Dav y C.H. Cheng, MD, MSc, FRCPC, FCAHS

• Distinguished University Professor and Chair
• Department of Anesthesia and Perioperative Medicine
• University of Western Ontario
• Chief of Anesthesia and Perioperative Medicine
• London Health Sciences Center and St. Joseph's Health Care
• London, Ontario, Canada

Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii Toxoplasma Gondii References 1041 genomes blood pressure jumps from high to low hydrochlorothiazide 12.5mg online. The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates blood pressure natural remedy purchase hydrochlorothiazide with amex. The generic genome browser: a building block for a model organism system database pulse pressure treatment discount hydrochlorothiazide 12.5mg fast delivery. As clinical disease in humans is extensively reviewed in Chapter 4 heart attack questions cheap 12.5 mg hydrochlorothiazide overnight delivery, Human Toxoplasma infection hypertension medication guidelines purchase hydrochlorothiazide overnight delivery, this chapter will focus on our mechanistic understanding of cerebral toxoplasmosis or T blood pressure under 120 cheap hydrochlorothiazide 25mg free shipping. An in-depth review of toxoplasmosis animal models, including cerebral toxoplasmosis is presented in Chapter 7, Toxoplasma animal models and therapeutics. The genes that account for the strain-specific differences in acute virulence in mice have been identified and are extensively covered in Chapter 14 "Toxoplasma secretory proteins and their roles in parasite cell cycle and infection"; Chapter 17 "Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages"; and Chapter 25 "Innate immunity to Toxoplasma gondii. In studies that have used more than one strain, strain-specific outcomes have been observed (Suzuki et al. Finally, in addition to differences between strain types, even two strains from the same strain type/clade can produce different outcomes (Blanchard et al. Collectively, these discrepancies mean that variations in experimental paradigms. The complexity of the brain includes its variability in cell types as well as how those cell types vary by brain region. Neurons are the major "signaling" cells of the brain that underlie the processing network that allows integration of complex information (from internal or external stimuli) to initiate simple and complex behavioral responses. Astrocytes, once considered simple support cells for neurons, are recognized to play many critical roles in the brain, including being an essential part of the brain immune response. Like neurons, astrocytes have different subtypes (Miller and Raff, 1984; Zamanian et al. These differences in distribution often lead to confusion about the number of glia to neurons. Careful counts in uninflamed brain suggest that this ratio varies across the brain, with white matter having a very high ratio of glia to neurons and the cerebral cortex having an almost 1:1 ratio across a range of mammals, including humans (Herculano-Houzel, 2014). Cerebral toxoplasmosis forms then differentiate into the rapidly dividing tachyzoite form, allowing the parasite to replicate within the small intestine (Gregg et al. In mice, within days after oral infection, extracellular and intracellular tachyzoites within infected monocytes are detected in the peripheral blood (Courret et al. In these studies, the mean circulation half-life of free tachyzoites in the blood was calculated to be approximately 3 minutes (Konradt et al. The blood vessels of this unique barrier comprise endothelial cells, which are surrounded by pericytes and astrocyte endfeet (Serlin et al. Collectively, these cellular structures form the neurovascular unit, a highly restrictive barrier that protects the brain by limiting the passage of immune cells, solutes, and toxins from the bloodstream into the brain during homeostasis. A key feature of the brain capillary endothelial cells is that they express tight junction proteins, such as occludin and claudin-5, thereby interconnecting the endothelial cells and contributing to the low permeability of this barrier (Potente and Makinen, 2017; Zhao et al. These features of activated endothelium allow immune cells and Toxoplasma Gondii 24. In order for extracellular parasites in the circulation to transmigrate into tissues, parasites must be able to attach to blood vessel endothelial cells in rapidly flowing blood. A recent study showed that during in vivo infection via oral or intraperitoneal administration of T. Consistent with these findings, tachyzoites have been shown to invade and replicate preferentially in human retinal vascular endothelial cells compared to dermal endothelial cells (Zamora et al. Cerebral toxoplasmosis endothelial cells were seeded into a transwell system, revealed that T. The hypermotility phenotype involves rapid actin cytoskeleton remodeling, integrin redistribution, and the loss of adhesive podosomes (Weidner et al. Based upon the studies with higher patient numbers (studies with data on $ 100 patients) (Arendt et al. In a small number of autopsies on severely immunocompromised patients, parasites/cysts were identified within astrocytes, neurons, oligodendrocytes, vascular endothelium, and pericytes (Bertoli et al. Huge variability exists in the identified regions that may be accounted for by differences in rodent model (rat vs mouse; immunocompetent vs immunocompromised); time postinfection analyzed; T. This study notes a high level of cysts in several areas of the cortex, basal ganglia, hippocampus, and amygdala and low levels of cysts in the cerebellum, the pons, caudate putamen, and white matter tracts. The findings of high cyst levels within the cortex and low levels within the brainstem/white matter tracts are consistent with human data and work from another group that localized neurons injected with T. At 3 weeks postinfection, the brain was harvested, sectioned into B200 m thick sections, processed to render the tissue optically clear, and then imaged at 40 3 on a confocal microscope. As very little is known about the role of oligodendrocytes in cerebral toxoplasmosis, oligodendrocytes are not discussed. For many years, neurons have been considered bystanders in most neuroinflammatory responses, though this view is changing as accumulating evidence in virology suggests that neurons do respond to inflammatory cytokines. Only one study has used conditional knockouts to look at the role of neuronal immune signaling in controlling cerebral toxoplasmosis in vivo (Handel et al. Collectively, these data suggest that neurons play a central role in the control of cerebral toxoplasmosis. Both human primary astrocytes and human astrocytoma (tumor) cell lines have been used in studies of the T. Further support for the essentiality of astrocytic responses in cerebral toxoplasmosis comes from studies in conditional and full knockout mice. This increased neuroinflammatory response correlated with increased neuronal stress and possible death at early and late time points (Cekanaviciute et al. The prevailing view for a large part of the 20th century was that microglia were neuroectodermal cells similar to neurons, astrocytes, and oligodendrocytes (Ginhoux et al. In vivo imaging has shown that microglial processes are highly dynamic and constantly survey the brain parenchyma (Nimmerjahn et al. Despite being called microglial nodules a variety of cells, including mononuclear cells, lymphocytes, and astrocytes, may also be part of these nodules that are associated with extracellular parasites (Frenkel, 1949; Stenzel et al. Within the nodules myeloid cells are hypertrophic and amoeboid, which is suggestive of microglia activation. Thus microglia/macrophages are found in association with parasites and may have an active role in clearing parasites from the infected brain (Conley and Jenkins, 1981; Frenkel, 1949; John et al. In addition, in vitro cultures of "microglia" are usually generated from neonatal mice, which do not resemble microglia isolated from the adult brain (Butovsky et al. The cultures are also purified in a way that brain perivascular macrophages might also be present and incorrectly identified as microglia. Thus, as newer techniques offer mechanisms for exclusively identifying or targeting microglia using Cre/loxP strategies in vivo and culturing microglia from adult mice in vitro (Ajami et al. With these caveats in place, below we summarize what is currently known about the role of microglia in cerebral toxoplasmosis, which primarily comes from studies of rodent microglia, most commonly mouse. Cerebral toxoplasmosis Microglial appear to help control cerebral toxoplasmosis through direct or cell-intrinsic mechanisms. In addition to the direct action on parasites, microglia may also produce cytokines and chemokines that affect the neuroinflammatory response. As demonstrated in the intestine, the resident macrophage population was insufficient to control T. However, as noted before, much of this knowledge has been gleaned from work unable to differentiate microglia from macrophages. Given the similarities of microglia and macrophages, these cell types may have similar mechanisms that contribute to