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Purpose For the diagnosis of acute appendicitis, the combination of clinical and laboratory variables achieves high diagnostic accuracy. Nevertheless, appendicitis can present with normal laboratory tests of inflammation. The aim of this study was to investigate the incidence of normal inflammatory markers in patients operated for acute appendicitis. Methods This is an analysis of data from a prospective, multicentre SNAPSHOT cohort study of patients with suspected acute appendicitis. Only patients with histopathologically proven acute appendicitis were included. Adult patients with acute appendicitis and normal preoperative inflammatory markers were explored further in terms of abdominal complaints, preoperative imaging results and intraoperative assessment of the degree of inflammation and compared to those with elevated inflammatory markers. Results Between June and July 2014, 1303 adult patients with histopathologically proven acute appendicitis were included. In only 23 of 1303 patients (1.8%) with proven appendicitis, both preoperative white blood cell count and C-reactive protein levels were normal. Migration of pain was reported less frequently in patients with normal inflammatory markers compared to those with elevated inflammatory marker levels (17.4% versus 43.0%, p = 0.01). Characteristics like fever, duration of symptoms and localized peritonitis were comparable. Only 4 patients with normal inflammatory markers (0.3% overall) had complicated appendicitis at histopathological evaluation. Conclusion Combined normal WBC and CRP levels are seen in about 2 per 100 patients with confirmed acute appendicitis and can, although rarely, be found in patients with complicated appendicitis.

Introduction In patients treated with an appendectomy for acute appendicitis, the specimen is generally sent for histological evaluation. In an era of increasing non-operative treatment for acute appendicitis, it is important to know the incidence, the diagnostic accuracy, and treatment consequences of appendicular neoplasms that are found in acute appendicitis. We hypothesize that pre- and intra-operative parameters might predict an appendicular neoplasm. Methods Data was used from our previous prospective observational cohort study. All patients undergoing surgery for suspected acute appendicitis were included. The primary outcome was the incidence of appendicular neoplasms in patients operated for acute appendicitis. Secondary outcomes were pre-operative diagnostics and imaging outcomes, intra-operative surgical judgment, and postoperative management and outcome. Possible predictors of an appendicular neoplasm were identified and used in multivariable logistic regression. Patients with an appendicular neoplasm were followed for 3 years after initial appendectomy. Results A total of 1975 patients underwent surgery for suspected acute appendicitis and in 98.3% (1941/1975) the appendix was removed. In 1.5% (30/1941) of these patients, an appendicular neoplasm was found. Among the malignant neoplasms, the majority were grade 1 neuroendocrine tumors (NET) in 65% (13/20). On pre-operative imaging, there was no suspicion of malignancy. In three cases, there was an intra-operative suspicion of malignancy. Multivariable analysis showed only age as an independent predictor for appendicular neoplasms. No recurrent or new malignancy was found during follow-up. Discussion The incidence of appendicular neoplasm in patients undergoing an acute appendectomy is very low and clinical risk factors could not be identified.

