Quantitative analysis of myocardial blood flow in surgically revascularized and not revascularized myocardial segments. A pilot PET study.

PURPOSE: To prospectively compare changes in myocardial blood flow (MBF) and myocardial flow reserve (MFR) in multivessel coronary artery disease (MVCAD) patients undergoing incomplete revascularization (IR) versus complete revascularization (CR) by coronary artery bypass grafting (CABG). METHODS: Seven male patients (age 68 ± 9 years) with MVCAD underwent myocardial perfusion PET/CT with [13N]ammonia before and at least 4 months after CABG. Segmental resting and stress MBF as well as MFR were measured. Resting and during stress left ventricle ejection fraction (LVEF) were also calculated. RESULTS: Three patients (43%) underwent CR and four (57%) IR. Among 119 myocardial segments, 101 (85%) were revascularized, and 18 (15%) were not. After CABG, stress MBF (mL/min/gr) and MFR are significantly increased in all myocardial segments, with a greater increase in the revascularized segments (p = 0.013). In both groups, LVEF significantly decreased during stress at baseline PET (p = 0.04), but not after CABG. CONCLUSION: Stress MBF and MFR significantly improve after CABG in both revascularized and not directly revascularized myocardial segments. IR strategy may be considered in patients with high surgical risk for CR.

Global longitudinal strain for prediction of mortality in ST-segment elevation myocardial infarction and aortic stenosis patients: two sides of the same coin.

OBJECTIVE: Global longitudinal strain (GLS) predicts major adverse events in ST-segment elevation myocardial infarction (STEMI) and aortic stenosis (AS). Different cut-off values and different end-points have been proposed for prognostic stratification. We aimed to verify whether a single GLS cut-off value can be used to identify increased risk of all-cause death in STEMI and AS. PATIENTS AND METHODS: One-hundred- seventeen successfully treated first STEMI (age 63.8±12.5 yrs, 70% men) and 64 AS (age 80.3±6.9 yrs, 44% men) patients, undergoing echocardiography before discharge and before AS treatment, respectively, were retrospectively analyzed. GLS was analyzed, together with pulmonary artery systolic pressure (PASP), Killip class and Genereux stage. End-point was all-cause death at 6-month follow-up. RESULTS: All-cause death occurred in 4 (3.4%) STEMI and 5 (7.8%) AS patients (p=ns). AS patients who died had GLS similar to died STEMI patients (9.7±2.1 vs. 11.3±1.7, p=ns). GLS cut-off ≤12% predicted death with 89% sensitivity and 70% specificity (AUC 0.84, p=0.001): STEMI and AS patients with GLS ≤12% had worse survival than STEMI and AS patients with GLS >12% (log-rank p=0.001). At multivariate Cox regression analysis, lower GLS values independently predicted death (HR 0.667, 95% CI 0.451-0.986, p=0.042), and the prediction model was improved when GLS was added to old age, significant comorbidities, PASP and Killip/Genereux stage (χ2 6.691 vs. 1.364, p=0.010). CONCLUSIONS: Died patients with STEMI and AS show similar values of GLS. A unique cut-off value of GLS can reliably be used to stratify the risk of all-cause death at 6-month follow-up in both two clinical settings.

Coronary plaque instability assessed by positron emission tomography and optical coherence tomography.

