Molecular and genetic mechanisms of acute and chronic pancreatitis

pancreatitis, and how the pancreas is normally protected from trypsin. The link between SPINK1 and International Congress Series 1255 (2003) 49–60 trypsin activation further supports these concepts. Hereditary pancreatitis also teaches us about chronic pancreatitis. In this case, chronic pancreatitis develops in patients with recurrent acute pancreatitis. The unifying sentinel acute pancreatitis event (SAPE) model is reviewed. There are several major unanswered questions. First, how are 20% of the individuals who inherit the hereditary pancreatitis gene protected from ever getting an attack of pancreatitis? Secondly, what is the cause of the pancreatitis in the 35–40% of patients who do not have trypsinogen mutations? Third, what determines the severity of pancreatitis in those who develop pancreatitis? Finally, with knowledge of the mechanisms leading to pancreatitis, what can be done to limit or prevent damage from this disease? D 2003 Published by Elsevier B.V. Keywords: Pancreatitis; Trypsin; CFTR; Pancreatic secretory trypsin inhibitor; SPINK1 Molecular and genetic mechanisms of acute and chronic pancreatitis David C. Whitcomb* Department of Medicine, Cell Biology and Physiology, and Human Genetics, University of Pittsburgh, VA Pittsburgh Health Care System, Pittsburgh, PA 15213, USA The VA Pittsburgh Health Care System, Pittsburgh, PA 15240, USA Received 27 February 2003; accepted 28 March 2003 Abstract Hereditary pancreatitis is a rare, autosomal dominant disorder characterized by acute pancreatitis, chronic pancreatitis and a very high risk for pancreatic cancer. Over 300 families have been identified, with the majority originating in Europe. The cause of most of the cases is from mutations in the cationic trypsinogen gene, or PRSS1. The three most common mutations are R122H, N29I and R122C. The mutations are thought to cause gain-of-function mutations so that trypsinogen is either more easily activated, or active trypsin has delayed degradation. Research focused on the amino acids mutated in hereditary pancreatitis teach us about the central role of trypsin in the initiation of acute 0531-5131/03 D 2003 Published by Elsevier B.V. doi:10.1016/S0531-5131(03)00674-5 Abbreviations: RAP, recurrent acute pancreatitis; SPINK1, serine protease inhibitor Kazal-type 1; SAPE, sentinel acute pancreatitis event; PRSS1, protease serine 1. * UPMC Presbyterian, M2, C-Wing (Gastroenterology), 200 Lothrop Street, Pittsburgh, PA 15213, USA. Tel.: +1-412-648-9604; fax: +1-412-383-7236. E-mail address: [email protected] (D.C. Whitcomb). The pancreas is a large gland located in the back of the upper abdomen that plays 3.1. Clinical aspects of acute pancreatitis D.C. Whitcomb / International Congress Series 1255 (2003) 49–6050 Pancreatitis has traditionally been divided into acute pancreatitis and chronic pancreatitis. Acute pancreatitis occurs with the sudden onset of pancreatic inflamma- tion associated with release of digestive enzymes into the blood stream and an intense inflammatory reaction. Clinically, an attack of acute pancreatitis is recognized by the sudden onset of pain, nausea and vomiting, elevation of circulating levels of digestive enzymes in the blood stream (where amylase and lipase are usually measured) and elevated levels of C reactive protein, reflecting the inflammatory response. Local and systemic injury can occur during an episode of acute pancreatitis depending on the extent of the initial injury. Local complications range from edema, fat necrosis, several key roles related to digestion and utilization of nutrients. The pancreas has three basic functions that are served by three corresponding cell types. First, the pancreas secretes a bicarbonate-rich solution into the proximal small intestine in response to acidification of the duodenum with gastric acid. The bicarbonate-rich solution originates in the duct cells, which line the duct system arborizing from the main pancreatic duct throughout the entire parenchyma. Secondly, the pancreas secretes an array of digestive enzymes which digest the large and complex nutrient molecules consumed with a meal, including proteins, carbohydrates, complex lipids, etc. Thirdly, the pancreas secretes regulatory hormones, including insulin, glucagons into the blood stream as part of the endocrine system. These hormones regulate the utilization of nutrients absorbed and stored by the major organs of the body, especially the liver, muscle and fat. The pancreas also secretes pancreatic polypeptide and somatostatin, which are important inhibitory regu- lators and modulators of pancreatic function. Dysfunction of any one of these cell types leads to disease of the entire pancreas. 