The potato tuber is a vegetative storage organ, which is initiated at the tip of an underground diagravitropic growing stem, called a stolon. The process of tuber formation comprises the induction, initiation and growth of a stolon, cessation of the longitudinal growth of the stolon, followed by the induction, initiation and growth of the tuber. Tuber initiation is characterized by swelling of the stolon tip. The formation of tubers is accompanied by a large accumulation of starch and of a specific set of storage proteins. How these major changes in carbohydrate metabolism interact and are coordinated with the developmental process of tuber formation is an important question from a fundamental point of view, not only for potato tubers, but also for the formation of starch-accumulating plant tissues in general. When a potato plant has started to form tubers, in principle all stolons present on the plant can be induced to initiate this developmental programme. However, this does not occur synchronously, creating problems for research as it is not possible to select externally those stolons, which are going to tuberize. Another problem is that the different developmental stages on one plant can influence each other. Therefore, for our research of tuberisation-related changes in carbohydrate metabolism, we have used a highly synchronous in vitro tuberisation system, providing plant material in well-defined developmental stages, which are not influenced by each other.Sucrose is the starting point of carbohydrate metabolism in growing stolons and tubers. It is synthesised in the leaves (source organs) and transported towards the underground parts via the phloem. When it arrives in a growing stolon it is most likely unloaded in an apoplastic way. In the apical region of the stolon a high invertase activity is present, which can be attributed to the cell wall-bound form (CWI). This apoplastic invertase isoform hydrolyses sucrose, resulting in the presence of glucose and fructose in the apoplast. The hydrolysis of sucrose steepens the sucrose gradient between phloem and the recipient cells, which is thought to play an important role in driving the unloading and short-distance transport of sucrose in apoplastically-supplied tissues. After the hexoses have entered the cells, they can be fed into carbohydrate metabolism by the activity of specific phosphorylating enzymes. During stolon growth, one of these enzymes, hexokinase (HK), shows a high correlation with CWI at the level of overall activity, suggesting a coordinated coarse control between these two enzymes.Furthermore, localisation of HK by specific enzyme activity staining showed, like CWI, a high activity in the apical region of the stolon. In this particular region of the stolon cell divisions occur. Therefore, the predominance of these two enzymes in this region in comparison with the rest of the stolon suggests a functional link with cell division.Cellular growth is sustained by a high flux of carbon through the respiratory(-related) pathways producing energy in the form of ATP, NAD(P)H, and carbon intermediates for important anabolic routes, such as the synthesis of DNA, RNA, proteins, lipids and cell wall-components. Measurements of activity of the main enzymes involved in glycolysis and the oxidative pentose phosphate pathway (OPPP) indicated that the extra demand for energy and carbon intermediates by dividing cells is accompanied with an elevated activity of these enzymes. Furthermore, from the patterns of activity of these enzymes it could be deduced that they are probably subject to a coordinated coarse control. Such a coordinated control mechanism may regulate the potential of respiratory(-related) pathways in response to the demand for their products. The high activities of CWI and HK in the mitotic-active region could ensure that sufficient carbon is directed into the respiratory(-related) pathways by enhancing the flow of carbon towards the dividing cells and by rapid conversion of sucrose into hexose-phosphates, the substrates for glycolysis and OPPP. In addition, these two enzymes may also be involved in the process of glucose-sensing, which is thought to mediate the transcriptional (and/or translational) regulation of genes involved in cell division. Interestingly, during stolon growth the enzyme alkaline pyrophosphatase (APPase) shows a similar pattern of overall activity as the main enzymes involved in glycolysis and OPPP.Moreover, in situ hybridisation with a RNA probe derived from a potato APPase revealed a clear presence of the corresponding transcript in all stolon tissues. This particular isoform has been proposed to be localised in the cytosol, which is in agreement with its temporal and spatial pattern of expression and with the pattern of overall APPase activity during stolon-to-tuber transition. In the dividing cells the biosynthesis of cell components produces a large amount of PPi, which is the substrate of this enzyme. Rapid conversion of PPi by a cytosolic APPase or another enzyme (e.g. tonoplast bound pyrophosphatase) is required to drive the biosynthetic reactions in