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Objective: To develop a minimally invasive, reproducible model of chronic severe mitral regurgitation (MR) that replicates the clinical phenotype of left atrial (LA) and left ventricular dilation and susceptibility to atrial fibrillation., Methods: Under transesophageal echocardiographic guidance, chordae tendinae were avulsed using endovascular forceps until the ratio of regurgitant jet area to LA area was ≥70%. Animals survived for an average of 8.6 ± 1.6 months (standard deviation) and imaged with monthly transthoracic echocardiography (TTE). Animals underwent baseline and preterminal magnetic resonance imaging. Terminal studies included TTE, transesophageal echocardiography, and rapid atrial pacing to test inducibility of atrial tachyarrhythmias., Results: Eight dogs underwent creation of severe MR and interval monitoring. Two were excluded-one died from acute heart failure, and the other had resolution of MR. Six dogs underwent the full experimental protocol; only one required medical management of clinical heart failure. MR remained severe over time, with a mean terminal regurgitant jet area to LA area of 71 ± 14% (standard deviation) and regurgitant fraction of 52 ± 11%. Mean LA volume increased over 130% (TTE: 163 ± 147%, P  = .039; magnetic resonance imaging: 132 ± 54%, P  = .011). Mean left ventricular end-diastolic volume increased by 38 ± 21% ( P  = .008). Inducible atrial tachyarrhythmias were seen in 4 of 6 animals at terminal surgery, and none at baseline., Conclusions: Within the 6 dogs that successfully completed the full experimental protocol, this model replicated the clinical phenotype of severe MR, which led to marked structural and electrophysiologic cardiac remodeling. This model allowed for precise measurements at repeated time points and will facilitate future studies to elucidate the mechanisms of atrial and ventricular remodeling secondary to MR and the pathophysiology of valvular atrial fibrillation., (© 2023 The Author(s).)

Background: The cyclical nature of gene expression in the intraerythrocytic development cycle (IDC) of the malaria parasite, Plasmodium falciparum, confounds the accurate detection of specific transcriptional differences, e.g. as provoked by the development of drug resistance. In lab-based studies, P. falciparum cultures are synchronized to remove this confounding factor, but the rapid detection of emerging resistance to artemisinin therapies requires rapid analysis of transcriptomes extracted directly from clinical samples. Here we propose the use of cyclical regression covariates (CRC) to eliminate the major confounding effect of developmentally driven transcriptional changes in clinical samples. We show that elimination of this confounding factor reduces both Type I and Type II errors and demonstrate the effectiveness of this approach using a published dataset of 1043 transcriptomes extracted directly from patient blood samples with different patient clearance times after treatment with artemisinin., Results: We apply this method to two publicly available datasets and demonstrate its ability to reduce the confounding of differences in transcript levels due to misaligned intraerythrocytic development time. Adjusting the clinical 1043 transcriptomes dataset with CRC results in detection of fewer functional categories than previously reported from the same data set adjusted using other methods. We also detect mostly the same functional categories, but observe fewer genes within these categories. Finally, the CRC method identifies genes in a functional category that was absent from the results when the dataset was adjusted using other methods. Analysis of differential gene expression in the clinical data samples that vary broadly for developmental stage resulted in the detection of far fewer transcripts in fewer functional categories while, at the same time, identifying genes in two functional categories not present in the unadjusted data analysis. These differences are consistent with the expectation that CRC reduces both false positives and false negatives with the largest effect on datasets from samples with greater variance in developmental stage., Conclusions: Cyclical regression covariates have immediate application to parasite transcriptome sequencing directly from clinical blood samples and to cost-constrained in vitro experiments., (© 2022. The Author(s).)

Background: Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations., Methods: Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples., Results: 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of - 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date., Conclusions: eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

Introduction: Breast cancer is the most diagnosed and second leading cause of cancer deaths in the U.S. female population. An estimated 5 to 10 percent of all breast cancers are inherited, caused by mutations in the breast cancer susceptibility genes (BRCA1/2). As many as 90% of all mutations are nonsense mutations, causing a truncated polypeptide product. A popular and low cost method of mutation detection has been the protein truncation test (PTT), where target regions of BRCA1/2 are PCR amplified, transcribed/translated in a cell-free protein synthesis system and analyzed for truncated polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. We previously reported a novel High Throughput Solid-Phase PTT (HTS-PTT) based on an enzyme-linked immunosorbent assay (ELISA) format that eliminates the need for radioactivity, SDS-PAGE and subjective interpretation of the results. Here, we report the next generation HTS-PTT using triple-epitope-tagged proteins and demonstrate, for the first time, its efficacy on clinical genomic DNA samples for BRCA1/2 analysis., Methods: Segments of exons 11 of BRCA1/2 open reading frames were PCR amplified from either blood derived genomic DNA or cell line mRNA. PCR primers incorporate elements for cell-free transcription/translation and epitope tagging. Cell-free expressed nascent proteins are then antibody-captured onto the wells of a microtiter plate and the relative amount of truncated polypeptide measured using antibodies against the N- and C-terminal epitope tags in an ELISA format., Results: 100% diagnostic sensitivity and 96% specificity for truncating mutations in exons 11 of BRCA1/2 was achieved on one hundred blood-derived clinical genomic DNA samples which were previously assayed using the conventional gel based PTT. Feasibility of full gene coverage for BRCA1/2 using mRNA source material is also demonstrated., Conclusions: Overall, the HTS-PTT provides a simple, quantitative, objective, low cost and high throughput method for analysis of truncating mutations as an alternative to gel based PTT for BRCA analysis. The technology is readily accessible to virtually any laboratory, with the only major instrumentation required being a PCR thermocycler and a basic micro-well plate reader. When compared to conventional gel based PTT, the HTS-PTT provides excellent concordance.