the control chronic T. As noted above, dissecting out the role of macrophage versus microglia is challenging at this time but new techniques may allow future experiments that specifically target each population individually. For cerebral toxoplasmosis, more work is required to fully determine the importance of antigen presentation in the brain and which cell types play a critical role. Typically, neutrophil infiltration is associated with an inability to control parasite replication or inflammation in the brain. As noted above, recent studies have also demonstrated that neutrophil depletion affects monocyte recruitment during chronic T. Mast cells reside in the meninges and may shape the inflammatory T cells are required to control T. Following T-cell depletion, mice rapidly succumb to infection and are unable to control parasite reactivation within the brain (Gazzinelli et al. Perforin knockout mice succumb to infection in the chronic phase and have significantly higher parasite burden in the brain than wild-type mice (Denkers et al. Surprisingly, the migration pattern was not mediated by chemokine signals, but rather, chemokine signals enhanced the speed of T cells migrating in the brain, which was predicted to increase the capacity of T cells to encounter rare infected cells in the brain (Harris et al. The increase in Treg number correlated with a protection from lethal immunopathology but increased parasite cyst burden in the brain during chronic infection (Oldenhove et al. This study highlights that Tregs are involved in maintaining a balance between protective immunity and immunopathology. A major question regarding the role of Tregs during infection is whether or not the Tregs are specific for the pathogen. Future studies are required to determine the specific roles of Tregs once they reach the brain using depletion studies and cell-type-specific knockout mice. Together, these studies highlight the potential importance of B cells during chronic T. The inconsistencies in the findings may arise from many factors, such as rodent or parasite strain, mode or timing of infection, or behavior assessed, but the lack of consistency makes it difficult to draw definitive conclusions about the extent to which T. Despite this inconsistency, several hypotheses for the cause of these behavioral changes have been suggested. These hypotheses [as reviewed in Tedford and McConkey (2017)] include (1) the location of persistent cysts; (2) changes in neurotransmitter levels, especially dopamine; (3) parasites producing excess dopamine; (4) neuroinflammation affecting specific pathways; and (5) infection-associated hormonal changes. While these hypotheses are not mutually exclusive and some data underlie each one, contradictory data for most hypotheses also exist. Similarly, two studies identified increases in dopamine or its metabolites in the brains of T. Thus, at this time, no definitive mechanisms have been identified to explain the T. Though these findings are most easily explained by a loss of parenchymal cells, histology on the mice showed no loss of neurons, axonal injury, or "extensive" demyelination (Hermes et al. One of the major issues with these studies is that, by necessity, they rely upon a correlation between T. Correlations do not prove causation, and it is equally plausible that the personality traits or disease process studied increases the propensity for acquiring a T. In addition, many of the studies have relatively small numbers of people (Kusbeci et al. The findings described above reveal our current understanding of the complex interactions between the immune system, the parasite, and the brain, which govern T. Future work will undoubtedly address many of these outstanding questions such as determining regional susceptibility to inflammation, the immune capabilities of neurons, segregating the roles of microglia versus infiltrating peripheral monocytes/macrophages, and defining antigen presentation in the brain. Chronic Toxoplasma infection modifies the structure and the risk of host behavior. Prevalence of Toxoplasma gondii infection in brain and heart by immunohistochemistry in a hospital-based autopsy series in Durango, Mexico. Toxoplasma gondii infection and mixed anxiety and depressive disorder: a case-control seroprevalence study in Durango, Mexico. A spectrum in the pathology of toxoplasmosis in patients with acquired immunodeficiency syndrome. Infection of human astrocytes and glioblastoma cells with Toxoplasma gondii: monocyte chemotactic protein-1 secretion and chemokine expression in vitro. Neurons are the primary target cell for the brain-tropic intracellular parasite Toxoplasma gondii. Dissecting amyloid beta deposition using distinct strains of the neurotropic parasite Toxoplasma gondii as a novel tool. Homeostatic interferon expression in neurons is sufficient for early control of viral infection. Regulatory T cells selectively preserve immune privilege of selfantigens during viral central nervous system infection. Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism. Immunohistological study of the anatomic relationship of toxoplasma antigens to the inflammatory response in the brains of mice chronically infected with Toxoplasma gondii. Toxoplasma gondii disrupts 1 integrin signaling and focal adhesion formation during monocyte hypermotility. Motile invaded neutrophils in the small intestine of Toxoplasma gondiiinfected mice reveal a potential mechanism for parasite spread. Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Antiparasitic effector mechanisms in human brain tumor cells: role of interferon-gamma and tumor necrosis factor-alpha.

Islet culture supplements Research efforts have been dedicated to identifying the right combination of islet culture medium blood pressure percentile cheap hydrochlorothiazide 12.5mg online, supplements and oxygenation strategies to implement alternative islet culture protocols that stimulate and preserve islet mass and function for longer periods during storage and transportation prior to transplantation pulse pressure klabunde buy hydrochlorothiazide 25mg otc. The use of a serum-free heart attack or pulled muscle discount 12.5mg hydrochlorothiazide amex, hormone- and factorsupplemented arrhythmia beta blocker order hydrochlorothiazide 25mg without a prescription, defined medium has been shown to support cell culture and cultures of beta cells and cell lines blood pressure medication during pregnancy order generic hydrochlorothiazide online. Among the most common ones are vitamin E (alphatocopherol) blood pressure 5332 buy cheap hydrochlorothiazide 12.5mg on-line, deferoxamine and nicotinamide. An elegant study has shown that the activation of beta cells by pyruvate suppresses the mitochondrial rise in Ca++ and glucagon secretion in alpha cells, an example of the paracrine activity of beta cells on alpha cells in response to a single agent. Pancreas and islet preservation Cryopreservation Preserving the structural integrity and function of islets that endured the stresses of isolation and purification is central to islet transplantation. Cryopreserving high quality islets for extended periods would facilitate conducting quality control tests, pooling islets from different donors, and transport and shipping to remote locations to benefit a larger population of T1D diabetic patients. However, the efficiency of the process needs to be considered given the potential for significant cell losses during the disaggregation/reaggregation process. Additional studies with whole islets and single cells from disaggregated islets are warranted to better document the recovery efficiency and quality of islets rendered by current and new methods in different species. Protocols able to cryopreserve islets for weeks or months are highly desirable as they provide a much-needed flexibility to: (1) islet banking, (2) pooling islets from different donors if needed, and (3) running a complete battery of quality assessment tests before transplantation. Emphasis should be placed on developing alternative, less disruptive methods to enzymatic digestion and distention, as structural damage to the islet vasculature and 3D cytoarchitecture occurs mainly during these steps. The transplantation field would also vastly benefit from adopting current and novel methodologies for oxygenation of whole pancreata prior to isolation and islets during isolation, culture and shipment. Islet allo-transplantation References 519 macroencapsulation devices following transplant into mice. Human embryonic stem cell derived islet progenitors mature inside an encapsulation device without evidence of increased biomass or cell escape. Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts. Risks and benefits of transplantation in the cure of type 1 diabetes: whole pancreas versus islet transplantation. Successful simultaneous islet-kidney transplantation using a steroid-free immunosuppression: two-year follow-up. Long-term improvement in glucose control and counterregulation by islet transplantation for type 1 diabetes. New insights into the architecture of the islet of Langerhans: a focused cross-species assessment. The islet transplantation field is accelerating the transition from a therapeutic approach that is currently focused on a subpopulation of few selected T1D patients and involves a high donor to recipient ratio to treating larger typical T1D patient populations using islets from single donors. The generation of human stem-cell derived islets that can be expanded in the lab in unlimited quantities, and the development of approaches (such as cell encapsulation) that eliminate the need for immunosuppression may in the near future result in a truly effective and scalable treatment for insulin dependent patients including children. Methods for insulin delivery and glucose monitoring in diabetes: summary of a comparative effectiveness review. Insulin pump therapy is associated with less post-exercise hyperglycemia than multiple daily injections: an observational study of physically active type 1 diabetes patients. Outcomes of simultaneous pancreas-kidney transplantation from brain-dead and controlled circulatory death donors. Simultaneous pancreas-kidney transplantation from donation after cardiac death: successful long-term outcomes. Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats. Maturation and function of human embryonic stem cell-derived pancreatic progenitors in 19. Chronic anemic hypoxemia attenuates glucose-stimulated insulin secretion in fetal sheep. Hypoxia-inducible factor-1alpha regulates beta cell function in mouse and human islets. Advances and challenges in islet transplantation: islet procurement rates and lessons learned from suboptimal islet transplantation. Oxygen environment and islet size are the primary limiting factors of isolated pancreatic islet survival. Effect of oxygen supply on the size of implantable islet-containing encapsulation devices. Liver transplantation using organ donation after cardiac death: a clinical predictive index for graft failure-free survival. Clinical islet isolation and transplantation outcomes with deceased cardiac death donors are similar to neurological determination of death donors. Long-term outcomes of clinical transplantation of pancreatic islets with uncontrolled donors after cardiac death: a multicenter experience in Japan. Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Calpain mediates ischemic injury of the liver through modulation of apoptosis and necrosis. Calpain is a mediator of preservation-reperfusion injury in rat liver transplantation. Evidence for role of cytosolic free calcium in hypoxia-induced proximal tubule injury. Vascular adaptations to hypoxia: molecular and cellular mechanisms regulating vascular tone. Protection by glycine against hypoxic injury of rat hepatocytes: inhibition of ion fluxes through nonspecific leaks. Alteration of Na+ homeostasis as a critical step in the development of irreversible hepatocyte injury after adenosine triphosphate depletion. Ischaemia-reperfusion injury and hyperbaric oxygen pathways: a review of cellular mechanisms. Pancreatic cellular injury after cardiac surgery with cardiopulmonary bypass: frequency, time course and risk factors. Impairment of microcirculation in the early reperfusion period predicts the degree of graft pancreatitis in clinical pancreas transplantation. Ischemia reperfusion of the pancreas: a new in vivo model for acute pancreatitis in rats. The role of nitric oxide in the physiology and pathophysiology of the exocrine pancreas. Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival. Impaired glucose homeostasis after a transient intermittent hypoxic exposure in neonatal rats. Insulin production hampered by intermittent hypoxia via impaired zinc homeostasis. The rat pancreas is not an appropriate model for testing the preservation of the human pancreas with the two-layer method. Histidine-tryptophan-ketoglutarate solution for organ preservation in human liver transplantation-a prospective multi-centre observation study. Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation. Increased pancreatitis in allografts flushed with histidine-tryptophan-ketoglutarate solution: a cautionary tale. Histidine-tryptophanketoglutarate and University of Wisconsin solution demonstrate equal effectiveness in the preservation of human pancreata intended for islet isolation: a large-scale, single-center experience. Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution. Pancreas preservation with University of Wisconsin and Celsior solutions: a single-center, prospective, randomized pilot study. Evaluation of Institut Georges Lopez-1 preservation solution in pig pancreas transplantation: a pilot study. Comparative impact on islet isolation and transplant outcome of the preservation solutions Institut Georges Lopez-1, University of Wisconsin, and Celsior. Role of adenosine in preservation by the two-layer method of ischemically damaged canine pancreas. In vitro alteration of hematological parameters and blood viscosity by the perfluorocarbon: oxycyte. Perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules as potential artificial oxygen carriers: preclinical safety and biocompatibility. Functionality of albumin-derived perfluorocarbon-based artificial oxygen carriers in the Langendorff-heart (dagger). Possibility of islet transplantation from a nonheartbeating donor pancreas resuscitated by the two-layer method. Long-term preservation of the pig pancreas by a onelayer method for successful islet isolation. Successful pancreas preservation by a perfluorocarbon-based onelayer method for subsequent pig islet isolation. Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage in a split lobe model. Oxygenation of the portal vein by intraperitoneal administration of oxygenated perfluorochemical improves the engraftment and function of intraportally transplanted islets. Polyethylene glycols: an effective strategy for limiting liver ischemia reperfusion injury. Polyethylene glycol reduces the inflammatory injury due to cold ischemia/reperfusion in autotransplanted pig kidneys. Development of pancreas storage solutions: initial screening of cytoprotective supplements for beta-cell survival and metabolic status after hypothermic storage. Pancreatic L-glutamine administration protects pig islets from cold ischemic injury and increases resistance toward inflammatory mediators. The benefits of hypothermic machine perfusion are enhanced with Vasosol and alpha-tocopherol in rodent donation after cardiac death livers. Hypothermic machine preservation facilitates successful transplantation of "orphan" extended criteria donor livers. Efficient suppression of murine intracellular adhesion molecule-1 using ultrasoundresponsive and mannose-modified lipoplexes inhibits acute hepatic inflammation. Small molecule signaling agents: the integrated chemistry and biochemistry of nitrogen oxides, oxides of carbon, dioxygen, hydrogen sulfide, and their derived species. Islet allo-transplantation References 523 limitations of a modified experimental preparation. In vitro insulin productivity of preserved pancreas: a simple test to assess the viability of pancreatic allografts. Different dynamics of insulin secretion in the perfused pancreas of mouse and rat. Experimental renal preservation by normothermic resuscitation perfusion with autologous blood. Normothermic machine perfusion of deceased donor liver grafts is associated with improved postreperfusion hemodynamics. Hypothermic perfusion preservation of pancreas for islet grafts: validation using a Split Lobe Porcine Model. Ex vivo porcine organ perfusion models as a suitable platform for translational transplant research. Ex vivo normothermic porcine pancreas: a physiological model for preservation and transplant study. The fluosol-perfused isolated canine pancreas: a model for the study of blood component effects in acute pancreatitis. Use of carbon monoxide in minimizing ischemia/reperfusion injury in transplantation. Crucial role for neuronal nitric oxide synthase in early microcirculatory derangement and recipient survival following murine pancreas transplantation. Impact of venous systemic oxygen persufflation supplemented with nitric oxide gas on cold-stored, warm ischemia-damaged experimental liver grafts. Machine perfusion versus cold storage for preservation of kidneys from expanded criteria donors after brain death. Advances in machine perfusion graft viability assessment in kidney, liver, pancreas, lung, and heart transplant. Pancreaticoduodenal preservation by hypothermic pulsatile perfusion for twenty-four hours. An ex-vivo model for hypothermic pulsatile perfusion of porcine pancreata: hemodynamic and morphologic characteristics. Development of pancreatic machine perfusion: translational steps from porcine to human models. Pancreatic allograft thrombosis: donor and retrieval factors and early postperfusion graft function. Islet isolation from juvenile porcine pancreas after 24-h hypothermic machine perfusion preservation. The survival of mammalian tissues perfused with intravascular gas mixtures of oxygen and carbon dioxide. Maintenance of the heart beat by perfusion of the coronary circulation with gaseous oxygen. Adding pulsatile vascular stimulation to venous systemic oxygen persufflation of liver grafts.