BACKGROUND: Non-ST-elevation myocardial infarction (NSTEMI) and unstable angina (UA) are caused often by destabilization of non-flow limiting inflamed coronary artery plaques. 18F-fluorodeoxyglucose (FDG) uptake with positron emission tomography/computed tomography (PET/CT) reveals plaque inflammation, while intracoronary optical coherence tomography (OCT) reliably identifies morphological features of coronary instability, such as plaque rupture or erosion. We aimed to prospectively compare these two innovative biotechnologies in the characterization of coronary artery inflammation, which has never been attempted before. METHODS: OCT and FDG PET/CT were performed in 18 patients with single vessel coronary artery disease, treated by percutaneous coronary intervention (PCI) with stent implantation, divided into 2 groups: NSTEMI/UA (n = 10) and stable angina (n = 8) patients. RESULTS: Plaque rupture/erosion recurred more frequently [100% vs 25%, p = 0.001] and FDG uptake was greater [TBR median 1.50 vs 0.87, p = 0.004] in NSTEMI/UA than stable angina patients. FDG uptake resulted greater in patients with than without plaque rupture/erosion [1.2 (0.86-1.96) vs 0.87 (0.66-1.07), p = 0.013]. Among NSTEMI/UA patients, no significant difference in FDG uptake was found between ruptured and eroded plaques. The highest FDG uptake values were found in ruptured plaques, belonging to patients with NSTEMI/UA. OCT and PET/CT agreed in 72% of patients [p = 0.018]: 100% of patients with plaque rupture/erosion and increased FDG uptake had NSTEMI/UA. CONCLUSION: For the first time, we demonstrated that the correspondence between increased FDG uptake with PET/CT and morphology of coronary plaque instability at OCT is high.

Diagnostic role of echocardiography in patients admitted to the emergency room with suspect no-ST-segment elevation acute myocardial infarction.

OBJECTIVE: We investigated whether echocardiography may help identify, among patients admitted with a suspect of non-ST-segment elevation acute myocardial infarction (NSTEMI), those with athero-thrombotic coronary artery disease (CAD). PATIENTS AND METHODS: We studied consecutive patients admitted with a clinical suspect of first NSTEMI. Echocardiography was assessed within 24 hours from admission. Patients were divided into two groups, according to the results of coronary angiography: 1) patients with obstructive stenosis (≥ 50%) and/or images of thrombosis in one or more coronary arteries (CAD group); 2) patients with no evidence of obstructive coronary arteries (NOCAD group). RESULTS: Of 101 patients enrolled in the study, 53 (52.5%) showed obstructive CAD and 48 (47.5%) NOCAD. At echocardiographic examination, regional wall motion abnormalities were found in 52.8% of patients in the CAD group and 43.7% in the NOCAD group (p=0.43). Left ventricle ejection fraction was 56.4±6.8 vs. 54.7±9.8% (p=0.30) and wall motion score index was 1.16±0.26 vs. 1.21±0.32 (p=0.39) in the two groups, respectively. A multivariable logistic regression independent predictors of obstructive CAD included age, male gender, typical angina, diabetes and hypertension. CONCLUSIONS: Our data showed that, in patients with acute chest pain and increased serum troponin T concentration, routine standard echocardiography does not significantly improve the diagnostic accuracy for the presence of obstructive CAD.

Hypoallergenic variants of the Parietaria judaica major allergen Par j 1: a member of the non-specific lipid transfer protein plant family.

BACKGROUND: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. METHODS: Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. RESULTS: The disruption of Cys14-Cys29 and Cys30-Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4-Cys52 bridge (PjC mutant) and the latter Cys50-Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4-Cys52 and Cys50-Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. CONCLUSION: Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.

The IgE-binding epitopes of rPar j 2, a major allergen of Parietaria judaica pollen, are heterogeneously recognized among allergic subjects.

Pollen allergens are multivalent proteins that cross-link IgE antibodies on mast or basophil cells, inducing secretion of biologic mediators, and resulting in various allergic symptoms. The IgE-binding regions of the Parietaria judaica (Pj) pollen major allergen rPar j 2 were investigated. Twenty-nine single sera from Pj-allergic subjects were tested by Western blot against five recombinant peptides. At least four putative IgE-binding epitopes were identified. The analysis of their diffusion suggested a heterogeneous IgE-binding response. In fact, 75% of the sera reacted with peptide 1-54, 48% with peptide 48-101, 24% with peptide 1-30, 7% with peptide 29-54, and none with peptide 48-76. These five peptides were analyzed with the histamine-release assay. Only peptide 48-101 was capable of inducing degranulation and release of histamine. These results suggest that the recombinant rPar j 2 allergen contains IgE epitopes that are heterogeneously recognized by sensitive patients, and that therefore the therapeutic approach based on the use of haptenic peptides needs a careful evaluation.