3. Acute pancreatitis 1. Introduction Recent advances in genetics, and especially the human genome project, provided the tools to pinpoint the molecular cause of model diseases, with broad application to similar diseases. This is especially true in acute and chronic pancreatitis where breakthrough understanding came from work on hereditary pancreatitis. Once the key gene in this disease was known, the mechanisms leading to acute and chronic pancreatitis became clear. This paper examines the problems in understanding acute and chronic pancreatitis, the mechanism of hereditary pancreatitis, and the implications for developing new models of pancreatic disease. 2. The function of the pancreas hemorrhage, pseudocysts formation, pancreatic necrosis (with or without superinfec- D.C. Whitcomb / International Congress Series 1255 (2003) 49–60 51 tion), splenic vein thrombosis and injury to adjacent organs including the duodenum and colon. Systemic complications reflect both the release of digestive enzymes and inflammatory mediators into the blood stream from the pancreas and the systemic response to severe inflammation. The organs most commonly injured include the lung, kidneys and cardiovascular system. The clinical course of acute pancreatitis is quite variable, with most cases resolving over 2–3 days with pancreatic rest. More severe cases may take weeks to resolve. However, the high mortality rates reported a decade or more ago appear to be diminishing with more aggressive and intensive support and treatment. 3.2. Gallstone pancreatitis Acute pancreatitis most commonly occurs in the context of gall stone disease. The pancreatic duct and common bile duct share a common channel just before emptying into the duodenum. The opening is controlled by the muscular sphincter of Oddi. If a gallstone that is traveling down the common bile duct becomes impacted at the sphincter of Oddi, it often causes an episode of acute pancreatitis. The exact link between this event and the initiation of acute pancreatitis remains controversial, but the association is unquestioned. 3.3. Acute alcoholic pancreatitis The second major causes of acute pancreatitis alcoholic ingestion. Again, the exact mechanisms remain controversial because alcohol causes acute pancreatitis only very rarely in less than 5% of people drinking alcohol heavily and regularly. Furthermore, animal models of alcohol ingestion are not associated with pancreatitis. Thus, the exact link between alcohol ingestion and acute pancreatitis remains obs- cure. 3.4. Rare causes of acute pancreatitis Less common causes of acute pancreatitis are also seen. They include hypercalcemia, hyperlipidemia, drug reactions, occasional infections or ‘‘idiopathic’’ cases of acute pancreatitis. As above, the actual intracellular or extracellular mechanisms remained unknown. 3.5. Theories on the origin of acute pancreatitis Because of the obscure location of the pancreas, little was known about this organ until recently. Prior to the 20th century, acute pancreatitis was thought to be some type of infection. In 1896, Chiari [1] challenged this dogma by hypothesizing that acute pancreatitis actually represented pancreatic autodigestion by the pancreatic digestive enzymes. Variations of this theory were proposed, e.g. that lipase leaking out of the duct would hydrolyze peripancreatic fat and initiate an inflammatory response. However, this theory was never seriously challenged or proven. D.C. Whitcomb / International Congress Series 1255 (2003) 49–6052 4. Chronic pancreatitis 4.1. Clinical features of chronic pancreatitis Chronic pancreatitis refers to a syndrome of destructive, inflammatory conditions that encompasses the many sequelae of long-standing pancreatic injury [2]. It has also been defined as a continuing inflammatory disease of the pancreas characterized by irreversible morphologic changes that typically cause pain and/or permanent loss of function [3–5]. Histologically, this process changes the normal pancreatic architecture into irregular fibrosis, acinar cell loss, islet cell loss and inflammatory cell infiltrates [2]. The diagnosis of chronic pancreatitis is usually made with abdominal imaging studies such as CT scanning which demonstrates parenchymal atrophy, sclerosis and distortion, intrapancre- atic calcifications, dilated pancreatic ducts, pseudocysts, common bile duct structuring and associated problems. Functionally, chronic pancreatitis is associated with maldigestion because of the loss of acinar cells to synthesize pancreatic enzymes and later diabetes, as the islet cells are lost. Severe, constant and unrelenting pain is common among patients with end stage chronic pancreatitis. 