the synthetic direction. The Pi released by this activity may serve as a metabolic pool of Pi for the conversion of ADP into ATP. In this way the observed apparent coordination between level of APPase activity and of respiratory(-related) pathways could be explained.When the cells have left the cell cycle and are no longer involved in cell division, their main cellular activity comprises enlargement, which continues until the cells are fully elongated and mature. In the region just below the apical part of the stolon, where cell divisions no longer occur, less invertase and HK staining is present. The reduction in the levels of activity of CWI and HK in this region, can also be deduced from their patterns of overall activity during stolon growth. The activity of a soluble vacuolar invertase, however, does not decrease but remains rather constant throughout the whole growing stolon. In these elongating parts of the stolon the combined activity of the soluble invertases will establish the sink strength, i.e. the capacity to draw sucrose from the phloem. Besides this function in maintaining a sucrose gradient between phloem and elongating cells and allowing sucrose to enter carbohydrate metabolism, the vacuolar invertase may also be involved in the process of cell elongation. Cell elongation is mechanistically driven by enlargement of the vacuole, which depends on the osmotic pressure of the vacuole. The hydrolysis of one osmotically-active molecule into two by the activity of this vacuolar isoform will enhance the osmotic pressure and the water influx into the vacuole. The elongation zone of the stolon probably has a lower requirement for energy and carbon intermediates in order to sustain the cellular growth than the mitotically active apical region, which is reflected by the decline in the overall activity of enzymes involved in glycolysis and OPPP during the period of consecutive stolon growth in our in vitro system.Tuber initiation is accompanied by a clear change in the patterns of several enzymes. In situ hybridisation revealed that the transcripts corresponding to the acid invertases progressively disappear from the parenchymatic tissues of the tuber. As a result, the overall activities of these two isoforms declined to very low levels, which was also indicated by the in situ activity staining. In contrast, the enzyme sucrose synthase (Susy) increases rapidly to high levels in the parenchymatic tissues of the initiated tuber as indicated by the in situ analysis at mRNA level and activity level and by overall activity measurements. This developmental change in the sucrose-degrading pathway is clearly associated with tuberisation and the onset of massive starch accumulation as it was absent in the two non-tuberising controls. Sucrose synthase catalyses in vivo a reversible, low-affinity reaction, which is subject to a high degree of metabolic regulation. Sucrose degradation by invertases is irreversible, not subject to metabolic regulation, and it occurs already at a low sucrose level. These differences between the two different sucrolytic pathways make the Susy-catalysed pathway probably more suitable than invertases for the complex regulation of sucrose degradation in coordination with tuber growth, symplastic sucrose unloading, and starch synthesis, as is explained in chapters 2 and 5. Furthermore, there are indications that a certain sucrose influx and/or sucrose levels are required for the upregulation of the expression of genes involved in sucrose-starch conversion. The effectiveness of this apparent sucrose-modulated mechanism depends largely on the type of sucrolytic route predominantly present in the cell. The down-regulation of CWI and the switch of an invertase-catalysed sucrose-degradation route into a Susy-mediated sucrolytic system allow import of carbon in the form of sucrose and allow high sucrose levels in the cytosol. Moreover, hexoses released by the activity of invertases appear to have an inducing effect on respiratory(-related) metabolism, which is thought to occur at the expense of the carbon flux toward starch synthesis.Concomitant with the developmental switch in the sucrose-degrading pathway an increase in the overall activity of fructokinase can be observed. In situ analysis at transcript level and activity level indicated that the spatial and temporal pattern of this enzyme show a close resemblance to that of Susy. The apparently closely coordinated coarse control indicates a functional linkage between these two enzymes. As already mentioned Susy catalyses a reversible reaction, which is severely inhibited by accumulation of fructose. Fructokinase may downregulate the fructose content, thereby allowing a large net flux of sucrose degradation through the Susy-catalysed reaction. The other product of the degrading reaction of Susy is UDPGlucose (UDPGlc). UDPGlc can be converted by the enzyme UGPase, which does not show a development-related increase in our in vitro system, but is present at a high activity throughout the whole period of culture. The coarse control of this enzyme, just like that of the enzyme PFP, seems