Restoration of Hydrogen Sulfide Production in Diabetic Mice Improves Reparative Function of Bone Marrow Cells.

BACKGROUND: Bone marrow cell (BMC)-based treatment for critical limb ischemia in diabetic patients yielded a modest therapeutic effect resulting from cell dysfunction. Therefore, approaches that improve diabetic stem/progenitor cell functions may provide therapeutic benefits. Here, we tested the hypothesis that restoration of hydrogen sulfide (H2S) production in diabetic BMCs improves their reparative capacities. METHODS: Mouse BMCs were isolated by density-gradient centrifugation. Unilateral hind limb ischemia was conducted in 12- to 14-week-old db/+ and db/db mice by ligation of the left femoral artery. The H2S level was measured by either gas chromatography or staining with florescent dye sulfidefluor 7 AM. RESULTS: Both H2S production and cystathionine γ-lyase (CSE), an H2S enzyme, levels were significantly decreased in BMCs from diabetic db/db mice. Administration of H2S donor diallyl trisulfide (DATS) or overexpression of CSE restored H2S production and enhanced cell survival and migratory capacity in high glucose (HG)-treated BMCs. Immediately after hind limb ischemia surgery, the db/+ and db/db mice were administered DATS orally and/or given a local intramuscular injection of green fluorescent protein-labeled BMCs or red fluorescent protein-CSE-overexpressing BMCs (CSE-BMCs). Mice with hind limb ischemia were divided into 6 groups: db/+, db/db, db/db+BMCs, db/db+DATS, db/db+DATS+BMCs, and db/db+CSE-BMCs. DATS and CSE overexpression greatly enhanced diabetic BMC retention in ischemic hind limbs followed by improved blood perfusion, capillary/arteriole density, skeletal muscle architecture, and cell survival and decreased perivascular CD68+ cell infiltration in the ischemic hind limbs of diabetic mice. It is interesting to note that DATS or CSE overexpression rescued high glucose-impaired migration, tube formation, and survival of BMCs or mature human cardiac microvascular endothelial cells. Moreover, DATS restored nitric oxide production and decreased endothelial nitric oxide synthase phosphorylation at threonine 495 levels in human cardiac microvascular endothelial cells and improved BMC angiogenic activity under high glucose condition. Last, silencing CSE by siRNA significantly increased endothelial nitric oxide synthase phosphorylation at threonine 495 levels in human cardiac microvascular endothelial cells. CONCLUSIONS: Decreased CSE-mediated H2S bioavailability is an underlying source of BMC dysfunction in diabetes mellitus. Our data indicate that H2S and overexpression of CSE in diabetic BMCs may rescue their dysfunction and open novel avenues for cell-based therapeutics of critical limb ischemia in diabetic patients.

Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction.

RATIONALE: Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes, however, their effect in the context of the heart is unknown. OBJECTIVE: Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit(+) cardiac progenitor cells (CPCs) function can be enhanced with ESC exosomes. METHODS AND RESULTS: This study demonstrates that mouse ESC-derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival, and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented CPC survival, proliferation, and cardiac commitment concurrent with increased c-kit(+) CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290-295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression, and proliferation. CONCLUSIONS: mES Ex provide a novel cell-free system that uses the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES-derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC-based repair programs in the heart.

Negative Regulation of miR-375 by Interleukin-10 Enhances Bone Marrow-Derived Progenitor Cell-Mediated Myocardial Repair and Function After Myocardial Infarction.