4.2. Theories on the cause of chronic pancreatitis The etiology of chronic pancreatitis remained as enigmatic as that of acute pancreatitis. Theories centered on the contents and obstruction of the small intrapancreatic ducts by protein and calcium carbonate stone plugs [6]. A protein was even incriminated, termed pancreatic stone protein and later lithostathine, that was thought to inhibit calcium carbonate crystal growth [7]. However, it was never clear if the plugs and duct dilation came before or after the chronic pancreatitis developed, and if it was the cause or consequence. It was determined, however, that lithostathine expression was unrelated to chronic pancreatitis [8], and that the protein had no unique anti-crystal forming properties [9]. Independent of this debate was that opinion that chronic pancreatitis was distinct from acute pancreatitis, based on the available evidence at several international conferences 1963 [3,10], 1984 [11] and 1988 [12]. The most frequently cited etiology for chronic pancreatitis is chronic, excessive alcohol use in 50 to 70% of patients, followed by ‘‘idiopathic’’ (f 20%) and the rest miscellaneous [13–15]. By 1995, it was conceded by in an expert review that ‘‘chronic pancreatitis remains an enigmatic process of uncertain pathogenesis, unpredictable clinical course and unclear treatment’’ [14]. 4.3. Alcoholic chronic pancreatitis Even though alcohol consumption is listed as the major contributing factor in chronic pancreatitis [13,16], the association is weak and the mechanism unclear. For example, why do only about 10% of heavy alcohol drinkers ever suffer from clinically recognized pancreatic disease [17,18]. Secondly, although the risk of chronic pancreatitis increases as a function of the quantity of alcohol consumption, there is no apparent lower threshold of toxicity [19]. Even epidemiological associations between alcohol consumption and chronic pancreatitis are weak compared to other common alcohol related problems [20]. discovered that the disease gene was cationic trypsinogen. This discovery was the key that unlocked the puzzle of acute and chronic pancreatitis. D.C. Whitcomb / International Congress Series 1255 (2003) 49–60 53 5.3. Trypsin biology and biochemistry Trypsinogen is the precursor for trypsin, the most important digestive enzyme produced by the pancreas. In addition to being a major enzyme for dietary protein digestion at arginine and lysine residues, trypsin serves at least three special functions. First, trypsinogen is the only proenzyme that is specifically activated by a special brush border enzyme (enterokinase) within the duodenum. Secondly, trypsin is the only enzyme involved in the digestive enzyme activation cascade, which changes all of the other pancreatic proenzymes into their active form within the intestine. Finally, trypsin plays a Laboratory studies also raise major questions because long-term, high-dose alcohol feeding of animals fails to cause chronic pancreatitis [21,22]. 4.4. Other factors associated with chronic pancreatitis Besides alcohol, the origin of chronic pancreatitis is even more obscure, as suggested by the second largest category, ‘‘idiopathic’’ chronic pancreatitis. In about 10% of cases, chronic pancreatitis has been linked with familial or hereditary causes, hyperlipidemia, tumors, trauma, etc. Thus, the relationship between various potential insults such as alcohol or other toxins and the process leading to chronic pancreatitis remained obscure. 5. Hereditary pancreatitis 5.1. Clinical features of hereditary pancreatitis Hereditary pancreatitis is a genetic disorder of recurrent acute and chronic pancreatitis beginning in childhood, with the trait being transmitted in an autosomal dominant pattern and associated with a high disease penetrance so that about 80% of individuals developing pancreatitis at some time in their life [23–25]. The attacks of acute pancreatitis appear identical to acute pancreatitis from other causes, with all of the associated complications [26]. About half of the patients with acute pancreatitis develop chronic pancreatitis, usually within 10 years of the onset of acute pancreatitis. Hereditary pancreatitis was thought to be relatively rare, accounting for about 2–3% of the cases of chronic pancreatitis. However, it is important because it appeared to be a good model for both acute and chronic pancreatitis. Furthermore, since the trait has a strong genetic basis, the underlying problem could be exactly identified through genetic linkage studies. 