Search

Background and Purpose: The ionic mechanisms underlying nitrergic inhibitory junction potentials (IJPs) in gut smooth muscle remain a matter of debate. Recently, it has been reported that opening of TWIK-related K(+) channel 1 (TREK-1) K(+) channels contributes to the nitrergic IJP in colonic smooth muscle. We investigated the effects of TREK-1 channel blockers on nitrergic neurotransmission in mouse and opossum lower oesophageal sphincter (LOS) circular smooth muscle (CSM).Experimental Approach: The effects of TREK-1 channel blockers were characterized pharmacologically in murine and opossum gut smooth muscle using conventional intracellular and tension recordings.Key Results: In LOS, L-methionine depolarized the resting membrane potential (RMP) but did not inhibit the nitrergic IJP. Cumulative application of theophylline hyperpolarized the RMP and inhibited the nitrergic IJP concentration dependently. The induced membrane hyperpolarization was prevented by pre-application of caffeine, but not by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. 8-Br-cAMP significantly hyperpolarized membrane potential and increased the amplitude of the nitrergic IJP. In opossum LOS muscle strips, L-methionine increased resting tone but had no effect on nerve-mediated LOS relaxation. On the other hand, theophylline markedly inhibited tone. In CSM from mouse proximal colon, L-methionine caused modest inhibition of nitrergic IJPs.Conclusions and Implications: TREK-1 channels were not involved in the nitrergic IJP in LOS CSM. Not only does L-methionine have no effect on the nitrergic IJP or LOS relaxation, but the effect of theophylline appears to be due to interruption of Ca(2+)-releasing pathways (i.e. caffeine-like effect) rather than via blockade of TREK-1 channels. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Primary central nervous system vasculitis (PCNSV) is an uncommon condition that affects the brain and spinal cord. This study was undertaken to evaluate the clinical features and outcomes among patients with PCNSV who presented with prominent gadolinium meningeal enhancement on magnetic resonance imaging (MRI). METHODS: Through retrospective review using the Mayo Clinic medical records linkage system, we identified 101 consecutive patients with PCNSV based on brain biopsy or conventional angiography (or both) between January 1, 1983, and December 31, 2003. We evaluated data on demographics, clinical findings, laboratory studies, imaging, biopsy of brain or spinal cord (or both), treatment, and neurologic outcome. RESULTS: MRIs showed prominent leptomeningeal enhancement in 8 of 101 patients with PCNSV. In 6 of those 8, cerebral angiography or magnetic resonance angiography results were normal, but biopsy of the brain or spinal cord showed vasculitis in all 8. Granulomatous vascular inflammation was found in 6 specimens and was associated in 4 cases with vascular deposits of beta-amyloid peptide. All 8 patients had a prompt response to therapy, with resolution of the MRI meningeal enhancement. Although 3 of the 8 patients had relapses during followup, the overall outcome was favorable. Patients with meningeal enhancement, compared with patients without enhancement, more commonly had substantial abnormalities of cerebrospinal fluid (100% versus 58%; P = 0.02) and amyloid angiopathy (50% versus 12%; P = 0.03). CONCLUSION: Prominent gadolinium leptomeningeal enhancement on MRI may point to a distinct subtype of PCNSV with small leptomeningeal artery vasculitis and rapid response to therapy. [ABSTRACT FROM AUTHOR]

We report an unusual renal complication of cocaine abuse in a young man who developed loin pain, renal vein thrombosis, and acute renal failure. Cocaine abuse should be considered in the differential diagnosis of renal vein thrombosis in young adults.Copyright © 2007 by National Kidney Foundation, Inc. [ABSTRACT FROM AUTHOR]

Several factors are responsible for the occurrence of cardiac complications after bone marrow transplantation (BMT). These factors include the cardiotoxic effects of radiation therapy, antineoplastic and immunosuppressive drugs, abnormal immunologic reactions associated with graft-vs-host disease, and infectious agents. We report the case of a 45-year-old woman with T-cell prolymphocytic leukemia and no prior risk factors for coronary artery disease in whom sudden cardiac death occurred 2 1/2 years after allogeneic BMT from an unrelated male donor. Autopsy revealed severe 3-vessel coronary disease with grade 4/4 stenosis. This process was primarily nonatherosclerotic, with intimal hyperplasia of undetermined etiology. Furthermore, fluorescence in situ hybridization to identify the donor Y chromosome with simultaneous immunofluorescence labeling of smooth muscle actin suggested the presence of donor cells that transformed into myocytes. Coronary artery disease is an important, albeit rare, complication of BMT. Donor hematopoietic cells may contribute to its pathogenesis. [ABSTRACT FROM AUTHOR]

