Gordon L. Todd

Isoproterenol-induced myocardial necrosis and membrane permeability alterations in the isolated perfused rabbit heart.

Abstract The quantitative effects of isoproterenol on myocardial necrosis and its effect on membrane permeability using the tracer horseradish peroxidase were studied in isolated, Langendorff-perfused rabbit hearts. The hearts were perfused at a column height of 70 cm with a modified Krebs — Henseleit bicarbonate buffer gassed with 95% O 2 : 5% CO 2 . Isoproterenol was infused at constant rates with separate molar concentrations of 10 −6 , 10 −5 , 10 −4 , and 10 −3 M . Horseradish peroxidase was infused manually for 3 min at the end of the experiment and reacted with Hanker — Yates reagent. Two predominant forms of “contraction band” lesions were observed: small “paradiscal” lesions involving clumping of a few sarcomeres adjacent to the intercalated disc and “holocytic” contraction band lesions involving clumping of myofilaments throughout the myocardial cell. Each dose of isoproterenol produced significantly increased numbers of both types of lesions. Incubation of tissue for horseradish peroxidase produced significant accumulation within myocardial cells damaged with “contraction band” lesions. This accumulation was occasionally seen in “normal” appearing cells with isoproterenol, but not in control hearts. The results support the use of quantitation of lesions as an index of severity of isoproterenol cardiotoxicity and documented the early involvement of membrane permeability alterations in the pathogenesis of these “contraction band” lesions.

31P nuclear magnetic resonance measurement of cardiac pH in perfused guinea-pig hearts ☆

Abstract Cardiac pH was measured by 31 P nuclear magnetic resonance spectroscopy. Measurements were made on 23 control hearts perfused with Krebs-Henseleit bicarbonate buffer containing potassium phosphate (1.2 m m ) at pH 7.4, but with no external bathing medium. Only one very weak peak could be observed in the orthophosphate region at pH 7.0±0.1. Previous reports have shown a relatively strong line in this region at pH 7.4. The pH 7.0 value was confirmed by perfusion with Krebs-Henseleit bicarbonate-phosphate at pH 7.5, but this time in the presence of an external bathing medium composed of the same buffer. Two peaks were observed in the orthophosphate region. One was at pH 7.0 while the other was at pH 7.5. Replacement of the external bathing medium and perfusate by one free of phosphate (Krebs-Henseleit bicarbonate-Tris-HCl, 1.2 m m Tris plus 1.2 m m KCl, pH 7.4) caused the pH 7.5 peak to disappear but did not affect the pH 7.0 line. Thus, cardiac pH in well-oxygenated, Krebs-Henseleit bicarbonate-phosphate perfused hearts is 7.0, not 7.4. In addition, the magnitude of the observed acidosis associated with coronary artery ligation was about two times smaller (∼0.4 pH units) than that seen in the other studies owing to this difference in control pH. The current value of cardiac pH determined by nuclear magnetic resonance is now in excellent agreement with the values determined by more conventional methods.

The endocytic recycling regulator EHD1 is essential for spermatogenesis and male fertility in mice.

BackgroundThe C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1-/- mice using a Cre/loxP system.ResultsBoth male and female Ehd1-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1-/- adult males were infertile and displayed decreased testis size, whereas Ehd1-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1-/- mice.ConclusionsContrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.

Effects of Topical and Systemic Nicotine on Gingival Blood Flow in Dogs

It has been suggested that due to its vasoconstrictive action, nicotine may have a deleterious effect on the periodontium. This study examined the effects of topical and systemic nicotine administration on gingival blood flow. Eighteen young adult dogs were divided into three groups receiving the following treatments for 28 days: topical nicotine in orabase, systemic nicotine via osmotic mini-pumps, and topical orabase or systemic saline via osmotic mini-pumps. Blood flow to the gingiva was measured (at days 0 and 28) by the radiolabeled microsphere method. Blood flow was consistently increased from day 0 to day 28 in the nicotine-treated animals. Comparison of days 0 and 28 blood-flow values demonstrated a statistically significant change (p<0.05) in the anterior regions of the topical-nicotine group as compared with the control group. The increased flow may be a reflection of the mode of nicotine delivery and timing of the blood-flow determination procedures.