The data presented in this chapter characterize trends related to the number of pancreas transplants performed in the United States by type of the procedure blood pressure chart hong kong purchase hydrochlorothiazide 12.5 mg with mastercard. The data also include characteristics of the recipients and donors of pancreas transplants pulse pressure 90 generic hydrochlorothiazide 12.5mg without a prescription. In addition arrhythmia originating in the upper chambers of the heart buy hydrochlorothiazide 12.5 mg overnight delivery, there is significant heterogeneity in the presence of pancreas transplant pro- grams within transplant centers in the United States hypertension 34 weeks pregnant buy hydrochlorothiazide 12.5 mg free shipping. The variation in the presence of pancreas programs and volume of pancreas transplants may affect access to care for end-stage organ disease patients that may benefit from the procedure arrhythmia for dummies hydrochlorothiazide 25 mg sale. This article depicts the variation in the size of transplant centers performing pancreas transplantation in the United States blood pressure medication names discount hydrochlorothiazide 25 mg without a prescription. Finally, outcomes following transplantation have also changed over time and the results of the short- and long-term graft and patient survival are described in the chapter. Cumulatively, the results describe the past and current landscape of transplantation in the United States. Number of pancreas transplant recipients Between 1995 and 2017, 27,297 pancreas transplants were performed in the United States. The remainder included pancreas-liver-intestine (n = 979), pancreas-intestine (n = 158), and other organ combinations (n = 54). In contrast the proportion of annual transplant recipients who were Black or other race/ethnic groups has significantly increased over time. Similarly, the proportion of patients with other race/ethnic groups increased over time. For pancreas transplants alone, private insurance was most common, however this proportion decreased and Medicare recipients comprised a larger proportion of patients over time. Donors with cerebrovascular event as a cause of death have significantly declined. The two centers with the largest number of transplants have performed more than the 80 centers with the fewest number of transplants combined. In 2017, 115 centers performed at least 1 transplant, 65 centers performed at least 5 transplants, and 32 centers performed at least 10 transplants. Similarly, in 1995, 14% of pancreas alone donors were over age 40 as compared to 4% in 2017. The number of transplant procedures has significantly changed over time including an increase in procedures between the mid1990s and the mid-2000s and a decline to present day. In addition to the overall number of transplants performed, the characteristics of pancreas transplant recipients and donors A. Trends in pancreas transplantation in the United States Recipient-pancreas alone 45. This includes a rise in recipient age and a decline in donor age among pancreas transplants. The racial/ethnic composition, body mass index, and donor race and causes of death have also changed over time. There is a wide variation in the size of pancreas transplant programs in the United States. There are a number of centers performing a small number of pan- creas transplants per year and a few centers performing relatively large numbers per year. Despite the relatively significant changes in characteristics and incidence of transplantation, outcomes following the procedure continue to improve. This may be due to improved technical skills and maintenance therapies as well as improved selection of both transplant candidates and donors. Trends in pancreas transplantation in the United States have been significant changes in treatment and outcomes for the general diabetic population in the United States. Dramatic secular changes in prognosis for kidney transplant candidates in the United States. Early studies by Billingham, Brent, Medawar, and Woodruff with fetal mice and tissue transplantation contributed greatly to the study of acquired immunity. Small animal models are ideally suitable for the study of diabetes mellitus and pancreas transplantation due to their short breeding cycle and wide range of genetic models. Large animal models were initially crucial in our understanding of the pathophysiology of diseases which eventually led to the treatment of end-stage organ failure through solid organ transplantation. The preeminent physiologist of the 19th century, Claude Bernard, used a dog model to study the role of the pancreas in digestion and described the glycogenic function of the liver. His and other studies in large animals led to a pivotal experiment by Minkowski and von Mering in 1889 in which a dog developed polyuria and diabetes after a total pancreatectomy, thereby identifying the central role of the pancreas in glucose metabolism. Experimental pancreas transplantation infrarenal aorta is most commonly used, but the use of the carotid artery has also been described. It can be directed enterically with a duodenal patch or duodenojejunostomy, or it can be diverted to the urinary system by anastomosing the donor duodenum to the bladder. Studies involving decellularized pancreatic transplants, reseeded with endothelial progenitor cells showed new vessel formation when transplanted to host rats, which presents as a potential further investigation of the use of scaffolds. Correlation studies are often performed between pancreas and different organ systems to evaluate the organ-specific immunogenicity. Compared to corneal transplants, pancreatic and skin grafts were rapidly rejected. The latter include technical complications, posttransplant infections, and graft thrombosis. Early on, the method of organ preservation can affect overall organ function, and determine the success of the transplant. Various methods of organ preservation have been studied in small animal models to reduce the incidence and magnitude of pancreatic reperfusion injury. It was also shown that reperfusion injury was greater due to ischemic changes than the insult of dissection. Whole pancreas allo-transplantation Small animal models 367 prostaglandin and interleukin levels as well as with pancreatic injury and apoptosis. The first and most commonly utilized method of organ preservation is the use of cold storage. Described in 1984, warm ischemia time of up to 2 h did not alter islet function, and hypothermic preservation could be tolerated up to 24 h. Ischemic preconditioning involves short periods of warm ischemia followed by reperfusion before extended cold ischemic preservation. Murine models are frequently utilized for demonstrating effectiveness of a therapeutic agent. Tetrahydrobiopterin, a cofactor for nitric oxide synthase, is depleted in reperfusion injury. Xenotransplant studies are often performed using a rat model as the recipient of pancreatic tissue from different donors such as pig, dog, fish, human, and mice. The fetal pancreas lacks exocrine tissue, and its proliferation potential makes it an ideal model for transplantation. While there is a scarcity of human fetal pancreatic tissue, porcine fetal tissues can be used with little difficulty. Fetal pancreatic transplantation is a nonvascular transplant, and depends on the blood supply of the engraftment site. Successful fetal porcine cells have been transplanted in the omentum and mesentery of rat models without requiring immunosuppression and normalization of glucose. While the results were encouraging when engrafted into the kidney and pancreas, the results were poor when engrafted in lung, liver, and spleen. This combined therapy showed prolonged graft survival in rat-to-mice islet xenotransplants, with higher rates of graft acceptance achieved with combination therapy of human anti alpha-antitrypsin. Different methods of islet xenotransplants have also been used to prevent rejection in small animal models. The purpose of xenotransplantation is to overcome the limitation of organ availability through inter-species transplantation. Rejection Small animal models have played an important role in the study of graft rejection including the use of biomarkers. Experimental pancreas transplantation found to be elevated in rats with graft failure because of the presence of hyperglycemia. The different stages were correlated with increased lymphocyte counts in the pancreatic juice. They transplanted the pancreas of Norway rats into Lewis rats after diabetes induction with streptozotocin. After initial immunosuppression with cyclosporine, each group was given different lengths of immunosuppressant-free days (2, 4, 6, 8, 9, and 11 days). A fraction of the animals from each time point were euthanized and the degree of rejection was histologically graded. The remainder of the group resumed immunosuppression using methylprednisolone and cyclosporine. The murine model is commonly used to study the effect of immunosuppressive agents on rejection and outcome. Uremic recipients of pancreas transplants in both pancreas transplant alone and simultaneous pancreas and kidney transplant had an increase in graft survival of endocrine and exocrine function. It also showed an increase in mean graft survival of endocrine and exocrine function in simultaneous pancreas and kidney transplantation compared to pancreas transplantation alone. Pharmacological agents such as monoclonal antibodies, antimetabolites, Il-2 inhibitor, and macrolide antibiotics have all been studied in murine models. The use of rapamycin has also been studied in rat models and showed improvement in pancreas allograft survival when combined with cyclosporine. They have also greatly improved our