Characterization of the autoimmune response against the nerve tissue S100ß in patients with type 1 diabetes.

Type 1 diabetes results from destruction of insulin-producing beta cells in pancreatic islets and is characterized by islet cell autoimmunity. Autoreactivity against non-beta cell-specific antigens has also been reported, including targeting of the calcium-binding protein S100ß. In preclinical models, reactivity of this type is a key component of the early development of insulitis. To examine the nature of this response in type 1 diabetes, we identified naturally processed and presented peptide epitopes derived from S100ß, determined their affinity for the human leucocyte antigen (HLA)-DRB1*04:01 molecule and studied T cell responses in patients, together with healthy donors. We found that S100ß reactivity, characterized by interferon (IFN)-γ secretion, is a characteristic of type 1 diabetes of varying duration. Our results confirm S100ß as a target of the cellular autoimmune response in type 1 diabetes with the identification of new peptide epitopes targeted during the development of the disease, and support the preclinical findings that autoreactivity against non-beta cell-specific autoantigens may have a role in type 1 diabetes pathogenesis.

Analysis of different routes of administration of heterologous 5-azacytidine-treated mesenchymal stem cells in a porcine model of myocardial infarction.

Stem cell therapy constitutes an exciting, powerful therapy to repair the heart. Nevertheless, there are numerous doubts about the best route of stem cell administration to achieve implantation into the injured myocardium. Development of a preclinical, large animal model may be useful to obtain a better approach to clinical situations. The aim of this work was to study the effectiveness of various routes of heterologous bone marrow mesenchymal stem cell (MSCs) administration in a porcine model of myocardial infarction. MSC treated with 5-azacytidine were stained with a fluorescent compound (DiO) before their administration to previously infarcted pigs via 3 routes: intracoronary (IC), intramyocardial (IM), or endocardial (EC; n = 5 each group). Healthy, noninfarcted animals were used as a control group. At 30 days after delivery, hearts were divided into 12 parts: infarcted zone (1-6), right-left atria, interatrial and interventricular septa, and right-left ventricles. In each zone we looked for and quantified, injected fluorescence-stained cells. In the animals in which presence of DiO-stained cells was detected, cells were located preferentially in the infarcted zone and not in the atria, ventricles, or septa. Comparing various administration routes, the mean number of engrafted cells within the infarct zone was significantly greater after IC infusion than either IM or EC injection. Fluorescent cells were not observed in healthy zones of the myocardium or in healthy animals.

Comparison of gene expression profiles in a porcine infarct model after intracoronary, transthoracic, or transendocardiac injection of heterologous bone marrow mesenchymal cells.

An in vivo porcine model of myocardial infarction was developed with the aim of comparing the effectiveness for cardiac repair of intracoronary, transthoracic, or transendocardial delivery strategies for bone marrow mesenchymal stem cells (BMMSC) using an analysis of expression levels of transcripts related to various cellular processes at 8 heart regions using quantitative reverse transcriptase polymerase chain reaction. We observed significant rises in cardiomyogenic markers Mef2C, Gata4 and Nkx2.5, and contractibility marker Serca2A at infarcted regions for cell-treated pigs. We also observed differences in Sdf1 expression related to the organ stress response between delivery strategies. Unexpectedly, increased expression of Col1A1 was detected in 2 cell-treated groups at various heart regions. Our results suggest improvements in both contractility and cardiomyogenic capability of damaged tissue after BMMSC injection, but also warned us about the relevance of the chosen delivery strategy and potential undesired effects like increasing fibrosis after treatment.

Late steroid withdrawal after heart transplantation and incidence of acute rejection.