Isolation and characterization of two cDNA clones coding for isoforms of the Parietaria judaica major allergen Par j 1.0101.

Two cDNA clones named P9* and P1* of 794 and 631 bp, respectively, were isolated from a lambda ZAP cDNA expression library using Parietaria judaica (Pj) pollen-specific IgE antibodies from a pool of sera (n = 23) of patients allergic to Pj. Sequence analysis showed open reading frames of 176 and 138 amino acids. Both clones contain a putative signal peptide giving two mature processed proteins named Par j 1.0102 of 14,726 D and Par j 1.0201 of 10,677 D. These proteins represent isoallergenic forms of the major Pj allergen Par j 1.0101 (clone P5) previously reported. The Par j 1.0102 shared 98% amino acid sequence homology with the P5, while the Par j 1.0201 shared 89% homology. Since P1, P5 and P9 clones were expressed in Escherichia coli, and since the three allergenic proteins shared a very high degree of sequence identity and comparable binding to the Pj-specific IgE, we decided to analyze in more detail the immunological properties of only one allergen, the recombinant Par j 1.0101. The allergenic activity determined by the histamine release assay ranged between 9 and 56%, depending on the allergic patient analyzed, while it blocked approximately 40% of all the Pj-specific IgE antibodies, as detected after ELISA and cross-absorption analysis.

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen.

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj-allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj-allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross-inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10-14 kDa native allergens and preincubation of sera from Pj-allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10-14 kDa native allergen region, suggesting that these two allergens contributed to the region.

Isolation of proteins and nucleic acids by electrophoresis on disposable gel columns.

A simple and cheap one-step method to isolate proteins or nucleic acids by electrophoresis in disposable gel columns is reported. A disposable syringe was modified to host a gel column and an elution chamber. Starting from a crude extract of E. coli, the laboratory-made devise allowed the isolation of the maltose binding protein (MBP) fused to a recombinant allergenic molecule with a molecular mass of 58 kDa, from a mixture of several proteins. Also, plasmid DNA could be isolated from a mixture containing chromosomal DNA and RNA, avoiding the use of organic solvents. Electrophoresis was performed at 150 V, 35 degrees C, pH 8.0 and 8.3 for protein and DNA, respectively. The protein or the DNA obtained showed a yield of 80% and a purity grade of 90%, as estimated by densitometry.

Influence of oral contraceptives on fasting gallbladder volume.

A total of 615 healthy fertile women (518 pill users and 97 non-users) were examined by real-time ultrasonography for fasting gallbladder volume, gallstones and biliary dysmorphism. None of the six examined combinations of oral contraceptives appeared to influence fasting gallbladder volume significantly. When fasting gallbladder volumes were reanalyzed according to the presence or absence of recognized biliary risk factors, significant modifications were detected, in both pill users and non-users. These changes related only to age and parity. Relative risks of cholelithiasis and biliary dysmorphism were not affected by contraceptive treatment.

PIP: In Italy, researchers used real-time ultrasonography for fasting (no solid food for 24 hours) gallbladder volume to compare data on 518 women using low-dose, combined oral contraceptives (OCs) for 12-120 months with data on 97 women who had never used OCs to examine the effect of OCs on the gallbladder. The 615 healthy, fertile 16-52 year old women were clients of Contraceptive Counseling Services of the Department of Obstetrics and Gynaecology at the University of Ferrara and the Local Sanitary Unit. No significant difference in fasting gallbladder volumes existed between cases and controls. OC use was not associated with fasting gallbladder volume in relation to duration of treatment, family cholelithiasis, smoking, or age. As parity increased so did fasting gallbladder volume in both groups (p = 0.04 for controls and p = 0.01 for cases). Fasting gallbladder volume increased with age in both groups but not significantly so. Biliary dysmorphism existed in 17.8% of OC users and 24.7% of controls. Cholelithiasis was present in 2.9% of OC users and 3.1% of controls. OC use did not affect the relative risk of biliary dysmorphism and cholelithiasis (0.66 and 0.95, respectively). In conclusion, low-dose OCs do not affect gallbladder motility.