The in vitro transcription (IVT) of messenger ribonucleic acid (mRNA) from the linearized deoxyribonucleic acid (DNA) template of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) was optimized for total mRNA yield and purity (by percent intact mRNA) utilizing machine learning in conjunction with automated, high-throughput liquid handling technology. An iterative Bayesian optimization approach successfully optimized 11 critical process parameters in 42 reactions across 5 experimental rounds. Once the optimized conditions were achieved, an automated, high-throughput screen was conducted to evaluate commercially available T7 RNA polymerases for rate and quality of mRNA production. Final conditions showed a 12% yield improvement and a 50% reduction in reaction time, while simultaneously significantly decreasing (up to 44% reduction) the use of expensive reagents. This novel platform offers a powerful new approach for optimizing IVT reactions for mRNA production.

The rise of mRNA as a novel vaccination strategy presents new opportunities to confront global disease. Double-stranded RNA (dsRNA) is an impurity byproduct of the in vitro transcription reaction used to manufacture mRNA that may affect the potency and safety of the mRNA vaccine in patients. Careful quantitation of dsRNA during manufacturing is critical to ensure that residual dsRNA is minimized in purified mRNA drug substances. In this work, we describe the development and implementation of a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) to quantitate nanogram quantities of residual dsRNA contaminants in mRNA process intermediates using readily available commercial reagents. This sandwich ELISA developed in this study follows a standard protocol and can be easily adapted to most research laboratory environments. Additionally, a liquid handler coupled with an automated robotics system was utilized to increase assay throughput, improve precision, and reduce the analyst time requirement. The final automated sandwich ELISA was able to measure <10 ng/mL of dsRNA with a specificity for dsRNA over 2000-fold higher than mRNA, a variability of <15%, and a throughput of 72 samples per day.

Objectives: To describe mismatch repair (MMR) and microsatellite instability (MSI) testing practices in laboratories using the College of American Pathologists (CAP) MSI/MMR proficiency testing programs prior to the 2022 publication of the MSI/MMR practice guidelines copublished by CAP and the Association of Molecular Pathology (AMP)., Methods: Data from supplemental questionnaires provided with the 2020-B MSI/MMR programs to 542 laboratories across different practice settings were reviewed. Questionnaires contained 21 questions regarding the type of testing performed, specimen/tumor types used for testing, and clinical practices for checkpoint blockade therapy., Results: Domestic laboratories test for MSI/MMR more often than international laboratories (P = .04) and academic hospitals/medical centers test more frequently than nonhospital sites/clinics (P = .03). The most commonly used testing modality is immunohistochemistry, followed by polymerase chain reaction, then next-generation sequencing. Most laboratories (72.6%; 347/478) reported awareness of the use of immune checkpoint inhibitor therapy for patients with high MSI or MMR-deficient results., Conclusions: The results demonstrate the state of MMR and MSI testing in laboratories prior to the publication of the CAP/AMP best practice guidelines, highlighting differences between various laboratory types. The findings indicate the importance of consensus guidelines and provide a baseline for comparison after their implementation., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The Banff Heart Concurrent Session, held as part of the 16th Banff Foundation for Allograft Pathology Conference at Banff, Alberta, Canada, on September 21, 2022, focused on 2 major topics: non-human leukocyte antigen (HLA) antibodies and mixed rejection. Each topic was addressed in a multidisciplinary fashion with clinical, immunological, and pathology perspectives and future developments and prospectives. Following the Banff organization model and principles, the collective aim of the speakers on each topic was to • Determine current knowledge gaps in heart transplant pathology • Identify limitations of current pathology classification systems • Discuss next steps in addressing gaps and refining classification system., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Polysorbate-80 (PS-80) is a common surfactant used in biologics formulations. However, the tendency of oxidation to PS-80 when exposed to stainless steel surfaces brings various challenges during manufacturing processes, such as inconsistent shelf-life of PS-80 solutions, which can further impact the biologics and vaccines production. In this work, the root causes of PS-80 oxidation when in contact with stainless steel conditions were thoroughly investigated through the use of various complementary analytical techniques including U/HPLC-CAD, LC-MS, ICP-MS, peroxide assay, and EPR spectroscopy. The analytical tool kit used in this work successfully revealed a PS-80 degradation mechanism from the perspective of PS-80 content, PS-80 profile, iron content, peroxide production, and radical species. The combined datasets reveal that PS-80 oxidative degradation occurs in the presence of histidine and iron in addition to being combined with the hydroperoxides in PS-80 material. The oxidative pathway and potential degradants were identified by LC-MS. The PS-80 profile based on the U/HPLC-CAD assay provided an effective way to identify early-signs of PS-80 degradation. The results from a peroxide assay observed increased hydroperoxide along with PS-80 degradation. EPR spectra confirmed the presence of histidine-related radicals during PS-80 oxidation identifying how histidine is involved in the oxidation. All assays and findings introduced in this work will provide insight into how PS-80 oxidative degradation can be avoided, controlled, or detected. It will also provide valuable evaluations on techniques that can be used to identify PS-80 degradation related events that occur during the manufacturing process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Magee Equations™ (ME) are multivariable models that can estimate oncotype DX ® recurrence score. One of the equations, Magee Equation 3 (ME3) which utilizes only semi-quantitative receptor results has been shown to provide chemopredictive value in the neoadjuvant setting in a single institutional study. This multi-institutional study (seven institutions contributed cases) was undertaken to examine the validity of ME3 in predicting response to neoadjuvant chemotherapy in estrogen receptor positive, HER2-negative breast cancers. Stage IV cases were excluded. The primary endpoint was the pathologic complete response (pCR) rate in different categories of ME3 scores calculated based on receptor results in the pre-therapy core biopsy. A total of 166 cases met the inclusion criteria. The patient age ranged from 24 to 83 years (median 53 years). The average pre-therapy tumor size was 3.9 cm, and axillary lymph nodes were confirmed positive by pre-therapy core biopsy in 85 of 166 cases (51%). The pCR rate according to ME3 scores was 0% (0 of 64) in ME3 < 18, 0% (0 of 46) in ME3 18-25, 14% (3 of 21) in ME3 > 25 to <31, and 40% (14 of 35) in ME3 score 31 or higher (p value: <0.0001). There were no distant recurrences and no deaths in the 17 patients with pCR. In the remaining 149 cases with residual disease, ME3 score of >25 was significantly associated with shorter distant recurrence-free survival and showed a trend for shorter breast cancer-specific survival. The results of this multi-institutional study are similar to previously published data from a single institution (PMID: 28548119) and confirm the chemo-predictive value of ME3 in the neoadjuvant setting. In addition, ME3 may provide prognostic information in patients with residual disease which should be further evaluated.