understanding of organ preservation, graft rejection, and transplant pharmacology. Current small animal research has moved from whole-organ pancreas transplantation to islet allo- and xenotransplantation. Whole-organ and segmental pancreas transplants as well as islet transplants have been successfully performed in both species, with studies focusing on technical aspects, graft outcomes, early and late technical and immunological complications, immunosuppressive regimens, and graft preservation. Canine model In the canine model, two types of pancreas transplants have been studied: whole-organ and segmental pancreas transplants. Whole-organ pancreas transplants in a canine model preserve the entire gland and are technically more challenging when compared to segmental pancreas transplants. In addition, cholecystoduodenostomy, gastroduodenostomy, free exocrine drainage into the peritoneal cavity, and ligation of the exocrine duct have also been described. The transected pancreatic duct can be anastomosed to the bowel, the bladder, the ureter, or the stomach. The anastomoses are performed to the external iliac artery and vein; the exocrine secretions are drained into the bladder or into the bowel. The donor portal vein is anastomosed to the recipient distal vena cava or common iliac vein. Segmental pancreas transplants involve the body and tail with transection of the pancreatic duct. Variations in the arterial supply to the body and tail include the following: a splenic artery, a pancreatic artery, which emerges from the splenic artery, or a pancreatic artery from the hepatic artery. It is performed in an en bloc fashion with the donor aorta (including the celiac axis, superior mesenteric artery, and left renal artery) anastomosed to the recipient aorta. The portal vein is anastomosed to the distal vena cava and the renal vein is anastomosed to the left common iliac vein. This en bloc technique has resulted in decreased preservation time, operation time, and clamp time. The theoretical disadvantage of systemic venous drainage is that it bypasses the initial hepatic metabolism of insulin (first-pass effect), thereby diverting the regulatory effect of the liver in endogenous glucose production. In canine studies, a common transplant model used to study this effect has been through segmental pancreatectomy and autotransplantation of the pancreas with portal venous drainage or with systemic venous drainage (anastomosis between the pancreaticoduodenal and gastrosplenic vein to the inferior vena cava). Not surprisingly, in systemically diverted canines, basal insulin levels were noted to be elevated, with an increase in the basal glucose metabolic clearance rate. With insulin infusion, systemically diverted subjects decrease in glucose metabolic clearance rate from a mean of 15. Agents such as octreotide and aprotinin showed no significant benefit in preventing graft pancreatitis. Functional outcome including glucose levels, pancreatic juice flow, and amylase levels was studied to evaluate the endocrine and exocrine dysfunction. In this study, the two-layer cold storage method showed preservation of almost normal architecture after 72 h of storage. Since the very first pancreas transplants in humans, rejection has been one of the major causes of posttransplant morbidity and mortality. Large animal models have been used to study the diagnosis, histopathology, immunology, and treatment of pancreas graft rejection. Detection of rejection was followed with implementation of antirejection therapy using cyclosporine and methylprednisolone. The time of rejection was noted to be earlier in fine-needle biopsy at a median of 5 days compared to urinary amylase levels, which led to earlier initiation of antirejection therapy. The use of fine needle biopsy to diagnose and treat rejection improved allograft survival from 9 days to 32 days.

Autophagy is generally seen as a prosurvival pathway which is important to maintain cellular homeostasis (Moreau et al hypertension vasoconstriction cheapest hydrochlorothiazide. For instance pulse pressure readings purchase hydrochlorothiazide visa, under physiological conditions arrhythmia young purchase hydrochlorothiazide mastercard, autophagy has a number of vital roles such as maintenance of the amino acid pool during starvation pulse blood pressure normal purchase hydrochlorothiazide 12.5 mg amex, clearance of damaged organelles blood pressure 7550 25 mg hydrochlorothiazide amex, and remodeling during cellular differentiation arteria jejunalis hydrochlorothiazide 25mg on line. In addition, in complex eukaryotes such as mammals, it has also evolved specialized prosurvival functions for the clearance of intracellular microbes and the regulation of innate and adaptive immunity (Mitchell and Isberg, 2017). Electron microscopy observation of intracellular parasites grown in diluted medium (Ghosh et al. Arrowheads show multimembrane compartments containing cytoplasmic material corresponding to autophagosomes. In mammals, fusion between the autophagosome and lysosomes (which may also involve an intermediate fusion between the autophagosome and an endosome to generate a transient structure called the amphisome) leads to the formation of a hybrid compartment called the autolysosome. In yeast, however, after fusion with the larger lytic vacuole, the inner autophagosome vesicle is directly released into its lumen as an autophagic body. However, these autophagic vesicles which are more advanced in the degradation process are less unequivocally recognizable because part of their content, including the inner membrane of the original autophagosome, might have been degraded. Thus for more accuracy and a more quantitative method, molecular markers can also be used to confirm the morphological hallmarks of autophagy observed in stressed tachyzoites. Yet, extensive biochemical evidence of digestion and recycling of autophagy substrates are lacking in Toxoplasma. Yeast and mammalian cells benefit from a long-standing knowledge and identification of autophagy substrates and adapters, so there are a number of markers and assays to monitor autophagic flux in these models (Delorme-Axford et al. However, an indirect method based on assessing the turnover of labeled long-lived proteins, allowing quantitative determination of the bulk autophagic flux, has been successfully implemented in tachyzoites and also suggested that there is an autophagydependent degradation of proteins (Nguyen et al. The use of pharmaceutical agents can also trigger autophagy in tachyzoites, like with the ionophore monensin, a very potent antiparasitic drug that is a potential inducer of oxidative stress (Lavine and Arrizabalaga, 2012). Both starvation and the use of monensin can lead to a marked fragmentation of the mitochondrial network that can be prevented by using 3-methyladenine, an inhibitor of autophagy-promoting PtdIns3k, suggesting a form of autophagic cell death is involved (Ghosh et al. Excessive or uncontrolled levels of autophagy may lead to cell death, but this is still a controversial notion that remains to be confirmed in Toxoplasma by experiments showing increased viability in these conditions upon genetic inactivation of the autophagic pathway in the parasites. Thus it is still unclear whether excessive autophagy might be responsible for cell death or, on the contrary, may be increased as a mechanism trying to prevent the cellular demise. In any case, harsh starvation conditions lead to a significant loss of tachyzoite viability coincidentally with the appearance of autophagosomes (Nguyen et al. Although Toxoplasma may experience extracellular stress during its developmental cycle, it is mostly an obligate intracellular parasite. Despite being impaired in the canonical degradative function of autophagy as assessed by quantifying the turnover of long-lived proteins, this mutant is viable in normal growth in vitro conditions (like most eukaryotic autophagy mutants grown in nutrient-rich conditions). These findings suggest that components of both the autophagic and proteolytic machineries may act synergistically to help surviving specific stresses encountered during the course of infection. Examples include the turnover of organelles during hematopoietic differentiation, or paternal Toxoplasma Gondii 15. Like other apicomplexan parasites, Toxoplasma goes through a complex life cycle that includes several developmental forms. In the intermediate host alone, the parasite can switch from the rapidly dividing tachyzoite to the encysted quiescent bradyzoite form upon stress. This cellular differentiation is accompanied by some morphological changes and some extensive metabolic adaptation. It is, however, not known if autophagy following encystation would be required for the recycling of organelles for cellular remodeling purposes, removal of damaged organelles associated with cellular stress, or for adaptation in response to a potentially more limited access to nutrients from the host (as the parasite is enclosed in a potentially less permeable compartment). These findings imply that the turnover of autophagic material in bradyzoites is crucial for parasite survival following differentiation. However, during the course of evolution, this ancient cellular feature has been modified and adapted by a number of different eukaryotic lineages. For example, budding yeasts use autophagosomal proteins in the cytoplasm-to-vacuole targeting pathway, for delivering cargo proteins to the vacuole and not for degradation purposes (Lynch-Day and Klionsky, 2010). In mammalian cells, autophagy has evolved into a mechanism of host defense: the autophagy machinery targets intracellular pathogens for lysosomal degradation directly through autophagosome formation, or though