5.2. Discovery of the first hereditary pancreatitis gene The gene causing hereditary pancreatitis was independently localized to chromosome 7q35 by Le Bodic et al. [27] and Whitcomb et al. [28] in 1996. Whitcomb et al. [29] key role in controlling pancreatic exocrine function. If free trypsin activity in the D.C. Whitcomb / International Congress Series 1255 (2003) 49–6054 duodenum diminishes because of a meal or a trypsin inhibitor a trypsin sensitive peptide, cholecystokinin (CCK) releasing factor, avoids hydrolysis, accumulates and stimulates CCK release from specialized cells on the intestinal mucosa. The CCK then stimulates the pancreas to secrete more digestive enzymes until the meal is digested and basal levels of free enzyme activity return. Thus, trypsin plays a key role in protein digestion, proenzyme activation and feedback regulation of the pancreas. 5.4. Trypsin mutations in hereditary pancreatitis The initial mutation in the trypsin gene was in codon 122 which resulted in an arginine (R) to histidine (H) substitution [29] (R122H, also described as an R117H substitution using the chymotrypsinogen numbering system for serine proteases [25, 30]). A review of the literature on trypsin biochemistry and X-ray crystallography revealed that the mutation was in a critical regulatory region of the enzyme [29]. Trypsin consists of two globular domains that are connected by a single peptide chain, known as the autolysis loop. This connecting chain contains the R122 residue, which is the initial site of hydrolysis during autolysis. The R122H mutation would eliminate the autolysis target in the side chain, thereby preventing autolysis of trypsin by a second trypsin molecule [29,31]. This finding suggested that the reason individuals with hereditary pancreatitis developed attacks of acute pancreatitis was because this site is part of a key fail-safe protective mechanism to eliminate prematurely activated trypsin the would otherwise activate all of the other digestive enzymes, leading to pancreatic autodigestion and pancreatitis [29]. This discovery meant that a critical initial factor in acute pancreatitis was trypsin activation inside the pancreas [29,32]. Comparison of previous clinical studies of trypsin inhibitors that act inside the cell or only extracellular acting, and noting the importance of giving these inhibitors before pancreatitis is initiated suggested that in most cases acute pancreatitis begins within the acinar cells [32]. Additional studies even suggested which intracellular compartments are most important [33,34]. Investigation of additional families revealed additional mutations in the cationic trypsinogen gene [35], with the second most common being N29I [36]. These mutations are clustered around the activation peptide of the trypsinogen gene (A16V [37], D22G [38], K23R [39], N29I [36]), or within the autolysis loop (e.g. R122C [40–42]). Taken together, it appears that these limited mutations represent gain-of-function mutations that cause trypsin to be either prematurely activated, or prevents inhibition through autolysis. 5.5. Calcium and trypsin Another important feature of trypsin is a calcium-binding pocket near the autolysis loop. When calcium levels are elevated, trypsin becomes resistant to autolysis [43]. Inspection of the X-ray crystallography structure suggests that resistance to autolysis occurs because calcium in the binding pocket forms a bond with R122, thereby preventing the autolysis loop from flipping away from the surface of the trypsin molecule where it can be attacked by a second trypsin molecule. From a physiological perspective, this makes sense because active trypsin should be protected in the duodenum and jejunum where most of the complex proteins are broken down. Calcium is absorbed in the jejunum so that half of the individuals who suffer recurrent episodes of acute pancreatitis, and that the chronic pancreatitis develops well after the acute recurrent attacks begin. The discovery D.C. Whitcomb / International Congress Series 1255 (2003) 49–60 55 that the genetic defect caused a gain-of-function in trypsin provides compelling evidence that, in this case, chronic pancreatitis is linked to recurrent acute pancreatitis. Since chronic pancreatitis in these patients appears similar to alcoholic chronic pancreatitis, the chicken and the egg argument of whether protein plugs and stones can before or after the onset of chronic pancreatitis was answered—the protein plugs come afterwards. Indeed, it is now known that pancreatic stone protein (lithostathine) is actually a product of trypsin digestion of the pancreatitis-associated protein (PAP) and reg protein, and that the hydrolyzed products polymerize into oligomeric fibrillar structures, which spontaneously sediment in vitro [47]. Thus, it now appears that intrapancreatic trypsinogen activation must occur before there are any protein plugs. 