Influence of spontaneous and surgical menopause on atherogenic metabolic risk.

We evaluated serum total-, LDL-, HDL-, cholesterol and triglycerides in 858 women. Sixty-seven were in premenopause, 307 in perimenopause, 326 in spontaneous menopause, and 158 women underwent hysterectomy. Of these, 101 had bilateral ovariectomy, 24 with unilateral ovariectomy and 33 with retention of both ovaries. After correcting for chronological age and body mass index by variance analysis, it was found that total-, LDL- and HDL-cholesterol significantly increased during menopause without changes in the total cholesterol/HDL-cholesterol, LDL-cholesterol/HDL-cholesterol and triglycerides/HDL-cholesterol ratio. The changes gradually occurred during the months preceding spontaneous cessation of menses. Atherogenic metabolic risk (as relative risk of high total and LDL-cholesterol, estimated by logistic regression analysis) significantly increased in all groups of women in reference to premenopause. The risk observed in surgical menopause with bilateral ovariectomy was higher than in spontaneous menopause, and conservation of ovaries in hysterectomized women seems to protect only partially against such an increase.

Purification of Parj I, a major allergen from Parietaria, judaica pollen.

A major allergen, the Parj I, was purified to homogeneity from Parietaria judaica pollen by means of ultrafiltration dialysis, preparative polyacrylamide gel chromatography and affinity chromatography through a column of Sepharose-monoclonal antibody specific for Parj I. The homogeneity of the Parj I was assessed by one single arc of immunoprecipitation both in cross immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis, by one single band of radiostaining after a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose and by one single peak after a size exclusion chromatography on high-performance liquid chromatography (HPLC). The homogeneity was further supported by crossed Laurell immunoelectrophoretic analysis, in that only one arc of precipitation was magnified in CIE after addition of the purified allergen. The purified Parj I allergen was capable of interacting in vitro with 70% of the human IgE specific for a crude P. judaica extract, as determined by radioallergosorbent test inhibition. The purified Parj I was capable of inducing positive reactions in vivo in skin prick tests, and of inducing release of histamine from blood containing basophils as determined by a histamine release assay. The amino acid analysis of the Parj I showed 118 amino acid residues per monomer analyzed and, among other residues, three methionine residues were detected. The molecular weight of the Parj I estimated by HPLC and amino acid composition was 26 kilodaltons.

Enzyme-linked immunosorbent assay for Brucella melitensis-associated antigens.

An ELISA assay was performed to detect antigens of Brucella melitensis directly in the blood of patients affected by Brucellosis. Disposable polystyrene microtiter plates were coated with rabbit immunoglobulins anti-Brucella melitensis antigens and then incubated with sera of Brucellosis patients and sera of not infected normal subjects as a control, to standardize the conditions of the different steps of the assay. The level of the blood-containing Brucella antigen bound to the plate was measured by addition of anti-Brucella melitensis antiserum conjugated with alkaline phosphatase followed by incubation with the specific enzyme substrate. Sera from 9 Brucellosis patients not undergoing therapy were tested by this ELISA assay, and all showed values significantly higher than the control. A pool of 96 sera from normal subjects not infected with Brucella melitensis was used as a negative control. In addition, a different group of 8 sera from patients with Brucellosis undergoing therapy were also analyzed, but no difference in the ELISA value was observed between the two groups with or without therapy. The ELISA assay described in this paper could be a reproducible, sensitive and suitable test to detect Brucella-antigens in the blood of Brucellosis patients and it could be used in addition to the more common methods for a more thorough diagnosis of Brucellosis.