BACKGROUND: Steroid withdrawal (SW) after heart transplantation (HT) reduces steroid-associated side effects, although it can increase acute rejection episodes (ARE). Patient selection criteria for SW and the time elapsed after HT for this maneuver are controversial issues. The objective of this study was to assess the safety of late SW after HT with regard to the occurrence of ARE and to analyze risk factors resulting in a poor evolution. METHODS: We studied a cohort of 24 patients who underwent SW late after HT. All of them had gone at least 4 years without any ARE. Independent variables were time after HT, general recipient and donor data, risk factors for ARE, and immunosuppression. The dependent variables were occurrence of ARE (proven or not proven with endomyocardial biopsy) and time and severity of ARE. RESULTS: Among 24 HT patients including 96% men with an overall mean age of 57 years who underwent SW, the mean follow-up was 2.32 +/- 0.86 years. Six patients (25%) displayed an ARE >or=2R according to the International Society for Heart and Lung Transplantation (ISHLT) at 5 +/- 3 months after SW. There were no deaths. Time from the last rejection episode to SW was 6.6 +/- 2 years. All ARE were treated with steroid boluses (mean total dose 1583 +/- 1044 mg). Among the HT patients with ARE, 5 (85%) had never experienced ARE after HT. Upon long-term follow-up, there were 2 deaths: 1 sudden death at 30 months after SW and 1 due to allograft vasculopathy at 20 months post-SW. Currently 92% are New York Heart Association (NYHA) functional class I with a mean left ventricular ejection fraction of 67% +/- 10%. CONCLUSIONS: In our series of HT with late SW after HT (even among an HT population with a low risk of rejection), there was a 25% rate of ARE. This study did not allow us to identify risk factors for ARE after SW. We believe that based upon these observations SW should be implemented with caution.

Long-term results of heart transplant in recipients older and younger than 65 years: a comparative study of mortality, rejections, and neoplasia in a cohort of 445 patients.

BACKGROUND: Whether being older than 65 years should be considered an absolute counterindication to heart transplant (HT), as it is in some centers, is controversial. In our centre, patients older than 65 years are accepted for HT if they satisfy stringent conditions. The aim of this study was to examine whether heart recipients older than 65 years have a greater risk of rejection, neoplasia, or mortality than younger ones. METHODS: We studied 445 patients who underwent HT between April 1991 and December 2003, 42 of whom were older than 65 years and 403 who were 65 years or younger. The parameters evaluated were the cumulative incidences of neoplasias and rejections (ISHLT grade > or = 3A), and the survival rates 1 month, 1 year, and 5 years post-HT. RESULTS: The two groups had similar percentages of patients with at least one rejection episode (< or =65 years 56.9%, >65 years 51.3%; P > .05), and although there were proportionally almost twice as many tumors in the older group (14.2%) as in the younger (7.9%), this difference was not statistically significant either. Nor were there any significant differences in survival, the 1-month, 1-year, and 5-year rates being 87.8%, 82.1%, and 68.8%, respectively, in the younger group and 85.7%, 78.6%, and 73.4%, respectively, in the older. CONCLUSIONS: Among carefully selected patients aged more than 65 years, HT can be performed without incurring greater risk of rejection, malignancy, or death than is found among recipients younger than 65 years.

3 beta hydroxysteroid dehydrogenase autoantibodies in patients with idiopathic premature ovarian failure target N- and C-terminal epitopes.

The steroid cell enzyme 3 beta hydroxysteroid dehydrogenase (3 beta HSD) has been identified as a target of steroid cell autoantibodies, and autoantibodies to this enzyme are present in patients with premature ovarian failure and patients with autoimmune polyendocrine syndrome 1. The aim of the present study was to develop a radioligand binding assay for 3 beta HSD autoantibodies and to exploit this to examine regions of the molecule targeted by autoantibodies. We generated a construct of 3 beta HSD coupled to a luciferase fusion partner in order to maximize the yield of (35)S-radiolabeled protein. Labeled 3 beta HSD was then immunoprecipitated and the autoantibodies quantified by phosphoimaging. Autoantibodies to 3 beta HSD were detected in 12 of 100 (12%) idiopathic premature ovarian failure patients and 0 of 103 (0%) healthy age-matched controls (P < 0.0001). Three overlapping fragments of 3 beta HSD cDNA were cloned downstream of luciferase to examine autoantibody binding sites. Two of nine sera with 3 beta HSD autoantibodies (22%) displayed reactivity to the N terminus of 3 beta HSD, and seven (77%) showed reactivity to the C terminal; no sera reacted with the middle region. Our study demonstrates a markedly enhanced disease specificity of autoantibodies to 3 beta HSD detected using this novel assay and shows that distinct regions of the molecule are targeted.

