Platelet microvesicles are associated with the severity of coronary artery disease: comparison between peripheral and coronary circulation.

Microvesicles (MVs) have recently emerged as markers of thrombosis. Furthermore, there is an unexplained residual thrombotic risk is observed in patients with acute coronary syndrome (ACS) and/or stable coronary artery disease (CAD), despite treatment. We measured platelet (PMVs) and erythrocyte (ErMVs) in patients with ACS and stable CAD, both in the peripheral and coronary circulation. We studied consecutive eligible patients during a coronary angiography. Blood samples were collected from the stem of the left coronary artery and femoral artery. PMVs were significantly increased in CAD patients compared to controls. ACS patients had also increased PMVs in coronary and peripheral circulation, compared to controls. Furthermore, ACS patients exhibited increased PMVs in coronary compared to peripheral circulation. Lastly, coronary PMVs were associated with the severity of CAD based on the SYNTAX score. No significant differences were observed in the levels of ErMVs among groups. Therefore, PMVs emerge as novel markers of thrombosis in CAD, further augmenting the vicious cycle of inflammation and thrombosis during ACS. Importantly, coronary PMVs may reflect the severity of CAD in this population.

Synthesis, structure elucidation and biological evaluation of triple bridged dinuclear copper(II) complexes as anticancer and antioxidant/anti-inflammatory agents.

Seeking for copper based metallo-therapeutics, three triple bridged dinuclear copper(II) complexes [Cu2(µ2-L2)(bipy)2(µ2-OH)(µ2-H2O)](NO3)2·H2O (1·H2O), [Cu2(µ2-L2)(bipy)2(µ2-OH)(µ2-NO3)](NO3)·0.6MeOH·0.4H2O (2·0.6MeOH·0.4H2O) and [Cu2(µ2-L1)(bipy)2(µ2-OH)(µ2-NO3)(H2O)](NO3)·2H2O (3·2H2O) where L2=2-thiophene acetato, L1=2-thiophene carboxylato and bipy=2,2'-bipyridine were synthesized and structurally characterized. The complexes were subjected in vitro to a pharmacochemical evaluation for their antioxidant/anti-inflammatory activity, cytotoxicity and efficacy against human ovarian, lung, colon, breast, kidney and cervical cancer cell lines along with non tumor human lung and breast cell lines. The biological results support the structure related cytotoxic activity of the compounds. Complex 3 presented a combination of best anticancer and anti-inflammatory activities. A computational analysis over the LOX-3 protein structure 1JNQ was performed to support the possible mode of action.

Synthesis, structural characterization and biological studies of the triphenyltin(IV) complex with 2-thiobarbituric acid.

The reaction between 2-thiobarbituric acid (H(2)TBA), which was treated with an equimolar amount of potassium hydroxide, in a water with triphenytin chloride in methanol, results in the formation of the {[Ph(3)Sn(O-HTBA)]}(n) (1) complex. Crystals of the hydrated 1 with formula {[Ph(3)Sn(O-HTBA)]·0.7(H(2)O)}(n) were growth from methanol/acetonitrile solution, of the white precipitation, filtered off, from the reaction. The crystal structure of complex 1 has been determined by X-ray diffraction at 120 K. Complex 1 is polymeric. The geometry around the tin(IV) ions is trigonal bi-pyramidal with coordination to three C atoms from phenyl groups and one O atom from a de-protonated HTBA ligand. Complex 1 and the already known [(n-Bu)(3)Sn(O-HTBA)·H(2)O] (2) were evaluated for their in vitro cytotoxic activity (cell viability) against human cancer cell lines: HeLa (cervical), OAW-42 (ovarian), MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER negative), A549 (lung), Caki-1 (renal) and additionally, the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells) and normal immortalized human mammary gland epithelial cell line MTSV17 with a Trypan Blue assay. Moreover complex 1 was evaluated for its in vitro cell growth proliferation activity against leiomyosarcoma cells (LMS), MCF-7 and MRC-5 cells with a Thiazolyl Blue Tetrazolium Bromide (MTT) assay. The type of cell death caused by complexes 1 and 2 was also evaluated by use of flow cytometry assay. The results showed that these compounds mediate a strong cytotoxic response to normal and cancer cell lines tested through apoptosis and induce cell cycle arrest in S phase of the cell cycle, suggesting DNA intercalation (direct or indirect) with the complexes. Finally, the influence of these complexes 1 and 2 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied.

Preparation and pharmacochemical evaluation of mixed ligand copper(II) complexes with triethanolamine and thiophenyl-2 saturated carboxylic acids.