The XVth Banff Conference on Allograft Pathology meeting was held on September 23-27, 2019, in Pittsburgh, Pennsylvania, USA. During this meeting, two main topics in cardiac transplant pathology were addressed: (a) Improvement of endomyocardial biopsy (EMB) accuracy for the diagnosis of rejection and other significant injury patterns, and (b) the orphaned lesion known as Quilty effect or nodular endocardial infiltrates. Molecular technologies have evolved in recent years, deciphering pathophysiology of cardiac rejection. Diagnostically, it is time to integrate the histopathology of EMBs and molecular data. The goal is to incorporate molecular pathology, performed on the same paraffin block as a companion test for histopathology, to yield more accurate and objective EMB interpretation. Application of digital image analysis from hematoxylin and eosin (H&E) stain to multiplex labeling is another means of extracting additional information from EMBs. New concepts have emerged exploring the multifaceted significance of myocardial injury, minimal rejection patterns supported by molecular profiles, and lesions of arteriolitis/vasculitis in the setting of T cell-mediated rejection (TCMR) and antibody-mediated rejection (AMR). The orphaned lesion known as Quilty effect or nodular endocardial infiltrates. A state-of-the-art session with historical aspects and current dilemmas was reviewed, and possible pathogenesis proposed, based on advances in immunology to explain conflicting data. The Quilty effect will be the subject of a multicenter project to explore whether it functions as a tertiary lymphoid organ., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Context.—: The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility., Objective.—: To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology., Design.—: The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review., Results.—: The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities., Conclusions.—: WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology., (© 2020 College of American Pathologists.)

