Immunization with plasmids encoding M2 acetylcholine muscarinic receptor epitopes impairs cardiac function in mice and induces autophagy in the myocardium

Abstract

Autoantibodies against the M2 subtype of muscarinic acetylcholine receptors with functional activities have been found in the sera of patients with dilated cardiomyopathy (DCM), and the second extracellular loop has been established as the predominant epitope. However, it has been shown that the third intracellular loop is recognized by Chagas disease patients with severe cardiac dysfunction. In this work, BALB/c mice were immunized with plasmids encoding these two epitopes, and a control group received the empty plasmid (pcDNA3 vector). Serum from these DNA-immunized animals had elevated and persistent titres of antibodies against respective antigens. Heart echocardiography indicated diminished left ventricular wall thickness and reduced ejection fraction for both epitope-immunized groups, and ergospirometry tests showed a significant decrease in the exercise time and oxygen consumption. Transfer of serum from these immunized mice into naïve recipients induced the same alterations in cardiac structure and function. Furthermore, electron microscopy analysis of donor-immunized animals revealed several ultrastructural alterations suggestive of autophagy and mitophagy, suggesting novel roles for these autoantibodies. Overall, greater functional and structural impairment was observed in the donor and recipient epitope groups, implicating the third intracellular loop epitope in the pathological effects for the first-time. Therefore, the corresponding peptides could be useful for autoimmune DCM diagnosis and targeted therapy.     

The natural history of a genetic subtype of arrhythmogenic right ventricular cardiomyopathy caused by a p.S358L mutation in TMEM43

To determine the phenotype and natural history of a founder genetic subtype of autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVC) caused by a p.S358L mutation in TMEM43. The age of onset of cardiac symptoms, clinical events and test abnormalities were studied in 412 subjects (258 affected and 154 unaffected), all of which occurred in affected males significantly earlier and more often than unaffected males. Affected males were hospitalized four times more often than affected females (p ≤ 0.0001) and died younger (p ≤ 0.001). The temporal sequence from symptoms onset to death was prolonged in affected females by 1–2 decades. The most prevalent electrocardiogram (ECG) manifestation was poor R wave progression (PRWP), with affected males twice as likely to develop PRWP as affected females (p ≤ 0.05). Left ventricular enlargement (LVE) occurred in 43% of affected subjects, with 11% fulfilling criteria for dilated cardiomyopathy. Ventricular ectopy on Holter monitor was common and occurred early: the most diagnostically useful clinical test. No symptom or test could rule out diagnosis. This ARVC subtype is a sex‐influenced lethal arrhythmogenic cardiomyopathy, with a unique ECG finding, LV dilatation, heart failure and early death, where molecular pre‐symptomatic diagnosis has the greatest clinical utility.     

A Monte Carlo approach for improving transient dopamine release detection sensitivity

Current methods using a single PET scan to detect voxel-level transient dopamine release—using F-test (significance) and cluster size thresholding—have limited detection sensitivity for clusters of release small in size and/or having low release levels. Specifically, simulations show that voxels with release near the peripheries of such clusters are often rejected—becoming false negatives and ultimately distorting the F-distribution of rejected voxels. We suggest a Monte Carlo method that incorporates these two observations into a cost function, allowing erroneously rejected voxels to be accepted under specified criteria. In simulations, the proposed method improves detection sensitivity by up to 50% while preserving the cluster size threshold, or up to 180% when optimizing for sensitivity. A further parametric-based voxelwise thresholding is then suggested to better estimate the release dynamics in detected clusters. We apply the Monte Carlo method to a pilot scan from a human gambling study, where additional parametrically unique clusters are detected as compared to the current best methods—results consistent with our simulations.     