6. SPINK1 mutations In 2000, another genetic discovery greatly strengthened the findings in hereditary pancreatitis and strengthened the argument that trypsin is central to acute and chronic pancreatitis. The finding was the mutations in the trypsin inhibitor SPINK1 as associated with idiopathic pancreatitis in children [48] and in familial pancreatitis [49]. SPINK1 (also known as pancreatic secretory inhibitor, PSTI) is a specific trypsin inhibitor that is synthesized in the pancreatic acinar cells along with trypsin. SPINK1 appears to offer the first line of defense against prematurely activated trypsin in the pancreas by binding to the active site in a one-to-one ratio. However, since the number of trypsin molecules greatly outnumbers SPINK1 the inhibitory capacity is limited. Fortunately, the second line of defense—trypsin autolysis—remains intact to prevent widespread enzyme activation, pancreatic autodigestion and pancreatitis [29], unless the intracellular calcium levels are too high! Although there remain a number of questions about the biology of SPINK1, it is clear calcium levels fall and trypsin becomes susceptible to autolysis. This may explain why trypsin activity appears to disappear within the ileum. Although high calcium levels are beneficial in the duodenum, they can be deleterious in the pancreas. It has long been known that hypercalcemia was associated with acute pancreatitis, but the mechanism was unclear [44,45]. It now appears that hypercalcemia leads to acute pancreatitis through stabilizing trypsin. The link between intracellular hypercalcemia and initiation of acute pancreatitis becomes even more interesting with the recent observation that pancreatic acinar cells are very sensitive to bile acids, which cause intracellular hypercalcemia [46]. Thus, many of the older observations appear to converge on trypsin activation. 5.6. Chronic pancreatitis in hereditary pancreatitis The pathophysiologic mechanisms leading to chronic pancreatitis were also revealed by studies on hereditary pancreatitis. As noted above, chronic pancreatitis develops in about that mutations in SPINK1, especially N34S, are associated with idiopathic pancreatitis in macrophages and activated stellate cells. The presence of these cells is critical to development of chronic pancreatitis because the resident macrophages and other cells D.C. Whitcomb / International Congress Series 1255 (2003) 49–6056 suppress acute inflammation by the release of antiinflammatory cytokines, e.g. IL-10 and transforming growth factor beta (TGFh), which drives fibrosis. The stellate cells respond to TGFh and other antiinflammatory cytokines as part of the postinflammatory healing children [48], in familial pancreatitis [49], in tropical pancreatitis [50] (especially fibro- calculous pancreatic diabetes (FCPD) where diabetes mellitus is the first major clinical sign leading to diagnosis) and other pancreatic diseases. 7. The importance of a triggering injury—the SAPE hypothesis The link between acute pancreatitis and chronic pancreatitis was inferred from studies on hereditary pancreatitis. However, these disorders do appear to have some marked differences and the concept had not been extended to other forms of chronic pancreatitis. In order to understand and test possible mechanisms, we developed the SAPE hypothesis. SAPE is an acronym for sentinel acute pancreatitis event, which we believe is required to initiate the processes leading toward chronic pancreatitis [24,51]. The SAPE hypothesis is illustrated in Fig. 1. On the left side of the figure, the process is described as a pathway and on the right side this process is illustrated. At the top of the figure is a normal pancreas, illustrated on the right as a normal acinus with acinar cells, duct cells and quiescent (inactive) stellate cells [52,53]. If a risk factor such as alcohol (ETOH) is added, the acinar cells are injured and prone to metabolic/oxidative stress through a variety of mechanisms, but the acinar cells, and the rest of the pancreas, appear remarkably normal [51]. Fibrosis, inflammation and destruction of acinar cells, which are the characteristic features of chronic pancreatitis, do not occur. The middle section of the figure illustrates the Sentinel Event, an episode of acute pancreatitis. An episode of acute pancreatitis can be divided into two phases: an early, proinflammatory cytotoxic phase (left side of the middle acinus) and a later antiinflam- matory healing phase (right side of the middle acinus). The early phase is dominated by acinar cell injury, invasion of cytotoxic lymphocytes and monocytes/macrophages. Importantly, stellate cells are also activated [54,55]. In the late phase of acute pancreatitis, the immune system switches from a proinflammatory to an antiinflammatory process where the cytokines and inflammatory cells that serve to limit further pancreatic damage and allow the healing process to begin. As part of the healing process, active stellate cells lay down matrix proteins, which, if this process were to continue, become fibrosis [52,53]. The lower section of the figure illustrates divergent pathways. To the left reflects healing, with gradual diminution of the inflammatory cells and a return to normal. The pathway to the right illustrates progression to fibrosis and chronic pancreatitis. The difference in pathways is determined by the presence or absence of ongoing metabolic/ oxidative stress (e.g. by continued alcohol use), and/or recurrent acute pancreatitis (RAP) or the influence of major risk factors that result in ongoing pancreatic injury and stimulation of the inflammatory system. The difference between the acinus at the top of the figure and the bottom in the postinflammatory state is the presence of resident tissue process and they serve as the source of the abundant extracellular matrix proteins that D.C. Whitcomb / International Congress Series 1255 (2003) 49–60 57 characterize the fibrosis of chronic pancreatitis [52,53]. Thus, an appropriately severe acute pancreatitis episode serves a triggering role by initiating the critical process of recruiting the inflammatory cells into the pancreas and by activate the stellate cells. This model can be used to explain the similar features of the pathway leading from any type of injury or stress toward chronic pancreatitis based in the initiation of the process through recruitment of appropriate inflammatory cells and propagation of the process through continued metabolic stress or other injuries. 8. Unanswered questions Although investigations into the molecular mechanisms of hereditary pancreatitis have provided insights into acute and chronic pancreatitis, questions as to why about 20% of Fig. 1. The SAPE hypothesis mode. See text for details. From Ref. [51]. [1] H. Chiari, Ueber selbstverdauung des menschlichen pankreas, Zeitschrift fur Heilkunde 17 (1896) 69–96. [2] B. Etemad, D.C. Whitcomb, Chronic pancreatitis: diagnosis, classification, and new genetic developments, [5] J.E. Clain, R.K. Pearson, Diagnosis of chronic pancreatitis: is a gold standard necessary? Surgical Clinics of North America 79 (4) (1999) 829–845. D.C. Whitcomb / International Congress Series 1255 (2003) 49–6058 [6] H. Sarles, T.J. Camarena, S.C. Gomez, R. Choux, J. Iovanna, Acute pancreatitis is not a cause of chronic pancreatitis in the absence of residual duct strictures, Pancreas 8 (3) (1993) 354–357. [7] J.P. Bernard, Z. Adrich, G. Montalto, C.A. De, R.M. De, H. 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Harada, et al., The pancreatitis classification of Marseilles, Rome 1988, Scandinavian Journal of Gastroenterology 24 (1989) 641. [13] C. Owyang, M. Levitt, Chronic pancreatitis, in: T. Yamada (Ed.), Textbook of Gastroenterology, Lippincott, Gastroenterology 120 (2001) 682–707. [3] H. Sarles, Pancreatitis: Symposium of Marseille, 1963, Karger, Basel, 1965. [4] M. Sarner, Pancreatitis definitions and classification, in: E.P.D. Vay Liang W Go, J.D. Gardner, E. Lebenthal, H.A. Reber, G.A. Scheele (Eds.), The Pancreas: Pathobiology and Disease, 2nd ed., Raven Press, New York, 1993, pp. 575–580. patients are unaffected remain. We investigated seven sets of monozygotic twins in order to determine if modifier genes or major environmental factors could explain this phenomenon [56]. Amazingly, 11 of 14 subjects (79%) were affected and 3 (20%) unaffected. However, the three unaffected subjects each had an affected identical twin. 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Molecular and genetic mechanisms of acute and chronic pancreatitis Introduction The function of the pancreas Acute pancreatitis Clinical aspects of acute pancreatitis Gallstone pancreatitis Acute alcoholic pancreatitis Rare causes of acute pancreatitis Theories on the origin of acute pancreatitis Chronic pancreatitis Clinical features of chronic pancreatitis Theories on the cause of chronic pancreatitis Alcoholic chronic pancreatitis Other factors associated with chronic pancreatitis Hereditary pancreatitis Clinical features of hereditary pancreatitis Discovery of the first hereditary pancreatitis gene Trypsin biology and biochemistry Trypsin mutations in hereditary pancreatitis Calcium and trypsin Chronic pancreatitis in hereditary pancreatitis SPINK1 mutations The importance of a triggering injury-the SAPE hypothesis Unanswered questions Acknowledgements References