The dinuclear complex [Cu(2)(L(1))(2)(H(2)tea)(2)] (1) as well as the linear trinuclear complexes [Cu(3)(L(1))(4)(H(2)tea)(2)] (2), [Cu(3)(L(2))(4)(H(2)tea)(2)] (3) and [Cu(3)(L(1))(2)(H(2)tea)(2)(NO(3))(2)] (4) where L(1) = 2-thiophene carboxylato, L(2) = 2-thiophene acetato and H(2)tea = the single deprotonated form of triethanolamine have been prepared and pharmacochemically studied. The crystal structure of 1 is also reported. In vitro antioxidant activity of free ligands and their respective copper complexes includes: a) interaction with 1,1-diphenyl-2-picrylhydrazyl stable free radical, b) the ΗΟ˙ mediated oxidation of DMSO, c) scavenging of superoxide anion radicals, d) inhibition of lipid peroxidation and e) soybean lipoxygenase inhibition. The results indicate selectivity of the complexes to different free radicals as a consequence of their physichochemical features. The majority of the complexes 1, 2, 3, 4 effectively inhibit lipid peroxidation. The trinuclear complex 3 is by far the most active with IC(50)=10 µM, within the set, followed by complexes 1 and 2. The complexes were evaluated for their efficacy as anticancer agents against different cancer and normal human cell lines. Results showed that, these compounds mediate a moderate cytotoxic response to normal and cancer cell lines tested and induce cell cycle arrest in G2/M phase of the cell cycle. Flow cytometric analysis suggested that the tested compounds can induce apoptosis.

Validation of the dorsal air pouch model to predict and examine immunostimulatory responses in the gut.

AIMS: To validate the use of the air pouch system to predict and examine early immune responses induced by the presumptive probiotics Lactobacillus paracasei subsp. paracasei B112, DC205, DC215 and DC412 strains in the gut mucosa. METHODS AND RESULTS: Only the DC412 strain interacted strongly with the cells forming the air pouch lining tissue and induced early innate immune responses such as polymorphonuclear (PMN) cell recruitment, phagocytosis and tumour necrosis factor alpha (TNF-alpha) production that equal the respective responses induced by the probiotic Lactobacillus acidophilus NCFB 1748. The strains exhibiting strong immunoregulatory activity in the air pouch also interacted strongly with the gut-associated lymphoid tissue (GALT). The strain DC412 exerts its effect on the intestine through stimulation of Toll-like receptor (TLR)2/TLR4-mediated signalling events leading to secretion of a certain profile of cytokines in which gamma interferon (IFN-gamma), TNF-alpha, interleukin (IL)-6 and IL-10 are included. The probiotic Lact. acidophilus NCFB 1748 induces the same cytokine profile in addition to IL-12B, and this response is potentially mediated by the synergy of TLR2 and TLR9. CONCLUSION: The strain DC412 possesses the in vitro and in vivo characteristics of a probiotic micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The dorsal mouse or rat air pouch may be used as an alternative and rapid method for the initial discrimination and selection of potential probiotic Lactobacillus strains.

Extramedullary (EMP) relapse in unusual locations in multiple myeloma: Is there an association with precedent thalidomide administration and a correlation of special biological features with treatment and outcome?

Extramedullary relapse constitutes an uncommon manifestation of multiple myeloma (MM), characterized by highly malignant histology, special biological features, resistance to treatment and poor outcome. Its incidence has been increased during the last years, probably due to the introduction of novel strategies in the management of MM, including intensified treatment and immunomodulatory drugs. Here we report nine cases of extramedullary relapse of MM, presented in unusual locations, seven of which had previously been treated with thalidomide-containing regimens (TCR). Our aim was to explore the morphological, immunophenotypical, molecular and laboratory characteristics accompanying EMP-relapse and seek possible correlations with treatment and clinical outcome.

Recombinant human erythropoietin is associated with increased overall survival in patients with multiple myeloma.

BACKGROUND: Recombinant human erythropoietin (rhEPO) is effective for the treatment of anemia associated with multiple myeloma. Data from animal studies and case reports suggest that rhEPO has antineoplastic properties. METHODS: Two hundred and ninety-two patients enrolled on different chemotherapy clinical trials at the Cleveland Clinic Myeloma Program between 1997 and 2003 were the subjects of this study. Information on erythropoietin use as well as baseline prognostic variables were collected retrospectively. RESULTS: The population consisted of 257 patients with multiple myeloma treated at the Cleveland Clinic Foundation from 1997 to 2003 and followed for at least 1 month. Thirty-five patients were excluded from this analysis because information on erythropoietin use was not available. One hundred and twenty-seven patients received rhEPO for at least 1 month and the rest did not received rhEPO. On average, patients who received rhEPO were older, had a higher Southwest Oncology Group (SWOG) stage, higher serum creatinine, lower serum hemoglobin, higher beta2-microglobulin, lower platelet counts, and a longer time from diagnosis to enrollment at the myeloma program (p < 0.001 for all). After adjusting for age, months from diagnosis to enrollment, serum creatinine, hemoglobin, platelet count, and beta2-microglobulin, the use of rhEPO was associated with improved overall survival (hazard ratio = 0.6; 95% CI = 0.38-0.94) in patients with SWOG stages II, III and IV but not in patients with SWOG stage I. CONCLUSION: rhEPO was associated with improved overall survival in this population of anemic multiple myeloma patients with SWOG stages of II, III and IV. A prospective randomized trial is warranted to corroborate this finding.

