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Background Takotsubo cardiomyopathy (TTC) is an acute, life-threatening condition which is typically induced by stress and manifested by chest pain and ECG changes. The TTC prevalence among patients (pts) with acute coronary syndrome is 1.7–2.2%. The mortality rate from TTC is up to 8%. TTC is clinically indistinguishable from acute myocardial infarction (AIM). Differential diagnosis of TTC and AIM remains an unresolved problem. Recent studies have shown the differences in profiles of plasma microRNAs inTTC and AIM. Purpose To evaluate the possibility of differentiating TTC and AIM using a PCR-based semi-quantitative analysis of mRNAs, the plasma levels of which had been shown to be increased in patients with AIM (miR-1, miR-208a, miR-133a, miR-499a) and TTC (miR-16, miR-26a). Methods Plasma from 38 pts was used: 13 pts with confirmed TTC (12 women, 1 man), 25 pts with AIM (9w, 16m). For 10 pts with AIM, blood was collected twice: at 6 and 24 hours after the heart attack. The control arm comprised 40 healthy people from the same age group (12w, 28m). Plasma was obtained using the Cell-Free DNA blood collection tubes. Nucleic acids were separated using a modified Boom method. A semi-quantitative assessment of the mRNA levels was performed using dCq method with stem-loop qRT-PCR. Normalization for spiked synthetic cel-miR-39 and endogenous miR-23a was performed. Control of hemolysis was performed by measuring the ratio of miR-451 (specific for RBCs) and miR-23a (absent from RBCs). The statistical significance of differences between mRNA levels was assessed using Mann–Whitney test. Results No significant differences in the levels of miR-16, miR-26a and miR-208a in TTC group and control group have been found. Pts with AIM significantly differed from pts with TTC (p=0.0038 and 0.0002, respectively) and control pts (p=3.1x10–9 and 2,66x10–10) with the increased levels of cardiac-specific miR-1 and miR-133a. As compared to plasma levels at 6 hours after the heart attack, at 24-hour point the levels of these mRNAs were markedly reduced in 9 of 10 pts (mean reduction was 22.3-fold for miR-133a and 7.5-fold for miR-1). The correlation between changes in the levels of these mRNAs was high (Spearman correlation coefficient=0.89). Significant differences in plasma levels of miR-133a between pts with AIM and TTC were maintained even if blood was collected after 24 hours (p=0.007). For miR-16 and miR-26a, no significant differences between pts with AIM and TTC were found. The results of analysis of these mRNAs are affected to a substantial degree by the residual hemolysis due to their high content in blood cells. Conclusions It was shown that measuring the plasma levels of mRNAs miR-1 and miR-133a allows to distinguish TTC from AIM by excluding the diagnosis of TTC. The differential diagnosis is possible only within several hours after acute clinical symptoms and requires proper normalization and full compliance with the technical specifications.

Antimyosin Fab has been modified to carry highly negatively charged synthetic polymers containing DTPAs (DTPA-PL) as chelating agents, of starting molecular weights 3.3 and 17 kD. The immunoreactivities of the modified antibodies were unaffected by the modification procedure. The isoelectric points (PI) of unmodified antimyosin (AM) Fab (PI range 7-9, Mr = 52kD) were changed to PIs predominantly between 4 and 5 (Mr = 59 kD for DTPA-PL3.3kD-AM-Fab and 67kD for DTPA-PL17kD-AM-Fab). These AM-Fab preparations were tested for specific target localization and visualization in vivo in an experimental canine model of acute myocardial infarction. The charge-modified 111In-labeled AM-Fab preparations showed enhanced target (necrotic myocardium) visualization within 30 min of intravenous infusion and decreased background activity in normal myocardium (mean %ID/g +/- s.e.m., 0.0076 +/- 0.0006, n = 164, and 0.0056 +/- 0.0004, n = 92, for 111In-DTPA-PL3.3kD- and DTPA-PL17kD-AM-Fab respectively) relative to conventional 111In-DTPA-AM-Fab (0.0263 +/- 0.0037, n = 135) (p less than 0.001) or radioiodinated AM-Fab (0.0098 +/- 0.0006, n = 256) (p less than or equal to 0.001). Furthermore, the concentration of negatively charged 111In-labeled antimyosin Fab decreased in non-target organs such as the liver and kidneys. In diagnostic and therapeutic applications, charge-modified macromolecules may improve target localization and reduce non-target organ activity.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without any change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.

A murine monoclonal antibody against human angiotensin-converting enzyme was radiolabeled with 111In via diethylenetriaminepentaacetic acid without substantial loss of antigen-binding capacity. This monoclonal antibody designated 9B9 cross-reacted with rat and monkey angiotensin-converting enzyme. Indium-111-labeled 9B9 selectively accumulated 10-20 times greater in the lung than in blood or other organs following intravenous administration in rats. Kinetics of lung accumulation and blood clearance were studied for 111In-9B9-antibody and compared to that of 125I-labeled 9B9 in rat. Highly specific accumulation of 111In-9B9-antibody in the lung of Macaca Rhesus monkeys after intravenous injection was monitored by gamma-imaging. Images of 111In-labeled antibody 9B9 biodistribution in monkey lung noticeably differ from the images of biodistribution of 99mTc-labeled albumin microspheres. This difference may provide information concerning the state of the endothelium of lung capillaries, which is different from the blood flow characteristics determined with routine microsphere technique.

Methods of rapid blood clearance of 111In-labeled mouse monoclonal antibody 9B9 against angiotensin-converting enzyme were studied. Indium-111-9B9 is specifically accumulated in rat lung, but its blood clearance is relatively slow and target-to-blood radioactivity ratio/g tissue (localization ratio) increases from 11 to 30 only 48 hr postinjection. Injection of second (anti-mouse immunoglobulin) antibodies results in slight (1.8-fold) increase of 9B9 localization ratio. Chemical modification of 9B9 aminogroups with lactose results in enhanced liver uptake and rapid blood clearance of antibody. Blood radioactivity level decreases tenfold, and as a result localization ratio increases threefold (up to 38 in 30 min). Injection of avidin following the injection of biotinylated 9B9 results in rapid clearance of blood radioactivity with increased uptake in liver and spleen. Lung uptake is not changed. Localization ratio increases fivefold over the avidin-untreated animal value. Implications of these approaches for various applications in immunoimaging are discussed.