T cell activation by coxsackievirus B4 antigens in type 1 diabetes mellitus: evidence for selective TCR Vbeta usage without superantigenic activity.

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.

Comparison of two-dimensional echocardiography and pulsed Doppler tissue imaging during dobutamine-atropine stress testing to detect coronary artery disease.

In order to compare the diagnostic accuracy of two-dimensional (2-D) echocardiography and pulsed Doppler tissue imaging (pDTI) during dobutamine-atropine stress testing (DAST) to detect significant coronary lesions, 41 patients underwent DAST (up to 40 microg/k/min of dobutamine with additional atropine during submaximal heart rate responses) and coronary angiography. Pulsed Doppler tissue sampling of territories corresponding to the left anterior descending (LAD), left circumflex (LCx), and right coronary arteries (RCAs) were performed in the apical four-chamber plus aorta and two-chamber apical views. The measurements were repeated at rest, at low dose (10 microg/k/min), and at peak stress. Pulsed DTI measurements included peak early systolic (Vs), peak early diastolic (Ve), and peak late diastolic (Va) velocities. Harmonic 2-D echocardiography was recorded at rest, low dose, peak stress, and recovery, and compared with pDTI assessment. Positive 2-D echocardiography was considered as infarction or ischemic response. The results were evaluated for the prediction of significant coronary stenosis (50% luminal narrowing). Feasibility of pDTI was 100%, 95%, and 98% for the LAD, the LCx, and RCA territories, respectively. At rest, Vs in territories supplied by arteries with coronary artery disease (CAD) (6.3 +/- 2.0 cm/sec) was not different from those without (6.6 +/- 2.1 cm/sec). Vs increased less in territories supplied by arteries with than without CAD (75 +/- 107% vs 102 +/- 69%, P = NS). Ve was lower in territories with CAD at rest (6.0 +/- 2.1 cm/sec vs 8.2 +/- 3.4 cm/sec, P < 0.0001) and low dose (7.2 +/- 2.1 cm/sec vs 8.8 +/- 3.6 cm/sec, P < 0.01), but similar at peak stress (7.6 +/- 3.5 cm/sec vs 8.1 +/- 3.3 cm/sec). Ve increase was similar in territories with (36 +/- 74%) than without CAD (15 +/- 6 4%). Va was similar at rest and low dose in territories with and without CAD (9.2 +/- 2.7 cm/sec vs 9.1 +/- 2.3 cm/sec and 10.9 +/- 3.1 vs 10.3 +/- 3.6 cm/sec, respectively), but lower at peak stress in territories with CAD (13.3 +/- 4.6 cm/sec vs 15.3 +/- 4.5 cm/sec, P = 0.05). The Va increase was lower in territories with CAD (43 +/- 37% vs 77 +/- 72%, P < 0.05). In a territory-based analysis, a failure to achieve Vs > or =10.5 cm/sec at peak stress in the LAD and LCx, and > or =10.0 cm/sec in the RCA territory, was found to be the more accurate limit to detect CAD in the corresponding arteries: sensitivity (95% confidence intervals): 63% (55-71), P = NS vs 2-D echocardiography: 59% (51-67); specificity 76% (68-84), P < 0.01 vs. 2-D echocardiography: 95% (89-100); and accuracy 69% (63-75), P = NS vs 2-D echocardiography: 76% (70-82). Thus, pDTI is feasible during DAST but not more accurate than 2-D echocardiography for the detection of significant CAD in a territory-based study.

Ion interaction reagent reversed-phase high-performance liquid chromatography determination of anti-tuberculosis drugs and metabolites in biological fluids.