Background: Coronary artery stenting has become a common procedure and cardiovascular pathology specimens containing these metallic stents are accordingly becoming common. Histologic examination of stented vessels is imperative, but special techniques are needed due to the presence of metal within the tissue. We describe a rapid and inexpensive method for preparing stented vascular specimens for routine histology suitable for use in almost any histology laboratory., Design: After formalin fixation and decalcification, stented vascular segments were freeze-embedded and sectioned using a handheld power micro cutoff wheel tool into ~1 mm slices. Sections were allowed to thaw and the strut shards removed with fine forceps. No longer containing metal, the sections were processed for routine paraffin embedding, microtomy and staining., Results: Histologic sections showed only minor tissue disruption around the stent struts. In our experience with 25 stented arteries (mean interval from implantation 5.6 years), the mean subjective section quality score was 4.1 out of 5. The position of each strut could easily be determined, along with neointimal in-stent restenosis and thrombosis. Local reaction to each strut could be surmised even if minor tissue disruption occurred. The entire process was completed in 2-3 days. The incremental cost over that of routine histology is nominal., Conclusion: This method for examining stented vascular segments histologically could readily be applied in most pathology laboratories and serves as a highly practical solution to dilemma of examining stents histologically., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Previous studies have demonstrated the noninferiority of pathologists' interpretation of whole slide images (WSIs) compared to microscopic slides in diagnostic surgical pathology; however, to our knowledge, no published studies have tested analytical precision of an entire WSI system., Methods: In this study, five pathologists at three locations tested intra-system, inter-system/site, and intra- and inter-pathologist precision of the Aperio AT2 DX System (Leica Biosystems, Vista, CA, USA). Sixty-nine microscopic slides containing 23 different morphologic features suggested by the Digital Pathology Association as important to diagnostic pathology were identified and scanned. Each of 202 unique fields of view (FOVs) had 1-3 defined morphologic features, and each feature was represented in three different tissues. For intra-system precision, each site scanned 23 slides at three different times and one pathologist interpreted all FOVs. For inter-system/site precision, all 69 slides were scanned once at each of three sites, and FOVs from each site were read by one pathologist. To test intra- and inter-pathologist precision, all 69 slides were scanned at one site, FOVs were saved in three different orientations, and the FOVs were transferred to a different site. Three different pathologists then interpreted FOVs from all 69 slides. Wildcard (unscored) slides and washout intervals were included in each study. Agreement estimates with 95% confidence intervals were calculated., Results: Combined precision from all three studies, representing 606 FOVs in each of the three studies, showed overall intra-system agreement of 97.9%; inter-system/site agreement was 96%, intra-pathologist agreement was 95%, and inter-pathologist agreement was 94.2%., Conclusions: Pathologists using the Aperio AT2 DX System identified histopathological features with high precision, providing increased confidence in using WSI for primary diagnosis in surgical pathology., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Journal of Pathology Informatics.)

Methyl coenzyme M reductase (MCR) catalyzes the last step in the biological production of methane by methanogenic archaea, as well as the first step in the anaerobic oxidation of methane to methanol by methanotrophic archaea. MCR contains a number of unique post-translational modifications in its α subunit, including thioglycine, 1- N -methylhistidine, S -methylcysteine, 5-C-( S )-methylarginine, and 2-C-( S )-methylglutamine. Recently, genes responsible for the thioglycine and methylarginine modifications have been identified in bioinformatics studies and in vivo complementation of select mutants; however, none of these reactions has been verified in vitro Herein, we purified and biochemically characterized the radical S -adenosylmethionine (SAM) protein Ma Mmp10, the product of the methanogenesis marker protein 10 gene in the methane-producing archaea Methanosarcina acetivorans Using an array of approaches, including kinetic assays, LC-MS-based quantification, and MALDI TOF-TOF MS analyses, we found that Ma Mmp10 catalyzes the methylation of the equivalent of Arg 285 in a peptide substrate surrogate, but only in the presence of cobalamin. We noted that the methyl group derives from SAM, with cobalamin acting as an intermediate carrier, and that Ma Mmp10 contains a C-terminal cobalamin-binding domain. Given that Mmp10 has not been annotated as a cobalamin-binding protein, these findings suggest that cobalamin-dependent radical SAM proteins are more prevalent than previously thought., (© 2019 Radle et al.)

Purpose of Review: Defining criteria for antibody-mediated rejection (AMR) in heart transplantation were standardized a few years ago, but very little is known about asymptomatic cardiac AMR. We will start the review with a background summarizing the timeline of cardiac AMR. Then we will cover past and current knowledge about asymptomatic cardiac AMR and its impact on outcome after transplantation, with added insight from experience with other solid-organ transplants., Recent Findings: The incidence of asymptomatic cardiac AMR had likely been under-estimated because biopsy surveillance for it in the absence of clinical manifestation was not the norm. Recent data indicate that it may be more common especially when counting concomitant acute cellular rejection (mixed rejection). Also a higher risk of cardiac allograft vasculopathy (CAV) and cardiovascular mortality have been linked to it. The primary implication of these findings is whether therapeutic intervention is warranted, but the appropriate target patient population likely to benefit from treatment is yet to be determined., Summary: Asymptomatic cardiac AMR is not uncommon and it negatively impacts outcome after heart transplantation.

Sensitization, defined as the presence of circulating antibodies, presents challenges for heart transplant recipients and physicians. When present, sensitization can limit a transplantation candidate's access to organs, prolong wait time, and, in some cases, exclude the candidate from heart transplantation altogether. The management of sensitization is not yet standardized, and current therapies have not yielded consistent results. Although current strategies involve antibody suppression and removal with intravenous immunoglobulin, plasmapheresis, and antibody therapy, newer strategies with more specific targets are being investigated.

