose-dependent prolongation of clotting times appeared after application within minutes and returned to baseline values as a function of dose. Blood level of the inhibitors was determined by a bioassay. Unlike heparin, no higher starting dose was required to reach the anticoagulant threshold level, i.e. 0.03-0.1 microgram/ml whole blood. The peptides did not cause significant changes in platelet count and function or in hemodynamic parameters (blood pressure, heart rate and ECG) and in respiration. They blocked platelet aggregation induced by thrombin ex vivo specifically. No rebound effect or bleeding could be demonstrated even after subtoxic doses of the compounds. The onset of the anticoagulant and antithrombotic effect appeared within 60 min after single oral doses and lasted for 3-6 h. In close correlation with the anticoagulant effect a complete or significant inhibition of platelet aggregation induced by thrombin ex vivo could also be recorded by using 5-10 mg/kg doses.

Calcitonin reserve and the differentiated response to calcitonin in osteoporosis.

The authors followed up the effect of calcium and calcitonin administration on phosphorus-calcium balance and on calcium retention, in correlation with the level of endogenous calcitonin, in 20 patients of perimenopause age (40-50 years), with radiologically confirmed osteoporosis. Calcium retention after loading with calcium gluconate (180 mg in i.v. injections) was determined before and after administration of salmon calcitonin (100 IV in i.m. injections). Two types of responses were noted. In a group of patients calcitonin administration determined a rise of calcium retention at the same time with the improvement of other biochemical parameters as well as a normal calcitonin response. The presence of high levels of circulating calcitonin 24h after loading with exogenous calcitonin demonstrated a slower inactivation rate of the hormone in these patients. The other group showed no positive response to calcitonin. The proposed test is a criterion for the selection of the patients with osteoporosis in view of chronic calcitonin treatment.