L-arginine supplementation does not affect chemically induced carcinogenesis and tumor growth in BALB-c mice.

UNLABELLED: T-cell zeta-chain downregulation is common in various types of cancer and it is proposed as a mechanism of cancer immunosubversion. L-arginine consumption by arginase rich suppressor myeloid cells has been incriminated. The effect of L-arginine supplementation on chemically induced carcinogenesis and tumor growth in mice was evaluated. METHODS: Eight-week old female BALB-c mice were used. Ten mice were injected i.m. with 0.6 mg methylcholanthrene (MCA) once. Ten mice were injected with MCA once and were receiving L-arginine supplementation (5% in animal drinking water) continuously during the study. Mice with cancer were sacrificed 12 weeks after. RESULTS: From the 10 MCA injected mice 6 developed sarcoma. From the 10 MCA injected mice that were receiving L-arginine supplementation 7 developed sarcoma. L-arginine supplementation did not affect MCA induced carcinogenesis (p=1.0, Fisher's exact test). The weight of tumors was not different between the tumors derived from mice that were or were not receiving L-arginine supplementation (1088.3+/-590.2 mg vs. 969.6+/-608.1 mg respectively, p=0.729, unpaired t-test). CONCLUSION: L-arginine supplementation does not affect chemically induced carcinogenesis and tumor growth in BALB-c mice. Although zeta-chain downregulation could be a mechanism of cancer immunosubversion there are enough other cancer immunosubversion mechanisms that were not overwhelmed by L-arginine supplementation. Additionally, except cancer immunosubversion, cancer immunoselection is another, possibly more significant, mechanism of tumor escape from immunosurveillance.

Clinical, immunological, and immunogenetic aspects of autoantibody production against Ro/SSA, La/SSB and their linear epitopes in primary Sjögren's syndrome (pSS): a European multicentre study.

OBJECTIVES: To investigate the clinical and immunogenetic aspects of antibody formation against Ro/SSA and La/SSB as well as their linear B cell epitopes in patients with primary Sjögren's syndrome (pSS) from different European countries. PATIENTS AND METHODS: Ninety patients with pSS from six European centres were studied. Serum samples from all patients were tested in a control laboratory for anti-Ro/SSA and anti-La/SSB autoantibodies by RNA precipitation assay and autoantibodies to the previously reported B cell linear epitopes of Ro 60 kDa (p169-190aa and p211-232aa) and La/SSB (p147-154aa, p291-302aa, p301-318aa, and p349-364aa). DNA from 88 patients was used for the determination of HLA-DRB1, -DQA1, and -DQB1 genotypes. Analysis of the results was performed in the 88 patients who were genotyped and tested also for antipeptide antibodies. RESULTS: Antibodies to B cell epitopes of Ro 60 kDa were detected at a low frequency (range 10-37%). In contrast, B cell epitopes of La/SSB were detected frequently (range 58-86%) among the anti-La/SSB positive sera. Autoantibodies to the La/SSB epitope, p349-364aa, were significantly positively associated with longer disease duration (p<0.05), recurrent or permanent parotid gland enlargement (p<0.005), and a higher proportion of non-exocrine manifestations (p<0.005), compared with patients without autoantibodies. The presence of anti-Ro/SSA and anti-La/SSB autoantibodies was significantly associated with the presence of HLA-DRB1*03 and DQB1*02 (p=0.038 and p=0.034, respectively). This association was even more prominent and extended to HLA-DQA1*0501 when patients were stratified according the presence of autoantibodies to discrete La/SSB B cell epitopes in comparison with autoantibody negative patients (p<0.01). They were found also to be highly associated with the alleles HLA-DQB1*02 and HLA-DQA1*0501 as well as the presence of a shared amino acid motif in the region 59-69aa of DQB1 first domain (p<0.01, respectively). CONCLUSIONS: Autoantibodies against La/SSB, binding to four synthetic peptides, derived from the sequence of the La protein were identified with increased frequency in sera of patients with pSS. The formation of autoantibodies against B cell epitope analogues of La/SSB in European patients with pSS may be dependent on the presence of a permissive HLA-DQ heterodimer, most prominently represented by the HLA-DQA1*0501/DQB1*0201 heterodimer, suggesting that a model of HLA restricted presentation of La/SSB peptide determinants is crucial for the autoimmune response against La/SSB.