Background: Cardiac allograft vasculopathy (CAV) remains an important source of mortality after heart transplant. The aim of our study was to identify structural and microvasculature changes in severe CAV., Methods and Results: The study group included heart transplant recipients with severe CAV who underwent retransplantation (severe CAV, n=20). Control groups included time from transplant matched cardiac transplant recipients without CAV (transplant control, n=20), severe ischemic cardiomyopathy patients requiring left ventricular assist device implantation (ischemic control, n=18), and normal hearts donated for research (donor control, n=10). We collected baseline demographic information, echocardiography data, and performed histopathologic examination of myocardial microvasculature. Echocardiographic features of severe CAV included lack of eccentric remodeling and presence of significant diastolic dysfunction. In contrast, diastolic function was preserved in transplant control subjects. Histopathologic examination showed increased interstitial fibrosis among severe CAV, transplant controls, and ischemic control patients. Compared with transplant controls, severe CAV subjects had reduced capillary density and increased capillary wall thickness ( P<0.05)., Conclusions: Our results suggest that the marked diastolic dysfunction and resultant symptoms in patients with severe CAV may be secondary to the loss of microvasculature and remodeling of remaining microvessels rather than a consequence of interstitial fibrosis. The clinical significance and potential therapeutic implications of these unique microvasculature characteristics warrant further investigation.

The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic reactions. The MSP has been completely elucidated in aerobic organisms, and found to rely on molecular oxygen. Since anaerobic organisms do not use O2, an alternative pathway(s) must be operating. We sought to evaluate whether the functions of two annotated MSP enzymes from Methanocaldococcus jannaschii, a methylthioinosine phosphorylase (MTIP) and a methylthioribose 1-phosphate isomerase (MTRI), are consistent with functioning in a modified anaerobic MSP (AnMSP). We show here that recombinant MTIP is active with six different purine nucleosides, consistent with its function as a general purine nucleoside phosphorylase for both AnMSP and purine salvage. Recombinant MTRI is active with both 5-methylthioribose 1-phosphate and 5-deoxyribose 1-phosphate as substrates, which are generated from phosphororolysis of 5'-methylthioinosine and 5'-deoxyinosine by MTIP, respectively. Together, these data suggest that MTIP and MTRI may function in a novel pathway for recycling the 5'-deoxyadenosine moiety of SAM in M. jannaschii. These enzymes may also enable biosynthesis of 6-deoxy-5-ketofructose 1-phosphate (DKFP), an essential intermediate in aromatic amino acid biosynthesis. Finally, we utilized a homocysteine auxotrophic strain of Methanosarcina acetivorans Δma1821-22Δoahs (HcyAux) to identify potential AnMSP intermediates in vivo. Growth recovery experiments of the M. acetivorans HcyAux were performed with known and proposed intermediates for the AnMSP. Only one metabolite, 2-keto-(4-methylthio)butyric acid, rescued growth of M. acetivorans HcyAux in the absence of homocysteine. This observation may indicate that AnMSP pathways substantially differ among methanogens from phylogenetically divergent genera.

The objective of this study is to assess changes in cardiac deformation during acute cellular- and antibody-mediated rejection in pediatric HT recipients. Pediatric HT recipients aged ≤18 years with at least one episode of biopsy-diagnosed rejection from 2006 to 2013 were included. Left ventricular systolic S (SS) and SR (SSr) data were acquired using 2D speckle tracking on echocardiograms obtained within 12 h of right ventricular endomyocardial biopsy. A mixed effect model was used to compare cardiac deformation during CR (Grade ≥ 1R), AMR (pAMR ≥ 2), and mixed rejection (CR and AMR positive) versus no rejection (Grade 0R and pAMR 0 or 1). A total of 20 subjects (10 males, 50%) with 71 rejection events (CR 35, 49%; AMR 21, 30% and mixed 15, 21%) met inclusion criteria. The median time from HT to first biopsy used for analysis was 5 months (IQR 0.25-192 months). Average LV longitudinal SS and SSr were reduced significantly during rejection (SS: -17.2 ± 3.4% vs. -10.7 ± 4.5%, p < 0.001 and SSr: -1.2 ± 0.2 s - 1 vs. -0.9 ± 0.3 s - 1 ; p < 0.001) and in all rejection types. Average LV short-axis radial SS was reduced only in CR compared to no rejection (p = 0.04), while average LV circumferential SS and SSr were reduced significantly in AMR compared to CR (SS: 18.9 ± 4.2% vs. 20.8 ± 8.8%, p = 0.03 and SSr: 1.35 ± 0.8 s - 1 vs. 1.54 ± 0.9 s - 1 ; p = 0.03). In pediatric HT recipients, LV longitudinal SS and SSr were reduced in all rejection types, while LV radial SS was reduced only in CR. LV circumferential SS and SSr further differentiated between CR and AMR with a significant reduction seen in AMR as compared to CR. This novel finding suggests mechanistic differences between AMR- and CR-induced myocardial injury which may be useful in non-invasively predicting the type of rejection in pediatric HT recipients.