noncanonical modification of microbecontaining vacuoles (Mitchell and Isberg, 2017). Apicomplexan parasites have also evolved unexpected features for the autophagy machinery. As an evolutionary twist of fate of some sort, original players potentially involved in prokaryotic pathogen control have been repurposed for controlling replication of an organelle of prokaryotic origin. However, the Apicomplexa constitute a phylum that is very evolutionarily distant from most canonical eukaryotic models and as such, perhaps unsurprisingly, they present a number of divergent features. Thus they might have an apparently less complex machinery due to their parasitic lifestyle and the possibility to exploit their host for a number of functions. However, their adaptation to parasitism also leads to some extent to the evolution of lineage-specific proteins. Yet, another strategy for combining reduction of complexity with the capacity to generate novel features is the repurposing a Toxoplasma Gondii Glossary 733 number of preexisting proteins or pathways. Because they are quite remote from the canonical textbook models, deciphering some of these particularly original features represents a considerable challenge. Besides, some organelles, such as dense granules and micronemes, present discrete subpopulations with distinct protein contents (although it is currently unknown if such a diversity in composition is related to different specialized functions). As described in this chapter, a number of protein markers have now been identified. Glossary Acidocalcisomes small, dense, spherical, and acidic organelles containing high concentrations of calcium and phosphate Apicoplast a remnant nonphotosynthetic plastid that compartmentalizes key metabolic pathways Autophagosome a double-membrane structure formed by closure of the phagophore to encapsulate cargo Autophagy a process of encapsulating and delivering cellular proteins, organelles, and structures for lysosomal degradation Dense granules spherical secretory organelles that are distributed throughout the parasite cytoplasm. Dense granule proteins play many roles in nutrient acquisition, immune evasion, and manipulation of host processes Egress the process of parasite exit from infected cells Endocytosis a process of internalizing cell surface components and exogenous material from the environment exterior to the cell Toxoplasma Gondii 734 15. Endomembrane trafficking pathways in Toxoplasma Inner membrane complex a complex consisting of an intermediate filament-like cytoskeletal structure and flattened cisternae that are sutured together to create a double membrane platform underlying the parasite plasma membrane Micronemes cigar-shaped apical secretory organelles that contain adhesive and membrane disruptive proteins that are discharged in response to elevated cytosolic calcium within the parasite Micropore a putative site of endocytosis consisting of a discrete invagination of the midapical parasite plasma membrane Parasitophorous vacuole a compartment formed during invasion in which the parasite replicates intracellularly Phagophore a double-membrane cup formed as a means of encapsulating autophagic cargo Prerhoptries transient organelles that are observed during parasite cell division and serve as the precursors for rhoptries Rhoptries club-shaped apical secretory organelles that inject their contents into the host cell immediately prior to invasion. Rhoptry proteins function in various ways to mediate invasion and evade host cell intrinsic immune mechanisms Vacuolar compartment/plant-like vacuole a lysosome-like organelle that most often resides in the mid-apical region of the parasites. This organelle is a site for degradation of proteins and likely other macromolecules along with a site for storage of ions and water References Adisa, A. Re-assessing the locations of components of the classical vesicle-mediated trafficking machinery in transfected Plasmodium falciparum. Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis. Rab6 promotes insulin receptor and cathepsin trafficking to regulate autophagy induction and activity in Drosophila. Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution. The yeast Saccharomyces cerevisiae: an overview of methods to study autophagy progression. Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii. Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection. A druggable secretory protein maturase of Toxoplasma essential for invasion and egress. Reconstructing the evolution of the endocytic system: insights from genomics and molecular cell biology. A Toxoplasma gondii protein with homology to intracellular type Na 1 /H 1 exchangers is important for osmoregulation and invasion. The recycling endosome of MadinDarby canine kidney cells is a mildly acidic compartment rich in raft components. Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids. Targeting to rhoptry organelles of Toxoplasma gondii involves evolutionarily conserved mechanisms. Toxoplasma gondii Syntaxin 6 is required for vesicular transport between endosomallike compartments and the Golgi complex. A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum. Comparative genomic analysis of multi-subunit tethering complexes demonstrates an ancient pan-eukaryotic complement and sculpting in Apicomplexa. Cryptic organelle homology in apicomplexan parasites: insights from evolutionary cell biology. Resolving the homologyfunction relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology. Analysis of monensin sensitivity in Toxoplasma gondii reveals autophagy as a mechanism for drug induced death. Rab11 in recycling endosomes regulates the sorting and basolateral transport of Ecadherin. Characterization of the subpellicular network, a filamentous membrane skeletal component in the parasite Toxoplasma gondii. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Innate immunity to intracellular pathogens: balancing microbial elimination and inflammation. Are rhoptries in apicomplexan parasites secretory granules or secretory lysosomal granules A highly dynamic F-actin network regulates transport and recycling of micronemes in Toxoplasma gondii vacuoles. Subunit organization and Rab interactions of Vps-C protein complexes that control endolysosomal membrane traffic. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection. An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway. Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection. Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex. Spatial dissection of the cis- and trans-Golgi compartments in the malaria parasite Plasmodium falciparum. Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae. Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of hook localization. Phosphatidylinositol 3monophosphate is involved in toxoplasma apicoplast biogenesis. Evolutionary repurposing of endosomal systems for apical organelle biogenesis in Toxoplasma gondii. Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. Secretory organelle trafficking in Toxoplasma gondii: a long story for a short travel. Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Characterization of the chloroquine resistance transporter homologue in Toxoplasma gondii. Three asexual invasive "zoite" forms of Toxoplasma (sporozoites, tachyzoites, and bradyzoites) have similar morphological properties and are likely to share many cytoskeletal features (Chobotar and Scholtyseck, 1982). Genetic recombination in Toxoplasma is achieved by a sexual cycle that occurs in the intestinal epithelium of felids and consequently has not been exhaustively studied (Pelster and Piekarski, 1971; Scholtyseck et al. During this process, Toxoplasma macrogametes are fertilized by microgametes, which are the only developmental stage that constructs flagella to power motility (Ferguson et al. Because the tachyzoite form is most amenable to experimental manipulation, nearly all studies of the cytoskeleton have characterized this stage. Although it is likely that many elements of the tachyzoite cytoskeleton will be conserved with bradyzoites and sporozoites, some cytoskeletal components are expressed or essential in a developmentally specific fashion. Examples include the bradyzoite-enriched expression of the myosins TgMyoB and TgMyoD (Polonais et al. Alveolates have a system of flattened vesicles (alveoli) that closely underlie the plasma membrane, creating a pellicle structure that is composed of three unit membranes. This structure is integral to Toxoplasma replication, motility, and invasion of host cells. Spindle microtubules originate at poles located in the cytoplasm and penetrate to the interior of the nucleus through pores in a specialized region of the nuclear membrane termed the centrocone. A centriole pair, organized in parallel configuration, is located at each spindle pole. During replication, daughter parasite buds emerge above the spindle poles but are omitted from this image for reasons of clarity. The elongated shape of Toxoplasma zoites is maintained by the association of 22 evenly spaced subpellicular microtubules that interact with the cytosolic face of the pellicle (Nichols and Chiappino, 1987). Additional single particle rows are interspersed between the double particle rows. In order to accommodate increasing diameter along the long axis of the parasite, the distance between double particle rows increases, as does the number of intervening single particle rows, to maintain a constant spacing between 746 16. Integrity of the lattice is independent of microtubules and actin filaments, suggesting that other cytoskeletal filaments organize this structure. This network is formed from alveolins to provide tensile strength, akin to intermediate filament proteins found in metazoans.