Poor survival and function of transplanted cells in ischemic and inflamed myocardium likely compromises the functional benefit of stem cell-based therapies. We have earlier reported that co-administration of interleukin (IL)-10 and BMPAC enhances cell survival and improves left ventricular (LV) functions after acute myocardial infarction (MI) in mice. We hypothesized that IL-10 regulates microRNA-375 (miR-375) signaling in BMPACs to enhance their survival and function in ischemic myocardium after MI and attenuates left ventricular dysfunction after MI. miR-375 expression is significantly upregulated in BMPACs upon exposure to inflammatory/hypoxic stimulus and also after MI. IL-10 knockout mice display significantly elevated miR-375 levels. We report that ex vivo miR-375 knockdown in BMPAC before transplantation in the ischemic myocardium after MI significantly improve the survival and retention of transplanted BMPACs and also BMPAC-mediated post-infarct repair, neovascularization, and LV functions. Our in vitro studies revealed that knockdown of miR-375-enhanced BMPAC proliferation and tube formation and inhibited apoptosis; over expression of miR-375 in BMPAC had opposite effects. Mechanistically, miR-375 negatively regulated 3-phosphoinositide-dependent protein kinase-1 (PDK-1) expression and PDK-1-mediated activation of PI3kinase/AKT signaling. Interestingly, BMPAC isolated from IL-10-deficient mice showed elevated basal levels of miR-375 and exhibited functional deficiencies, which were partly rescued by miR-375 knockdown, enhancing BMPAC function in vitro and in vivo. Taken together, our studies suggest that miR-375 is negatively associated with BMPAC function and survival and IL-10-mediated repression of miR-375 enhances BMPAC survival and function.

Cytomorphology of unusual infectious entities in the Pap test.

Rare entities in the Pap test, including neoplastic and non-neoplastic conditions, pose challenges due to their infrequent occurrence in the daily practice of cytology. Furthermore, these conditions give rise to important diagnostic pitfalls. Infections such as tuberculosis cervicitis may be erroneously diagnosed as carcinoma, whereas others, such as schistosomiasis, are associated with squamous cell carcinoma. These cases include granuloma inguinale (donovanosis), tuberculosis, coccidioidomycosis, schistosomiasis, taeniasis, and molluscum contagiosum diagnosed in Pap tests. Granuloma inguinale shows histiocytes that contain intracytoplasmic bacteria (Donovan bodies). Tuberculosis is characterized by necrotizing granulomatous inflammation with Langhans-multinucleated giant cells. Coccidioidomycosis may show large intact or ruptured fungal spherules associated with endospores. Schistosoma haematobium is diagnosed by finding characteristic ova with a terminal spine. Molluscum contagiosum is characterized by the appearance of squamous cells with molluscum bodies. This article reviews the cytomorphology of selected rare infections and focuses on their cytomorphology, differential diagnosis, and role of ancillary diagnostic studies.

Identification and Comparison of Hyperglycemia-Induced Extracellular Vesicle Transcriptome in Different Mouse Stem Cells.

Extracellular vesicles (EVs) derived from stem /progenitor cells harbor immense potential to promote cardiomyocyte survival and neovascularization, and to mitigate ischemic injury. However, EVs' parental stem/progenitor cells showed modest benefits in clinical trials, suggesting autologous stem cell/EV quality might have been altered by stimuli associated with the co-morbidities such as hyperglycemia associated with diabetes. Hyperglycemia is a characteristic of diabetes and a major driving factor in cardiovascular disease. The functional role of stem/progenitor cell-derived EVs and the molecular signature of their secreted EV cargo under hyperglycemic conditions remain elusive. Therefore, we hypothesized that hyperglycemic stress causes transcriptome changes in stem/progenitor cell-derived EVs that may compromise their reparative function. In this study, we performed an unbiased analysis of EV transcriptome signatures from 3 different stem/progenitor cell types by RNA sequencing. The analysis revealed differential expression of a variety of RNA species in EVs. Specifically, we identified 241 common-dysregulated mRNAs, 21 ncRNAs, and 16 miRNAs in three stem cell-derived EVs. Gene Ontology revealed that potential function of common mRNAs mostly involved in metabolism and transcriptional regulation. This study provides potential candidates for preventing the adverse effects of hyperglycemia-induced stem/progenitor cell-derived EV dysfunction, and reference data for future biological studies and application of stem/progenitor cell-derived EVs.

Laboratory management curriculum for cytopathology subspecialty training.

Laboratory management should be an integral part of training in pathology residency and fellowships. Herein, we have outlined some basic laboratory management topics a graduating cytopathology fellow should be familiar with. An overview of regulatory agencies that have oversight over laboratory testing, cytopathology laboratory accreditation, pre-analytic, analytic and post-analytic quality assurance, billing/coding, basic statistics, verification/validation of testing, physician credentialing, board certification/maintenance of certification, and malpractice in cytopathology are addressed. This review is by no means all inclusive, but rather a guide to the basic management related topics to be covered during cytopathology subspecialty training.