Purpose Gene expression profiling assays are frequently used to guide adjuvant chemotherapy decisions in hormone receptor-positive, lymph node-negative breast cancer. We hypothesized that the clinical value of these new tools would be more fully realized when appropriately integrated with high-quality clinicopathologic data. Hence, we developed a model that uses routine pathologic parameters to estimate Oncotype DX recurrence score (ODX RS) and independently tested its ability to predict ODX RS in clinical samples. Patients and Methods We retrospectively reviewed ordered ODX RS and pathology reports from five institutions (n = 1,113) between 2006 and 2013. We used locally performed histopathologic markers (estrogen receptor, progesterone receptor, Ki-67, human epidermal growth factor receptor 2, and Elston grade) to develop models that predict RS-based risk categories. Ordering patterns at one site were evaluated under an integrated decision-making model incorporating clinical treatment guidelines, immunohistochemistry markers, and ODX. Final locked models were independently tested (n = 472). Results Distribution of RS was similar across sites and to reported clinical practice experience and stable over time. Histopathologic markers alone determined risk category with > 95% confidence in > 55% (616 of 1,113) of cases. Application of the integrated decision model to one site indicated that the frequency of testing would not have changed overall, although ordering patterns would have changed substantially with less testing of estimated clinical risk-high or clinical risk-low cases and more testing of clinical risk-intermediate cases. In the validation set, the model correctly predicted risk category in 52.5% (248 of 472). Conclusion The proposed model accurately predicts high- and low-risk RS categories (> 25 or ≤ 25) in a majority of cases. Integrating histopathologic and molecular information into the decision-making process allows refocusing the use of new molecular tools to cases with uncertain risk.

Background: C3 glomerulopathy (C3G) includes both C3 glomerulonephritis (C3GN) and dense deposit disease (DDD) and is defined by C3-dominant deposits on immunofluorescence. Dysfunction of the alternative pathway (AP) of complement is central to the pathophysiology of C3G and young patients often harbor genetic alterations of AP mediators. Recently, a link between C3G and paraproteinemia has been established. We performed this study to better characterize older patients with C3G where this association is more frequently seen., Methods: Fourteen biopsies from 12 patients meeting diagnostic criteria for C3G were identified in patients > 49 years of age from 2005 to 2015 after exclusion of cases containing masked monotypic immunoglobulin deposits. Pathologic and clinical features were reviewed., Results: The median age was 63.5 years and 75% of patients were male. All had renal insufficiency at presentation. Kidney biopsy showed DDD in three patients and C3GN in the remainder. Serum protein electrophoresis revealed a paraprotein in 10 patients, 8 of which had a plasma cell dyscrasia on bone marrow biopsy. A membranoproliferative pattern of glomerular injury was seen in 64% of biopsies, while mesangial proliferative and endocapillary proliferative patterns were seen less frequently. Among patients with at least 1 year of follow-up ( n = 9), five were on renal replacement therapy, three showed stable (but impaired) kidney function and one demonstrated improvement., Conclusions: C3G is an uncommon but important cause of kidney injury in older adults and associates with a high prevalence of paraproteinemia. In adult patients with C3G, prognosis is guarded as most patients showed either progression to end-stage kidney disease or stable but impaired kidney function.

Background and Purpose: MR imaging-detected carotid intraplaque hemorrhage indicates vulnerable plaque with high stroke risk. Angiotensin II stimulates intraplaque hemorrhage in animal models, and the angiotensin system is highly regulated by vitamin D. Our purpose was to determine whether low vitamin D levels predict carotid intraplaque hemorrhage in humans., Materials and Methods: In this cross-sectional study, 65 patients with carotid disease underwent carotid MR imaging and blood draw. Systemic clinical confounders and local lumen imaging markers were recorded. To determine the association of low vitamin D levels with MR imaging detected intraplaque hemorrhage, we performed multivariable Poisson regression by using generalized estimating equations to account for up to 2 carotid arteries per patient and backward elimination of confounders. MR imaging detected intraplaque hemorrhage volume was also correlated with vitamin D levels and maximum plaque thickness. Thirty-five patients underwent carotid endarterectomy, and histology-detected intraplaque hemorrhage was correlated with vitamin D levels and total plaque area., Results: Low vitamin D levels (<30 ng/mL, prevalence ratio = 2.05, P = .03) were a significant predictor of MR imaging detected intraplaque hemorrhage, along with plaque thickness (prevalence ratio = 1.40, P < .001). MR imaging detected intraplaque hemorrhage volume linearly correlated with plaque thickness (partial r = 0.45, P < .001) and low vitamin D levels (partial r = 0.26, P = .003). Additionally, histology-detected intraplaque hemorrhage area linearly correlated with plaque area (partial r = 0.46, P < .001) and low vitamin D levels (partial r = 0.22, P = .03). The association of intraplaque hemorrhage volume with low vitamin D levels was also higher with ischemic stroke., Conclusions: Low vitamin D levels and plaque thickness predict carotid intraplaque hemorrhage and outperform lumen markers of vulnerable plaque. This research demonstrates a significant link between low vitamin D levels and carotid intraplaque hemorrhage., (© 2016 by American Journal of Neuroradiology.)