New methods of ion interaction reagent (IIR) RP-HPLC are presented for the determination of anti-tuberculosis drugs and their metabolites, singly or in multi-component mixtures, in biological fluids. The following analytes are considered: isoniazid, ethionamide, pyrazinamide, morphazinamide, p-aminosalicylic acid, nicotinic and isonicotinic acids. Aqueous solutions of three different ion interaction reagents are alternatively or comparatively used as the mobile phases, namely: (A) 5.00 mM octylamine at pH 3.00 for o-phosphoric acid, (B) 5.00 mM octylamine at pH 8.00 for o-phosphoric acid, and (C) 5.00 mM 1,6 diaminohexane at pH 6.00 for o-phosphoric acid. The response linearity between peak area and analyte concentration is verified for all the analytes in the concentration range within the determination limits and 2.00 mg/l. Detection limits are always lower than 82 microg/l for standard solutions; in the analysis of samples of rat serum, rat plasma and human serum, the matrix effect is negligible, the detection limits are always lower than 94 microg/l and the average recovery yield is always greater than 96%.

T-Cell reactivity to the P2C nonstructural protein of a diabetogenic strain of coxsackievirus B4.

Enteroviruses are proposed as initiating factors in the etiology of Type 1 diabetes mellitus (Type 1 DM). Molecular mimicry between the autoantigen glutamic acid decarboxylase 65 (GAD65) and the coxsackievirus B4 (CVB4) nonstructural protein P2C is frequently cited as a mechanism by which this virus triggers the disease, but little is known about the immunogenicity of this viral protein in humans, mainly due to the problem of obtaining highly pure preparations of P2C. We generated large amounts of highly pure, soluble P2C protein, coupled to the fusion partner maltose binding protein (MBP-P2C) using the PMAL-c2 bacterial expression plasmid and a two-step purification system comprising amylose resin and ion exchange. Using purified viral protein we show that specific T-cell responses against P2C are detected in the blood of healthy donors and Type 1 DM patients. Proliferation responses to P2C were detected only in subjects also demonstrating T-cell proliferation to CVB4 Vero cell lysates. However, in additional cases T-cell responses to P2C were detectable through the release of interferon-gamma or interleukin-4 in individuals who did not make proliferative responses. Taken together, our data show that the P2C nonstructural protein of CVB4 is targeted by T cells during the antiviral immune response and may trigger the production of T helper 1 and T helper 2 cytokines. The availability of pure, immunogenic P2C should allow the putative role of antiviral responses in the development of autoimmune diabetes to be investigated.

Effect of oral aminophylline in patients with angina and normal coronary arteriograms (cardiac syndrome X).

BACKGROUND: Patients with syndrome X (exertional angina, positive exercise test, normal coronary arteriogram) have an altered perception of cardiac pain. This symptom may arise from increased sensitivity to adenosine. Previous studies suggest that intravenous aminophylline (an adenosine receptor blocker) improves exercise tolerance in patients with this disorder. OBJECTIVE: To examine the efficacy of oral aminophylline in syndrome X. METHODS: 13 patients (11 women and two men, mean (SD) 54 (6) years) with syndrome X were studied. Patients were randomised in a double blind crossover study to receive either oral aminophylline or placebo for three weeks. All patients underwent symptom limited exercise testing and ambulatory electrocardiography at the end of each three week period. RESULTS: 10 patients completed the study. The time to angina during exercise testing in patients who were given aminophylline was longer than for the placebo group (mean (SD) 632 (202) seconds v 522 (264) seconds, P = 0.004). Peak exercise ST depression did not differ significantly between patients who received aminophylline and those administered placebo (mean (SD) -1.9 (0.7) mm v -1.5 (0.8) mm). Six patients taking aminophylline reported a reduction in the total number of episodes of chest pain during the three weeks, but the frequency and duration of ST segment depression during Holter monitoring was unchanged. CONCLUSION: Oral aminophylline has a favourable effect on exercise induced chest pain threshold in patients with syndrome X. The disparate effects on symptoms and ST segment changes are intriguing and further study is warranted.

Differences in heart transplant physiology according to surgical technique.