µ-Calpain as a Novel Target for Impairment of Nitric Oxide-Mediated Vascular Relaxation in Diabetes: A Mini Review.

Diabetes is one of the most prevalent metabolic disorders. In diabetes, incidence of coronary artery diseases and peripheral vascular diseases is increased 2- to 4-fold and 10-fold, respectively, compared to healthy individuals. In spite of extensive studies, the underlying mechanisms of endothelial dysfunction (ED), an early event in the development of vascular diseases, remain incompletely understood in diabetes. This mini-review discusses the role and signaling pathways of calpains - a family of Ca2+-sensitive intracellular proteases in nitric oxide (NO)-mediated ED in diabetes. We conclude that activation of calpains, especially µ-calpain, plays an important role in the pathogenesis of NO-mediated ED and inflammatory responses in diabetes which is mainly via endothelial Nitric Oxide Synthase (eNOS) inactivation/degradation in macro- and micro-vasculature. We review existing literature demonstrating that hyperhomocysteinemia, elevated plasma homocysteine level, potentiates hyperglycemia-induced ED via µ-calpain/PKCß2 activation-induced eNOS-pThr497/495 and eNOS inactivation. µ-calpain may be a critical therapeutic target for NO-mediated ED in diabetes.

Five top stories in cytopathology.

CONTEXT: Cytology relies heavily on morphology to make diagnoses, and morphologic criteria have not changed much in recent years. The field is being shaped predominantly by new techniques for imaging and for acquiring and processing samples, advances in molecular diagnosis and therapeutics, and regulatory issues. OBJECTIVE: To review the importance of classical morphology in the future of cytopathology, to identify areas in which cytology is expanding or contracting in its scope, and to identify factors that are shaping the field. DATA SOURCES: Literature review. CONCLUSIONS: Five stories paint a picture in which classical cytomorphology will continue to have essential importance, both for diagnosis and for improving our understanding of cancer biology. New endoscopy and imaging techniques are replacing surgical biopsies with cytology samples. New molecularly targeted therapies offer a chance for cytology to play a major role, but they pose new challenges. New molecular tests have the potential to synergize with, but not replace, morphologic interpretation of thyroid fine-needle aspirations. Ultrasound-guided fine-needle aspiration performed by cytopathologists is opening a new field of "interventional cytopathology" with unique value. For the productive evolution of the field, it will be important for cytopathologists to play an active role in clinical trials that document the ability of cytology to achieve cost-effective health care outcomes.

Implementation of the Bethesda System for Reporting Thyroid Cytopathology: observations from the 2011 thyroid supplemental questionnaire of the College of American Pathologists.

CONTEXT: Although information about the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) has been widely disseminated since its inception in 2007, the extent of its implementation and impact on daily practice has not been formally evaluated. OBJECTIVES: To assess the extent of uptake of TBSRTC across pathology laboratories and to evaluate its impact on daily practice by collating participant responses to the 2011 supplemental thyroid questionnaire of the College of American Pathologists. DESIGN: A questionnaire was designed to gather information about various aspects of TBSRTC and mailed in June 2011 to 2063 laboratories participating in the College of American Pathologists cytopathology interlaboratory comparison program. The participating laboratories' answers were collated and summarized. RESULTS: Seven hundred and seventy-seven laboratories (37.6%) returned the survey. Although 60.9% (n = 451) and 17.1% (n = 127) of laboratories reported using TBSRTC or planning to use it in the near future, respectively, 22% (n = 163) had no plans to implement TBSRTC. Of the latter, 32% (n = 70) stated that they were unaware of this classification system. The majority (78.3%, n = 343) of the laboratories used TBSRTC as published in the Thyroid Bethesda System atlas, whereas 21.7% (n = 95) used it with minor modifications. Most reported that the use of TBSRTC had caused either no change (n = 67, 15.2%) or only minor changes (n = 353, 80.2%) in the terminology and diagnostic criteria previously used in their laboratories. CONCLUSIONS: According to the collected data, TBSRTC is generally well implemented in pathology laboratories. However, because approximately a third of those not using this terminology are not aware of it, additional educational efforts regarding TBSRTC are warranted.