In Plasmodium pulse pressure close together generic hydrochlorothiazide 12.5 mg on line, where coronin localization dynamics are comparable to Toxoplasma heart attack marlie grace order hydrochlorothiazide online pills, coronin is critical to organize F-actin for productive motility (Bane et al arrhythmia drugs discount hydrochlorothiazide 12.5 mg line. They consist of motor domain-containing heavy chains that power motility and regulatory light chains arteriogram procedure cheap 25 mg hydrochlorothiazide with visa. The first myosin-driven behaviors to be documented in Toxoplasma are the related processes of tachyzoite gliding motility and host cell invasion blood pressure medication what does it do purchase discount hydrochlorothiazide line. Tachyzoite invasion of host cells links gliding motility to a circular moving junction that is inserted into the host cell plasma membrane at the site of contact blood pressure chart emt generic 12.5 mg hydrochlorothiazide with amex. Remarkably, most moving junction components as well as the primary motor (MyoA) that drives penetration are conserved in apicomplexans. TgMyoA is critical for efficient gliding motility and host cell invasion: parasites lacking TgMyoA are Toxoplasma Gondii 766 16. The Toxoplasma cytoskeleton: structures, proteins, and processes nonmotile, exhibit reduced invasion and egress, and cannot form plaques in culture (Meissner et al. Coccidian parasites, including Toxoplasma, additionally require TgMyoH, a conoid-tethered myosin, to initiate invasion. TgMyoC is not essential and null tachyzoites do not have defects in replication or egress. Among these, MyoF is conserved across diverse apicomplexan parasites, while TgMyoI and TgMyoJ are restricted to the coccidian lineage (Foth et al. TgMyoF localizes to the cytoplasm and is enriched above the nucleus and at the pellicle (Jacot et al. During endodyogeny, it associates with the pellicle, within daughter buds, and at the extremities of the apicoplast. TgMyoF is required for correct positioning of the centrosomes and emerging buds to capture the apicoplast, micronemes, and rhoptries during daughter parasite formation. It is also required for directed transport of dense granules, secretory vesicles that release components that are required to form a mature parasitophorous vacuole (Heaslip et al. The Toxoplasma cytoskeleton: structures, proteins, and processes apicoplast segregation defects (Jacot et al. Cytochalasin D treatment increases the TgMyoJ ring diameter and localization of TgCen2, suggesting that actin, TgMyoJ and TgCen2 constrict the basal complex at the completion of cytokinesis. These defects uncover a role for soluble regulators or metabolites to coordinate cell cycle checkpoints during tachyzoite replication. While the functions of several Toxoplasma myosins remain to be dissected, two additional motors, TgMyoB and TgMyoD, have been studied in some detail (Delbac et al. Depletion of TgMyoD demonstrates that it is not essential for in vitro tachyzoite growth and gliding motility. Transcripts for TgMyoB, like those for TgMyoD, are more abundant in bradyzoite stage organisms, suggesting that both motors may have more significant roles in this stage. While the TgMyoC motor is known to be a component of the posterior polar ring glideosome (above), the role of TgMyoB is unclear. TgMyoB transcripts are significantly outnumbered by TgMyoC transcripts and its localization is complicated by low expression levels and a nonantigenic unique tail. Collectively, technical advances and biological discoveries have increased our understanding of the unconventional roles of actin and myosin in Toxoplasma tachyzoites. As described previously, Toxoplasma actin polymers are extremely short-lived and reliable detection of native F-actin has required expression of an actin chromobody, which detects cytosolic filaments and a network that connects sibling parasites within the parasitophorous vacuole (Periz et al. The close relationship between activity of formins, actin-binding proteins, F-actin, and myosins in tachyzoites suggests that most or all roles for F-actin are intimately associated with the activity of specific myosin motors. Endodyogeny involves a single nuclear division coupled to formation of two daughter parasites per replication cycle. In contrast, in endopolygeny, a multinucleated mother cell is created by several rounds of S-phase and mitosis (S/M) unlinked to cytokinesis prior to the synchronous emergence of many daughter parasites. By comparison, other apicomplexans replicate by schizogony, a process that involves disassembly of the mother pellicle prior to multiple rounds of nuclear division with subsequent budding of daughter parasites from the plasma membrane (Francia and Striepen, 2014). The uncoupling of cell division (cytokinesis) from S/M is coordinated by the unique bipartite centrosome structure (Chen and Gubbels, 2019, 2015; Suvorova et al. Since the nuclear cycle can proceed without activation of the outer-core, apicomplexan parasites can accommodate accumulation of polyploid intermediates. A mutant that disconnects the inner- and outer-cores confirms that each platform executes its function independently (Chen and Gubbels, 2019). Based on the nongeometric expansion of Plasmodium merozoites in the red blood cell, inner-core activation is controlled at the individual core- Toxoplasma Gondii 770 16. The Toxoplasma cytoskeleton: structures, proteins, and processes level (Arnot et al. At the conclusion of schizogony, all outer-cores are activated simultaneously, to permit synchronous budding of all nuclei produced during a final mitotic cycle. This indicates that the outer-core cannot be activated unless the inner-core is activated as well. It is clear that the centrosome serves as a physical and signaling hub to orchestrate the various modes of apicomplexan cell division. The tachyzoite nuclear membrane remains intact and chromosome segregation occurs without chromosome condensation. Preceding spindle assembly, the centrosome rotates from the apical to basal side of the nucleus where it duplicates (Chen et al. Assembly of spindle microtubules begins at the basally located, newly duplicated centrosomes, and these microtubules are stabilized by acetylation of -tubulin K40 upon return of the duplicated centrosomes to the apical side of the nucleus (Chen et al. The mitotic spindle traverses a membrane tunnel between the two centrocones to separate the spindle poles and associated centrosomes. The spindle poles never extend to the opposite ends of the nucleus but remain in close proximity at the apical side of the nucleus (pleuromitosis). Completion of spindle pole separation is marked by reorientation of polar spindle microtubules from an apical horizontal alignment to a nearly vertical orientation such that they project into the nucleoplasm (Chen et al. Although karyokinesis is not concluded until the enclosing daughter cytoskeletons are partially assembled, it is generally assumed that for signaling purposes (permitting initiation of daughter bud assembly) mitosis is complete at this time. Although several components of the eukaryotic chromosomal passenger complex are missing from the Toxoplasma genome, a complex containing the aurora kinase ortholog TgArk1 is critical for mitotic progression (Berry et al. Homologs of several spindle assembly checkpoint components are encoded in the genome but remain largely unstudied. Many questions regarding mitosis and nuclear division remain unanswered, including how chromosomes are captured (or remain clustered at the centrocone) and how the two sets of chromosomes are distinguished and segregated. Rather than partitioning cellular components by formation of a contractile ring immediately prior to abscission, Toxoplasma daughter buds form in a mother parasite which retains functional apical organelles, a pellicle and cortical cytoskeleton until daughters are sufficiently mature to emerge from the mother, adopting her plasma membrane as their own. Prior to S phase, the