A new cardiac transplantation technique that preserves the shape of the left atrium and leaves the right atrium intact has been introduced. To compare the new and the standard techniques, we studied cardiac physiology with Doppler echocardiography and catheterization in 26 patients who underwent operation with the standard technique (group A) and I1 who underwent operation with the new technique (group B). Right atrial dimensions were significantly lower in group B (right atrial area index 8.4 +/- 1.5 vs 14.5 +/- 1.9 cm2/m2, p < 0.001), whereas left atrial dimensions were slightly lower (left atrial area index 10.8 +/- 2.0 vs 16.4 +/- 7.0 cm2/m2, p = 0.07). Right atrial contraction, as reflected by peak late tricuspid velocity, was greater in group B (37 +/- 15 vs 30 +/- 10 cm/sec, p < 0.05). The subsequent systolic vena caval flow-velocity integral was also greater in group B at all respiratory phases (inspiration 10.0 +/- 4.0 vs 5.2 +/- 4.0 cm, p < 0.001; expiration 4.8 +/- 1.9 vs 2.9 +/- 1.4 cm, p < 0.001; apnea 5.3 +/- 2.0 vs 2.9 +/- 1.9 cm, p < 0.001) suggesting better atrial relaxation. Filling pressures on the right side of the heart were lower in group B (mean right atrial pressure 5.5 +/- 2.4 vs 6.6 +/- 2.8 mm Hg, p = 0.1; right atrial A wave 6.0 +/- 3.1 vs 8.3 +/- 3.2 mm Hg, p < 0.01; right atrial V wave 6.8 +/- 3.1 vs 9.2 +/- 3.2 mm Hg, p < 0.01; right ventricular end-diastolic pressure 5.6 +/- 3.2 vs 7.3 +/- 2.9 mm Hg, p < 0.05); however, no significant differences were found in left ventricular end-diastolic pressure or cardiac index. We conclude that patients undergoing the new technique exhibit cardiac physiologic improvements. Follow-up study is indicated to ascertain whether this finding implies improved long-term prognosis.

Unreliability of echocardiographic and Doppler indexes in the diagnosis of heart transplant acute rejection.

OBJECTIVE: To investigate the value of Doppler echocardiography to diagnose heart transplant acute rejection in both patients submitted to the standard surgical technique (Group A), and to a new technique that preserves the size of the left atrium and leaves the right atrium intact (Group B). METHODS: 122 Doppler echocardiographic studies and endomyocardial biopsies were performed on 27 group A and 11 group B patients. Systolic measurements included left ventricular shortening fraction, left ventricular endsystolic stress and tricuspid ring systolic displacement. Diastolic indexes investigated were left and right ventricle filling and superior vena caval flow parameters. RESULTS: As expected, right atrium was smaller in group B(p < 0.01). In group A, peak early to late mitral flow velocity increased, as did the rejection grade; whereas in group B it decreased. Pericardial effusion was seen more frequently in group B rejectors (> or = 3) than non-rejectors (63% vs 27%, p < 0.01). Right heart pressures (right ventricular end-diastolic and right atrial mean) were slightly higher for group A and B rejector patients (p < 0.05 when comparing right ventricular end-diastolic pressure in rejector to non-rejector group B patients). Left ventricular isovolumic relaxation time was reduced during subsequent rejection episodes in the same patient, but sensitivity of 15% left ventricular isovolumetric relaxation time reduction for rejection > or = 3 diagnosis was only 22% with a specificity of 73%. No significant intrapatient changes were found in other Doppler-derived systolic or diastolic indexes. CONCLUSION: Doppler echocardiography does not diagnose heart transplant acute rejection with enough reliability to avoid endomyocardial biopsies.

Plasma immunoreactive endothelin concentration correlates with severity of coronary artery disease in patients with stable angina pectoris and normal ventricular function.