Previously we have shown that CD8(+) T cells are critical for containment of simian immunodeficiency virus (SIV) viremia and that rapid and profound depletion of CD4(+) T cells occurs in the intestinal tract of acutely infected macaques. To determine the impact of SIV-specific CD8(+) T-cell responses on the magnitude of the CD4(+) T-cell depletion, we investigated the effect of CD8(+) lymphocyte depletion during primary SIV infection on CD4(+) T-cell subsets and function in peripheral blood, lymph nodes, and intestinal tissues. In peripheral blood, CD8(+) lymphocyte-depletion changed the dynamics of CD4(+) T-cell loss, resulting in a more pronounced loss 2 weeks after infection, followed by a temporal rebound approximately 2 months after infection, when absolute numbers of CD4(+) T cells were restored to baseline levels. These CD4(+) T cells showed a markedly skewed phenotype, however, as there were decreased levels of memory cells in CD8(+) lymphocyte-depleted macaques compared to controls. In intestinal tissues and lymph nodes, we observed a significantly higher loss of CCR5(+) CD45RA(-) CD4(+) T cells in CD8(+) lymphocyte-depleted macaques than in controls, suggesting that these SIV-targeted CD4(+) T cells were eliminated more efficiently in CD8(+) lymphocyte-depleted animals. Also, CD8(+) lymphocyte depletion significantly affected the ability to generate SIV Gag-specific CD4(+) T-cell responses and neutralizing antibodies. These results reemphasize that SIV-specific CD8(+) T-cell responses are absolutely critical to initiate at least partial control of SIV infection.

Here, we investigated the containment of virus replication in simian immunodeficiency virus (SIV) infection by CD8(+) lymphocytes. Escape mutations in Mamu-A*01 epitopes appeared first in SIV Tat TL8 and then in SIV Gag p11C. The appearance of escape mutations in SIV Gag p11C was coincident with compensatory changes outside of the epitope. Eliminating CD8(+) lymphocytes from rhesus monkeys during primary infection resulted in more rapid disease progression that was associated with preservation of canonical epitopes. These results confirm the importance of cytotoxic T cells in controlling viremia and the constraint on epitope sequences that require compensatory changes to go to fixation.

The simian immunodeficiency viruses (SIV) naturally infect a wide range of African primates, including African green monkeys (AGM). Despite moderate to high levels of plasma viremia in naturally infected AGM, infection is not associated with immunodeficiency. We recently reported that SIVagmVer90 isolated from a naturally infected vervet AGM induced AIDS following experimental inoculation of pigtailed macaques. The goal of the present study was to evaluate the replication of this isolate in two species of AGM, sabaeus monkeys (Chlorocebus sabaeus) and vervets (C. pygerythrus). Inoculation of sabaeus AGM with SIVagmVer90 resulted in low and variable primary and set-point viremia (<10(2) to 10(4) copies/ml). In contrast, inoculation of vervet AGM with either SIVagmVer90 or blood from a naturally infected vervet (Ver1) resulted in high primary viremia and moderate plateau levels, similar to the range seen in naturally infected vervets from this cohort. CD4(+) T cells remained stable throughout infection, even in AGM with persistent high viremia. Despite the lack of measurable lymphadenopathy, infection was associated with an increased number of Ki-67(+) T cells in lymph node biopsies, consistent with an early antiviral immune response. The preferential replication of SIVagmVer in vervet versus sabaeus AGM shows that it is critical to match AGM species and SIV strains for experimental models of natural SIV infection.

Purpose: To evaluate whether dynamic imaging of the coronary arteries can be performed with intracoronary infusion of low-dose gadolinium (Gd)-based contrast agent and assess the effect of long duration and multiple infusions on the image signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR)., Materials and Methods: Dynamic coronary magnetic resonance (MR) imaging (130 msec/image) and contrast agent first pass myocardial perfusion studies were performed with intracoronary infusions of low-dose Gd-based MR contrast agent on dogs (N = 4) using a fast multislice gradient recalled echo (GRE) sequence., Results: Contrast-enhanced coronary arteries were clearly imaged during infusion periods as long as 2.3 minutes. The SNR and CNR of the contrast-enhanced coronary arteries remained essentially unchanged over multiple consecutive angiographic sessions. In addition, we demonstrated that first pass studies performed with intracoronary injection of MR contrast agent can be used as a means of assessing regional myocardial perfusion., Conclusion: These studies demonstrated that, using intracoronary infusion of Gd, coronary magnetic resonance angiography (MRA) can be performed with high temporal resolution, and multiple low-dose slow infusions of Gd-based MR contrast agent can be performed without compromise of the vessel SNR and CNR., (Copyright 2002 Wiley-Liss, Inc.)