first indication that tachyzoites have committed to cell division is duplication of the Golgi apparatus (Nishi et al. Development of daughter buds occurs in a series of orderly steps that are intimately Toxoplasma Gondii 16. Each daughter is endowed with a complete set of apical complex organelles as well as a mitochondrion, Golgi apparatus, apicoplast, and nucleus that are acquired with characteristic timing during replication. All major events revolve around the assembling daughter cytoskeleton scaffold, which initiates at the centrosome outer-core (Anderson-White et al. Subsequently, bud subpellicular microtubules appear: nascent daughter buds are marked by tubulin arranged in five spots surrounding a central spot in flower petal-like arrangement (Nagayasu et al. Once the cap alveolus is established, the apical annuli are assembled at sutures between the cap and median alveoli (Hu et al. When the bud reaches its widest point, several events indicate a critical transition in the maturation state. The distinct composition of mother and daughters may contribute to differential stability during daughter emergence, although a critical function appears to be additional rigidification of the cytoskeleton (Dubey et al. It is not known whether maturation and recycling processes occur concurrently or are organized spatially. Although the maternal plasma membrane is inherited by emerging daughters, additional membrane is added in a Rab11a-dependent fashion to separate the lateral surfaces of daughter buds (Agop-Nersesian et al. This bridge must be severed prior to egress to activate parasite motility which requires the activity of an atypical guanylate cyclase fused to a flippase residing in the basal complex (Bisio et al. These proteins, together with many additional proteins with the same basal complex dynamics (Engelberg and Gubbels, unpublished data), suggest that this structure harbors additional functions. However, since these proteins are not essential for completion of the lytic cycle, their roles are nonessential or required in other developmental stages. One logical role for these proteins would be a structural role in cytoskeletal organization, but since not all apicomplexan zoites maintain a basal complex following Toxoplasma Gondii 16. F-actin forms a network that connects tachyzoites within the same vacuole (Periz et al. This network permits communication between parasites and keeps the cell division cycles between sibling parasites synchronized (Periz et al. In larger vacuoles and during bradyzoite replication these connections can get lost, likely due to mechanical forces on the infected cell, which additionally underscores that these connections are not essential. The strict need for this cytoplasmic bridge as well as the mature basal complex is unclear, although previous observations suggest a variety of roles for this portal. For example, host cell vesicles captured by the parasite and transported into the vacuole aggregate near the basal complex suggesting a putative uptake or digestive role (Romano et al. In addition, the basal complex of the parasites appears twisted and invaginated following invasion (Morisaki et al. Acquisition of gliding motility permitted ancestral apicomplexans to cross biological barriers, greatly expanding their host range and tissue access prior to diversification of this lineage (Leander, 2008). Gliding motility requires the transport of apically secreted, membrane-spanning microneme proteins to the posterior end of the parasite where they are released from the pellicle by the activity of a rhomboid protease (Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection). Tachyzoites are capable of moving external beads in an apical to posterior direction along their surface, revealing the underlying behavior of secreted adhesins (Stadler et al. Gliding requires adhesin-substrate engagement and the parasite always moves in an apical (forward) direction. In the artificial twodimensional context of protein-coated glass slides, tachyzoites exhibit three distinct modes of motility: circular gliding, helical gliding, and twirling (Hakansson et al. However, when tachyzoites are embedded in a three-dimensional matrix that better simulates a natural tissue environment, these behaviors are resolved as a uniform corkscrew motility pattern (Leung et al. Studies in Toxoplasma have demonstrated that gliding motility requires the concerted action of signaling molecules, cytoskeletal components, secreted proteins, and a rhomboid protease. Productive motility requires posterior proteolysis of microneme-secreted adhesins (see Chapter 14: Toxoplasma secretory proteins and their roles in parasite cell cycle and infection) to permit localized substrate release for continuous forward motion (Brossier et al. Conoid extrusion is a reversible Ca21-mediated process that occurs during gliding motility and at the time of host cell invasion. To date, the mechanism underlying conoid extrusion has not been identified, although the critical role of TgMyoH in initiating host cell invasion explains at least in part why extrusion is essential. Toxoplasma tachyzoites egress may occur after multiple rounds of replication as a consequence of parasite-mediated changes in the vacuolar compartment (Bisio et al. Both pathways involve signaling cascades to activate egress from the vacuole and host cell (Chapter 13 "Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii"). Egress requires microneme secretion to facilitate parasitophorous vacuole and host plasma membrane disintegration as well as host cell cytoskeleton remodeling (Blackman and Carruthers, 2013; Chandramohanadas et al. Gliding motility participates in effective egress to bring the lytic cycle to completion and to drive successive invasion events. Differences in component function likely contribute to the site-specific efficiency of the various glideosomes. A possible explanation for this difference relates to optimal curvature of the pellicle at the apical pole. Gliding progresses at the same speed for parasites relying on either TgMyoA or TgMyoC; however, host cell invasion is impaired and progresses in a stop-and-go fashion for TgMyoA-deficient tachyzoites that use TgMyoC in its place (Egarter et al. Although these proteins are functionally redundant, their presence and Ca21-bound state contribute to the stability of the TgMyoA lever arm, thereby regulating the quality, displacement, and speed of gliding (Williams et al. Data from two studies that captured Ca21dependent phosphorylation events suggest that TgMyoA glideosome activity is further regulated by phosphorylation (Nebl et al. Direct evidence for TgMyoA regulation by Ca21dependent phosphorylation is derived from a small molecule enhancer of motility and invasion (Tang et al. Reciprocally, parasites expressing a nonphosphorylatable mutant myosin exhibit slower host cell egress after treatment with calcium ionophore. In summary, assembly, localization, and specific components for distinct glideosomes combined with direct Ca21 binding and posttranslational modifications, such as phosphorylation, provide ample modulatory flexibility to tailor control of gliding at specific times, places, and functional niches. Relative to many eukaryotes, apicomplexan genomes contain genes for a limited set of actin-interacting proteins (Baum et al. Furthermore, differences in the requirement for these proteins throughout the parasite life cycle indicate that they are not equally critical during all cellular programs (Sattler et al. In vitro, Toxoplasma actin polymerizes into extremely short (100 nm), unstable filaments (Sahoo et al. Current observations are consistent with a model whereby filaments are formed at the conoid and spatiotemporal polymerization dynamics control the directionality and timing of motility as well as other processes requiring F-actin (Periz et al. During apical to basal transport, F-actin is stabilized by external contact of the pellicle.

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