Study of antibody and T cell responses in rabbits immunized with synthetic human B cell epitope analogues of La (SSB) autoantigen.

The aim of this study was to investigate the immunogenicity of four synthetic peptides, representing linear B cell epitopes of the human La/SSB autoantigen: 145-164 aa (p1), 289-308 aa (p2), 301-318 aa (p3) and 349-364 aa (p4), in rabbits. New Zealand White rabbits were immunized with each of the above peptides attached in four copies on tetrameric sequential oligopeptide carriers (SOC) in duplicate. Control immunizations were also performed (one rabbit each, immunized with Freud's complete adjuvant alone or with the SOC carrier alone). Animals were bled at regular intervals and sera were analysed for anti-La/SSB activity by ELISA assays using as antigen the various synthetic peptides, as well as the whole La/SSB protein. Four months after the last immunization, the animals were killed and peripheral blood mononuclear and spleen cells were co-cultured with either the peptides, the SOC carrier, or 27 peptides, covering the entire length of the human La/SSB molecule (23 amino acids long, overlapping by eight residues to each other). A specific, IgG, anti-peptide antibody response was detected, initially directed against the priming peptide, and subsequently expanded to the other La/SSB synthetic peptides. The antibody titres remained high, even 4 months after the last immunization. Sera from rabbits immunized with either p2 or p3 reacted also with the whole La/SSB protein, as was demonstrated by ELISA and immunoblot assays. No reactivities against either Ro60 or Ro52 autoantigen were found. Rabbit spleen cell reacted not only with the epitope used for the immunization but also with other La/SSB peptides. Immunization of rabbits with the major human La/SSB B cell antigenic determinants, linked to SOC carrier, induces strong and sustained antibody and T cell responses against multiple epitopes of the human La/SSB protein. Thus, La/SSB B cell linear epitopes are probably capable also of functioning as T cell epitopes, in this experimental animal.

The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods.

In a previous study it was shown that La/SSB contains four linear epitopes, p147-154, p291-302, p301-318 and p349-364. The aim of the present study was to investigate the value of the synthetic epitope analogues of the La/SSB autoantigen for the detection of antibodies to La/SSB, in comparison with recombinant La and fragments of this protein. A total of 122 sera with anti-La/SSB activity, from patients with primary Sjögren's syndrome (pSS) or systemic lupus erythematosus (SLE), were tested in various peptide-based assays. In addition, 62 sera from pSS or SLE patients with other autoantibody specificities and 95 sera from healthy individuals were used as controls. The autoantibody specificity was identified by counter immunoelectrophoresis and immunoblot. The peptide-based ELISA assays presented sensitivities ranging from 78% to 88-8% and specificities from 69% to 94-3%. Dot blot assays exhibited sensitivities ranging from 93-6% to 97%, but remarkably lower specificities from 56% to 88%. The most sensitive and specific peptide 349GSGKGKVQFQGKKTKF364 was synthesized and attached on a tetramer sequential oligopeptide carrier SOC4 and used for immunoassay development. Assays based on the recombinant native La protein, the La-C terminal (215 aa), and the N-terminal of La with a mutation at base pair 640 (nine adenines instead of eight) were also developed and compared with the SOC4 peptide-based assay. Of anti-La-positive sera, 88.1% were reactive with both the synthetic peptide SOC4-(349-364aa) and the recombinant La protein. Eighty-three percent of sera were reactive with the La N-terminus and 67.8% of sera were reactive with the La C-terminus. Using sera that were anti-Ro-positive but anti-La-negative, 37% were reactive with the recombinant protein, 26% with the La N-terminus, 33% with the La C-terminus and only 11 % with the synthetic peptide. Our results suggest that the synthetic peptide epitopes exhibit high sensitivity and specificity for the detection of anti-La/ SSB antibodies in ELISA and dot blot techniques. The peptide SOC4-(349-364aa) has the same sensitivity for the detection of anti-La/SSB antibodies as the recombinant protein.

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization.

The B cell epitope mapping of La/SSB was performed using 20mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145-164, 289-308, 301-320 and 349-368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes: 147HKAFKGSI154, 291NGNLQLRNKEVT302, 301VTWEVLEGEVEKEALKKI318 and 349GSGKGKVQFQGKKTKF364. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope 147HKAFKGSI154 presented 83.3% similarity with the 139HKGFKGVD146 region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope 147HKAFKGSI154 to 100% to epitope 349GSGKGKVQGKKTKF364. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope 147HKAFKGSI154 presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.