This study sought to investigate the effects of mechanical unloading on myocardial energetics and the metabolic perturbation of heart failure (HF) in an effort to identify potential new therapeutic targets that could enhance the unloading-induced cardiac recovery. The authors prospectively examined paired human myocardial tissue procured from 31 advanced HF patients at left ventricular assist device (LVAD) implant and at heart transplant plus tissue from 11 normal donors. They identified increased post-LVAD glycolytic metabolites without a coordinate increase in early, tricarboxylic acid (TCA) cycle intermediates. The increased pyruvate was not directed toward the mitochondria and the TCA cycle for complete oxidation, but instead, was mainly converted to cytosolic lactate. Increased nucleotide concentrations were present, potentially indicating increased flux through the pentose phosphate pathway. Evaluation of mitochondrial function and structure revealed a lack of post-LVAD improvement in mitochondrial oxidative functional capacity, mitochondrial volume density, and deoxyribonucleic acid content. Finally, post-LVAD unloading, amino acid levels were found to be increased and could represent a compensatory mechanism and an alternative energy source that could fuel the TCA cycle by anaplerosis. In summary, the authors report evidence that LVAD unloading induces glycolysis in concert with pyruvate mitochondrial oxidation mismatch, most likely as a result of persistent mitochondrial dysfunction. These findings suggest that interventions known to improve mitochondrial biogenesis, structure, and function, such as controlled cardiac reloading and conditioning, warrant further investigation to enhance unloading-induced reverse remodeling and cardiac recovery.

Background: Marfan syndrome (MFS) is a multisystem connective tissue disorder that can lead to aortic dilation requiring aortic root replacement. Neonatal MFS (nMFS) is a rare and severe form of MFS compared to classic MFS (cMFS). Aortic root histology in MFS is thought to demonstrate predominantly medial degeneration (MD) of a translamellar mucoid extracellular matrix accumulation (MEMA-T) vs. the intralamellar mucoid extracellular matrix accumulation (MEMA-I) seen in other aortopathies. The objective of this study was to describe the clinical and histopathologic features of nMFS and cMFS patients undergoing aortic root replacement., Methods: Children with MFS who underwent aortic root replacement between 2000 and 2012 at a single institution were included. Medical records including clinical details, aortic dimensions (Z scores), and histology including MD type were obtained. Statistics were descriptive with univariate analysis of age at surgery and type of MD., Results: Eleven patients, 3 (27%) with nMFS, were included. Root dilation at time of surgery was greater in nMFS compared to cMFS (Z=12.8 vs. 7.6, P=.005), and nMFS patients were younger at time of surgery (7.3 vs. 18.8 years, P=.002). Histology in the nMFS group demonstrated MEMA-I in one and no MD in two. In the cMFS group, there were three with MEMA-T, four with MEMA-I, and one with both types., Conclusion: In summary, nMFS has earlier root dilation often in the absence of MD. Both forms of MD were present in our cohort, and there was no correlation between age at surgery and type of MD., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Immune allosensitization can be triggered by continuous-flow left ventricular assist devices (CF LVAD). However, the effect of this type of allosensitization on post-transplant outcomes remains controversial. This study examined the post-transplant course in a contemporary cohort of patients undergoing transplantation with and without LVAD bridging., Methods: We included consecutive patients who were considered for cardiac transplant from 2006 to 2015. Serum alloantibodies were detected with single-antigen beads on the Luminex platform (One Lambda Inc., Canoga Park, CA). Allosensitization was defined as calculated panel reactive antibody (cPRA) > 10%. cPRA was determined at multiple times. LVAD-associated allosensitization was defined as development of cPRA > 10% in patients with cPRA ≤ 10% before LVAD implantation. Post-transplant outcomes of interest were acute cellular rejection (ACR), antibody-mediated rejection (AMR), and survival., Results: Allosensitization status was evaluated in 268 patients (20% female). Mean age was 52 ± 12 years, and 132 (49.3%) received CF LVADs. After LVAD implant, 30 patients (23%) became newly sensitized, and the level of sensitization appeared to diminish in many of these patients while awaiting transplant. During the study period, 225 of 268 patients underwent transplant, and 43 did not. A CF LVAD was used to bridge 50% of the transplant recipients. Compared with patients without new sensitization or those already sensitized at baseline, the patients with LVAD-associated sensitization had a higher risk of ACR (p = 0.049) and higher risk of AMR (p = 0.018) but a similar intermediate-term post-transplant survival. The patients who did not receive a transplant had higher level of allosensitization, with a baseline cPRA of 20% vs 6% in those who received an allograft and a high risk (40%) of death during follow-up., Conclusions: New allosensitization takes place in > 20% of patents supported with CF LVADs. Among patients who undergo transplant, this results in a higher risk of ACR and AMR, but survival remains favorable, likely due to the efficacy of current management after transplant. However, mortality in sensitized patients who do not reach transplant remains high, and new approaches are necessary to meet the needs of this group of patients., (Published by Elsevier Inc.)