The Influence of Surgical Demonstrations During an Anatomy Course on the Perceptions of First-Year Medical Students Toward Surgeons and a Surgical Career

OBJECTIVE We previously have demonstrated the educational benefits of surgical demonstrations to first-year medical students. The aim of this current study was to analyze the influence of these demonstrations on the perceptions of students toward surgeons and a possible career in surgery. METHODS A faculty member from the Department of Surgery provided an instruction on pancreatic malignancies and management to first-year medical students during their gross anatomy course. After this instruction, using a lightly embalmed cadaver, the clinically relevant anatomy was detailed and a pancreaticoduodenectomy was performed on the cadaver. Immediately after the demonstration, a brief survey was conducted to obtain feedback from the students about the experience. RESULTS A total of 170 students over 2 years returned the survey for a response rate of 69%. The demonstration provided 77% of students with a favorable impression of surgeons, and 90% of the students felt that this exposure gave them an understanding of the knowledge, skills, and qualities needed to become a surgeon. Additionally, 57% of respondents stated that watching the demonstration increased the likelihood of them pursuing a surgical career. For the 67% of students who were considering a surgery career, the demonstration reinforced their interest; however, for the students who were not interested in surgery, the demonstration did not alter their opinion. CONCLUSION The results of this study showed that surgical demonstrations to first-year medical students can influence their perceptions favorably about surgeons and a surgical career. This interaction provided students with information and motivation to pursue a career in surgery and also may counteract any negative stereotypes of the field that first-year students may have had.

Cardioprotective effects of diltiazem when given before, during or delayed after infusion of norepinephrine in anesthetized dogs☆

Catecholamine excess has been shown to produce 2 distinct forms of irreversible myocardial necrosis termed contraction band lesions. Calcium channel blocking agents provided a variable protective effect from these contraction band lesions. The purpose of this study was to determine the temporal responses of the most effective of these blocking agents, diltiazem, when given before, simultaneous with or after an initial exposure to a necrogenic infusion of norepinephrine (NE). Forty-one adult mongrel dogs were anesthetized with sodium pentobarbital (32 mg/kg) and infused with saline solution or NE (4 micrograms/kg/min) for 60 minutes or diltiazem at a rate of 20 micrograms/kg/min for the first 5 minutes and 10 micrograms/kg/min for the remaining 70 minutes. Diltiazem was infused as pretreatment 15 minutes before continued infusion with NE for 60 minutes, simultaneously infused with NE for 60 minutes or delayed 30 minutes after the start of NE infusion. Diltiazem alone exhibited no significant effect on hemodynamics, but pretreatment with diltiazem was able to moderate the rapid NE-induced increases in heart rate. NE infusion produced significant numbers of the 2 forms of contraction band lesions: (1) paradiscal contraction band lesions involving a small portion of the cell adjacent to the disc, and (2) holocytic contraction band lesions involving the entire cell. Diltiazem reduced the number of contraction band lesions, particularly the holocytic contraction band lesions, provided diltiazem was available before the insult and massive influx of calcium with a pharmacologic dose of NE. Although the exact mechanism of diltiazems cardioprotective properties is not known, the timing of drug administration does appear to affect the degree of protection.

Comparison of Phenotypic and Functional Dendritic Cells Derived from Human Umbilical Cord Blood and Peripheral Blood Mononuclear Cells