p64 flow diverter: Results in 108 patients from a single center

Background and purposeFlow diverters are increasingly used to treat intracranial aneurysms. We report the safety and efficacy of the p64 flow diverter, a resheathable and detachable device for intracranial aneurysms.Materials and methodsWe retrospectively reviewed 108 patients with 109 aneurysms treated with the p64 between March 2014 and July 2019. There were 87 women and 21 men, mean age 57 years. Of 109 aneurysms, 74 were discovered incidentally, 12 were symptomatic, 18 were previously treated, and five were ruptured dissection aneurysms. A total of 10 aneurysms were located in the posterior circulation. The mean aneurysm or remnant size was 8.1 mm.ResultsHemorrhage by perforation with the distal guidewire occurred in two patients with permanent neurological deficits in one. In one patient, acute in-stent occlusion caused infarction with a permanent deficit. Permanent morbidity was 1.9% (2 of 108, 95%CI 0.1–6.9%); there was no mortality. During follow-up, three in-stent occlusions occurred, all asymptomatic. There were no delayed hemorrhagic complications. At six months, 77 of 96 aneurysms (80.2%) were completely occluded, and at last follow-up, this increased to 93 of 96 aneurysms (96.9%). In-stent stenosis at any degree occurred in 11 patients, progressing to asymptomatic complete occlusion in one. In the other patients, stenosis resolved or improved at further follow-up.ConclusionThe p64 offers an effective and safe treatment option. Aneurysm occlusion rate was 97% at last follow-up, mostly achieved with a single device. There were no delayed hemorrhagic complications. Delayed in-stent stenosis infrequently progresses to occlusion but remains a matter of concern.     

Activated natural killer cells and interleukin-2 promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice

Purified populations of natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID). SCID spleen cells were cultured and activated with recombinant human interleukin-2 (rhIL- 2) in vitro. The activated NK cells were then transferred with syngeneic BALB/c bone marrow cells (BMC) and rhIL-2 into lethally irradiated syngeneic recipients to determine their effect on long-term hematopoietic reconstitution. On analysis, the transfer of rhIL-2- activated NK cells along with BMC resulted in significant increases in splenic and BM hematopoietic progenitor cells when compared with those for mice not receiving NK cells. Histologic and flow cytometric analysis showed a marked increase in granulocytic and megakaryocytic lineage cells present in the spleens of the mice receiving activated NK cells. Analysis of the peripheral blood indicated that the transfer of activated NK cells with BMC also significantly improved platelet and total white blood cell counts, with increases in segmented neutrophils. Erythroid recovery was not affected. Finally, lethally irradiated mice receiving activated NK cells and rhIL-2 along with limiting numbers of syngeneic BMC showed a marked increase in survival rate. These results show that the use of populations enriched for activated NK cells after syngeneic BM transplantation (BMT) has a profound enhancing effect on engraftment primarily affecting megakaryocytic and granulocytic cell reconstitution. Therefore, the transfer of activated NK cells and rhIL- 2 may be of clinical use to promote hematopoietic reconstitution after BMT.     

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.     

Incidence and risk factors for pulmonary tuberculosis among the poor in the northern region of Poland

SETTING: The falling trends in pulmonary tuberculosis (PTB) incidence observed in European countries may be due both to an improving epidemiological situation and to a shift of tuberculosis (TB) towards socially important subpopulations; this trend may cause some TB cases to go unnoticed. Identification of such risk groups should be the basis for prevention programmes aimed at containing the spread of the disease. OBJECTIVE: To evaluate the incidence and risk factors for PTB among the poor. DESIGN: The study material was based on the data of 7380 people living in poverty, including 243 homeless adults, aged between 18 and 96 years. Potential medical and socio-economic risk factors were evaluated with regard to PTB incidence. RESULTS: The TB incidence rate in the group studied was estimated at 730 per 100,000 population. The main risk factor was homelessness, with a TB incidence rate in the homeless group of 4290/100,000. According to our data, socio-economic factors correlated much more closely with a final TB diagnosis than subjective disease symptoms. CONCLUSIONS: Efficient TB control requires prevention programmes aimed at systematic monitoring of the homeless. A population with such a high proportion of TB patients is a dangerous source of TB.