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Surgical aortic specimens are usually examined in Pathology Departments as a result of treatment of aneurysms or dissections. A number of diseases, genetic syndromes (Marfan syndrome, Loeys-Dietz syndrome, etc.), and vasculopathic aging processes involved in vascular injury can cause both distinct and nonspecific histopathologic changes with degeneration of the media as a common denominator. Terminology for these changes has varied over time leading to confusion and inconsistencies. This consensus document has established a revised, unified nomenclature for the variety of noninflammatory degenerative aortic histopathologies seen in such specimens. Older terms such as cystic medial necrosis and medionecrosis are replaced by more technically accurate terms such as mucoid extracellular matrix accumulation (MEMA), elastic fiber fragmentation and/or loss, and smooth muscle cell nuclei loss. A straightforward system of grading is presented to gauge the extent of medial degeneration and synoptic reporting tables are provided. Herein we present a standardized nomenclature that is accessible to general pathologists and useful for future publications describing these entities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Little is known about mixed cellular and antibody-mediated rejection (MR) in heart transplantation. It remains unclear whether cardiac MR has distinctive pathologic and clinical features beyond those of simultaneous cellular rejection (CR) and antibody-mediated rejection (AMR). In this study we systematically explore the pathologic and clinical characteristics of MR in heart transplantation., Methods: The UTAH Cardiac Transplant Program database was queried for transplant recipients who survived long enough to have at least one endomyocardial biopsy (EMB) between 1985 and 2014. Only EMBs with both CR and AMR scores documented were included. In addition to detailed pathologic analyses, we also examined the incidence and prevalence of MR, the likelihood to transition from and to MR, and mortality associated with MR., Results: Patients (n = 1,207) with a total of 28,484 EMBs met the study inclusion criteria. The overall prevalence of MR was 7.8% and it was nearly twice as frequent within the first year post-transplant. Mild MR was by far the most common occurrence and was typically preceded by an immune active state. When CR increased in severity, AMR tended to follow, but the reverse was not true. On pathology, individual features of CR and AMR were more easily separated in cases of mild MR, whereas they substantially overlapped in more severe cases. MR was associated with a significant cardiovascular death risk that was incremental with severity., Conclusions: MR is not common, usually occurs early after transplant, and is associated with worse outcomes. MR reflects a complex interplay between cellular and humoral processes, which varies with rejection severity., (Copyright © 2016 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Antibody-mediated rejection (AMR) in cardiac transplant recipients is a serious form of rejection with adverse patient outcomes. The International Society of Heart and Lung Transplantation (ISHLT) has published a consensus schema for the pathologic diagnosis of various grades of antibody-mediated rejection (pathology antibody-mediated rejection [pAMR]). We sought to determine whether the ISHLT pAMR grading schema correlates with patient outcomes., Methods: Using our database, which contains a semi-quantitative scoring of all pathologic descriptors of pAMR, we retrospectively used these descriptors to convert the previous AMR categories to the current ISHLT pAMR categories. Cox proportional hazard models were fit with cardiovascular (CV) death or retransplant as the outcome. The pAMR value was included as a categorical variable, and cellular rejection (CR) values were included in a separate model., Results: There were 13,812 biopsies from 1,014 patients analyzed. The pAMR grades of pAMR1h, pAMR1i, and pAMR2 conferred comparable increased risk for CV mortality. Significantly increased risk of CV mortality was conferred by biopsies graded as severe AMR (pAMR3)., Conclusions: The new ISHLT pAMR grading schema identifies patients at increased risk of CV mortality, consistent with risks published from several programs before 2011. The current schema is validated by this analysis in a large biopsy database. Because pAMR1h, pAMR1i, and pAMR2 have similar CV risks associated with them, the threshold for a positive diagnosis of pAMR should be re-evaluated in future iterations of the ISHLT schema., (Copyright © 2016 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)