Dendritic cells (DC) are important accessory cells that are capable of initiating an immune response. Generation of functional DC has potential clinical use in treating diseases such as cancer. In this report, we have demonstrated the generation of functional DC from mononuclear cells isolated from human umbilical cord blood cells (UCBC) and peripheral blood cells (PBC) using a defined medium Prime Complete Growth Medium (PCGM) (GenePrime LLC, Gaithersburg, MD). DC generated using PCGM showed the typical phenotype of DC as determined by flow cytometry and electron microscopy. Further analysis of the DC using confocal microscopy showed localization of the antigen and major histocompatibility complex (MHC) molecules in the cytoplasm 3-5 days following tumor antigen loading into DC. Subsequently, the tumor antigen-MHC complex was localized on the surface of DC. DC generated from UCBC or PBC also increased (p < 0.001) the allogeneic mixed lymphocyte reaction, confirming their immune accessory functions compared to a control mixed lymphocyte reaction (MLR) without DC added. Interestingly, DC generated using PCGM medium also significantly enhanced the hematopoietic colony (CFU-C)-forming ability. Furthermore, addition of 5% DC derived from cord blood loaded with tumor antigen also significantly (p < 0.001) increased peripheral and cord blood-derived antigen-specific cytotoxic T lymphocyte (CTL)-mediated killing of human leukemic cells (K562) and breast cancer cells (MDA-231). Thus, these results show that functional DC generated from cord blood using a defined medium are a useful source of accessory cells for augmenting CTL-mediated cytotoxicity and have potential use in cellular therapy for human leukemia and breast cancer.

Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.

The relationship of the subarachnoid injection of blood and blood fractions with cardiac rate change and arrhythmias.

The relationship of subarachnoid hemorrhage and cardiac arrhythmias was studied utilizing a Sprague-Dawley rat model. A total of 30 male animals were divided into five groups and given subarachnoid injections of either blood, blood fractions, or control substances. Blood pressure, intracranial pressure, serum electrolytes, arterial blood gases, hypothalamic multiple unit activity and an electrocardiogram were concurrently monitored. Cardiac arrhythmias were graded on a 0 to 4 + objective scale. Control parameter values were similar for all animals. Arrhythmias, hypotension, and decreased hypothalamic multiple unit activity were seen with infusion of whole blood and packed red blood cells. Packed red blood cells were statistically demonstrated to have the most potent arrhythmogenic effect. Cardiac histopathology revealed myocardial contraction band lesions most predominant in the packed red blood cell group. In addition, significant QT interval prolongation was observed after subarachnoid injection of either whole blood or packed red blood cells. These findings indicate that packed red blood cells, or a component thereof, may play an important role in the etiology of immediate (i.e. acute) post subarachnoid hemorrhage induced cardiac arrhythmias.

Novel Treatment for Mantle Cell Lymphoma Including Therapy-Resistant Tumor by NF-κB and mTOR Dual-Targeting Approach

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell non-Hodgkin lymphomas with a median survival of approximately five years. Currently, there is no curative therapy available for refractory MCL because of relapse from therapy-resistant tumor cells. The NF-κB and mTOR pathways are constitutively active in refractory MCL leading to increased proliferation and survival. Targeting these pathways is an ideal strategy to improve therapy for refractory MCL. Therefore, we investigated the in vitro and in vivo antilymphoma activity and associated molecular mechanism of action of a novel compound, 13-197, a quinoxaline analog that specifically perturbs IκB kinase (IKK) β, a key regulator of the NF-κB pathway. 13-197 decreased the proliferation and induced apoptosis in MCL cells including therapy-resistant cells compared with control cells. Furthermore, we observed downregulation of IκBα phosphorylation and inhibition of NF-κB nuclear translocation by 13-197 in MCL cells. In addition, NF-κB–regulated genes such as cyclin D1, Bcl-XL, and Mcl-1 were downregulated in 13-197–treated cells. In addition, 13-197 inhibited the phosphorylation of S6K and 4E-BP1, the downstream molecules of mTOR pathway that are also activated in refractory MCL. Further, 13-197 reduced the tumor burden in vivo in the kidney, liver, and lungs of therapy-resistant MCL-bearing nonobese diabetic severe-combined immunodeficient (NOD/SCID) mice compared with vehicle-treated mice; indeed, 13-197 significantly increased the survival of MCL-transplanted mice. Together, results suggest that 13-197 as a single agent disrupts the NF-κB and mTOR pathways leading to suppression of proliferation and increased apoptosis in malignant MCL cells including reduction in tumor burden in mice. Mol Cancer Ther; 12(10); 2006–17. ©2013 AACR.