Antigens of Euparipha pisana (snail). I. Identification of allergens by means of in vivo and in vitro analysis.

The data obtained in this study suggest that eating Euparipha pisana (snail), a common food in Mediterranean countries, could give serious allergic reaction such as asthma. We describe here the identification and partial characterization of allergenic molecules form this new source. An aqueous extract of snail was obtained by homogenization in distilled water, centrifugation, dialysis and defatting with ethyl ether. Skin prick test (SPT) performed with the snail extract on 70 subjects allergic to the more common allergens of the Mediterranean area gave a SPT positivity in 61% of the subjects tested, with a mean value of histamine-equivalent prick (HEP) equal to 0.81 +/- 0.25 (n = 43), while no SPT-snail-positive reactions were obtained by using the same extract on 30 not allergic subjects. To ascertain if such a sensitivity was IgE-mediated, sera from SPT-snail-positive subjects were analyzed by RAST, coupling the snail extract to polystyrene balls and to paper discs. 19% of the sera tested were RAST-positive, mean value of binding 4.8 +/- 2.8% (n = 13), while when using sera from SPT-snail-negative subjects, the RAST mean value was 0.49 +/- 0.18% (n = 27). Histamine release (HR) was also performed. Basophils prepared from SPT-snail-positive subjects were incubated with a snail extract. All of the SPT-snail-positive subjects gave a significant value of HR, mean value 21.8 +/- 7% using 1 micrograms of snail extract (n = 16), while 1.41 +/- 1.1% (n = 10) was the mean value obtained when SPT-snail-negative subjects were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical identification of antigens of Brucella melitensis by means of CRIE.

A crude extract of Brucella melitensis was obtained by sonication, centrifugation and dialysis, and analyzed by quantitative immunoelectrophoresis. CIE analysis, using a high titer purified rabbit antibody anti-brucella fraction, showed that the Brucella melitensis extract contained at least 22 antigens that all moved towards the anode. The antigens of the crude extract able to interact with IgG of patients affected by brucellosis, were identified by means of CRIE using sheep 125I-anti human IgG. Fifteen sera of patients affected by brucellosis were analyzed by CRIE and three (Ag6, Ag12, Ag15) of the 22 antigens were able to bind specific human IgG to their corresponding immunoprecipitates. Ag6 was the only one present after one day of the CRIE plate exposure. After treatment of brucellosis patients with antibiotics for two weeks, the three antigens were still able to bind the specific IgG, after two months of treatment the antigens were reduced to two (Ag6, Ag15), while after ten months only one antigen (Ag6) was still able to bind specific human IgG. The brucellosis titer determined with common methods gave normal values after ten months of therapy. These results suggest that Ag6 is the most important antigen specific for human IgG. A preliminary determination of the molecular weight range of these antigens was carried out by a combination of size exclusion chromatography and CIE analysis. The Kav values were all above 66 kD.

Identification of a DNA polymerase beta-like form in Drosophila melanogaster adult flies.

DNA polymerase beta is widely distributed in the eukariotes. So far, few examples are known in which a DNA polymerase alpha -like form alone is reported. Surprisingly, DNA polymerase beta was not detected in Drosophila embryos, while it is present in the cells of multicellular species from sponge to mammals. In view of the relevance of Drosophila as a model biological system for studying the role of the various DNA metabolism enzymes in vivo we have reinvestigated the presence of the DNA polymerase beta-like form in Drosophila adult flies. Here we report the occurrence in Drosophila melanogaster adult flies of a DNA polymerase activity that, for its NEM(1) resistance, template specificity, sensitivity to ddTTP, sedimentation coefficient and nuclear localization can be classified as a beta-like form.