OBJECTIVES: The present study tested the hypothesis that plasma immunoreactive endothelin concentration correlates with the severity and extent of coronary atherosclerosis. BACKGROUND: Plasma endothelin-1 concentration is increased in patients with unstable coronary syndromes and advanced atherosclerosis. This finding, together with other clinicopathologic observations, suggests that endothelins may participate in the atherogenic process. However, the relation between plasma immunoreactive endothelin and coronary artery disease in patients with stable angina pectoris remains controversial. METHODS: Ninety consecutive patients undergoing coronary angiography for the investigation of exertional chest pain and 49 normal control subjects were prospectively studied. Eleven patients had normal coronary angiographic findings (group I), 65 had coronary artery stenoses (group II), and 14 had coronary artery disease plus symptoms indicating atheroma in other vascular territories (group III). Computerized angiography was used to determine the extent, severity and morphology of coronary stenoses. Plasma immunoreactive endothelin was measured by radioimmunoassay. RESULTS: Mean (+/- SD) plasma endothelin concentration (pg/ml) was significantly higher in patients than in control subjects (7.29 +/- 4.07 vs. 3.48 +/- 1.29, p < 0.0001). Endothelin levels were higher in patients of group III than in those of groups II and I (9.43 +/- 5.48, 7.20 +/- 3.72 and 4.94 +/- 2.89, respectively, p = 0.02). In patients of group II, plasma endothelin correlated with the maximal degree of stenosis in each patient (r = 0.25, p = 0.04) and with the number of stenoses with > or = 70% diameter narrowing (r = 0.36, p = 0.002). The highest plasma endothelin levels were found in patients with total occlusions (8.65 +/- 3.78 vs. 6.46 +/- 3.51 p = 0.02). CONCLUSIONS: Plasma immunoreactive endothelin concentration is increased in patients with chronic stable angina. The higher levels occur in patients with severe stenoses and total coronary occlusion.

Angiotensin-converting enzyme insertion/deletion polymorphism and restenosis after coronary angioplasty in unstable angina pectoris.

We studied the relation between angiotensin-converting enzyme insertion/deletion gene polymorphism and restenosis in Caucasian patients who underwent coronary angioplasty for management of unstable angina pectoris. Our results indicate that, in contrast to previous reports in Japanese patients, no association exists between angiotensin-converting enzyme gene polymorphism and the development of restenosis in Caucasian patients with acute coronary syndromes.)

Activation of endothelial guanylate cyclase inhibits cellular reactivity.

The study shows that endothelial cells from human umbilical veins have a soluble guanylate cyclase which can be activated by sodium nitroprusside (SNP), SIN-1 (3-morpholinosydnonimine) and S35b (4-methyl-3-phenylsulfonylfuroxan). Cells which were pretreated with these compounds showed an inhibition of thrombin-induced arachidonic acid release, PGI2 formation, PAF synthesis and PMNL adhesion. Endothelial guanylate cyclase can also be activated by nitric oxide (NO) which is generated in endothelial cells upon stimulation with thrombin or ionomycin. It is suggested that endogenously produced NO might control cell activation and endothelial function through a cGMP-dependent mechanism.

[The relationship between clinical events and the angiographic lesions in coronary atherosclerosis. Hypolipemic treatment and plaque stabilization]. / Relación entre eventos clínicos y lesiones angiográficas en la aterosclerosis coronaria. Tratamiento hipolipemiante y estabilización de placa.

Secondary prevention studies have shown that lipid-lowering therapy improve angiographic outcome and reduce mortality and incidence of ischemic clinical events in patients with coronary artery disease. The mechanism responsible for the improvement in prognosis seem to be subtle as this improvement cannot be explained by changes in the angiographic diameter of coronary arteries. Atherosclerotic plaque rupture and subsequent thrombosis are the central features in the pathogenesis of acute coronary events: unstable angina, myocardial infarction and sudden death. Cholesterol lowering might decrease the risk of plaque rupture and its thrombogenicity, as well as normalise the impaired endothelial function in hypercholesterolemic patients. In this report, the effects of lipid-lowering therapy on angiographic outcome and incidence of clinical events, in patients suffering from coronary artery disease, are reviewed, and the mechanisms that might explain these findings are discussed.