subject to modulation by high sugar levels. Both enzymes catalyse a reversible reaction, use PPi as an energy-donor and are able to convert the substrate of the sucrose synthesising enzyme SPS. In combination with Susy (and SPS), the reversible character of these two enzymes allows a process of sucrose/hexose-P and hexose-P/triose-P cycling, which offers the cell a mechanism to adjust rapidly and automatically the net rate of sucrose degradation in response to the supply of sucrose and demand for carbon in the respiratory and biosynthetic pathways in the tuber parenchyma cells.Measurements of the overall level of different hexose-P's during stolon-to-tuber transition indicated that tuber initiation and successive growth is accompanied with a decline in the hexose-P pool, which is in contrast with an expected increase due to the tuberisation-initiated rise in sucrose supply and degradation rate. A plausible explanation for this unexpected observation is that the level of the hexose-P pool has to remain low during tuber growth, because an increase in this pool might lead to an enhanced rate of sucrose resynthesis by Susy and SPS. Such an increase in unidirectional sucrose synthesis will lead to a decrease in the net rate of sucrose degradation, thereby lowering the net flux of carbon towards starch synthesis. Therefore, a low hexose-P pool might be an important prerequisite for a high, but adjustable net rate of sucrose degradation in Susy-mediated sucrolytic systems. A possible role for the enzyme PFP in the maintenance of a low hexose-P content is discussed in chapter 5.During successive tuber growth the overall activities of enzymes involved in respiratory(-related) metabolism declined, indicating that tuber growth and starch accumulation does not require an increase in the potential of respiratory(-related) metabolism. Concomitant with this decline the overall activity of APPase decreased also. In situ hybridisation with the presumed corresponding RNA probe revealed a substantial decline in mRNA content in the whole growing tuber. The Susy-mediated pathway of sucrose degradation depends on the availability of PPi, which is required by the UDPGlc-converting reaction of UGPase. The decline in activity of the assumed cytosolic APPase may prevent that the sucrose-degrading reaction of Susy becomes restricted by the accumulation of UDPGlc.Three other enzymes show a clear and large increase in activity after the onset of tuberisation, which is not observed in the non-tuberising controls. Two of these enzymes are known to be involved in starch synthesis. The enzyme ADPglucose pyrophosphorylase (AGPase) synthesises the precursor for starch synthesis and is thought to contribute to a large extent to the total flux control of carbon towards starch synthesis. The other enzyme is starch phosphorylase (STP), which catalyses a reversible reaction and may, therefore, be involved in the synthesis of starch or in its degradation. The patterns of both enzyme-activities correlate highly with the accumulation of starch in our in-vitro tuberisation system, which is consistent with their known role in the (cycling) process of starch synthesis. Starch accumulation seems to be a process of simultaneous synthesis and degradation. Two possible routes of degradation may exist. The first one can be catalysed by STP, the second one involves the activity of amylases. The (final) endproduct of amylases, glucose, can only be redirected into starch metabolism by the activity of a hexokinase. This enzyme shows a considerable increase in its level of activity after tuberisation onset. Activity staining revealed that this enzyme is present in the main starch-accumulating tissues of the tuber. The product of its reaction, Glc6P, has to be converted into Glc1P, which serves as the substrate for AGPase and STP.The enzyme responsible for this reaction, phosphoglucomutase (PGM) also shows a considerable increase in overall activity after the initiation of tuberisation, although not that large as AGPase, STP and HK. Furthermore, the tuberisation-related increase in Glc1P content in comparison with the decline in the content of the other hexose-P's, suggests that the reversible reaction catalysed by the plastidial isoform of PGM lies in the direction of Glc1P synthesis.From this study on changes of carbohydrate metabolism during stolon-to-tuber transition of potato, it has become clear that the sucrolytic switch itself forms a central and important step in the differentiation process of starch-storing tubers. The direct implications and consequences of this metabolic switch for the regulation of carbon fluxes towards starch synthesis and other carbon-demanding process are discussed in chapter 2, 3, 4 and 5. In addition in chapter 5 an effort has been made to place this tuberisation-related switch in a broader context of metabolic induction of growth-related and storage-associated differentiation processes, including the role and importance of glucose and sucrose as different signal-inducing molecules, modulating the expression of genes involved in cell division and respiratory(-related) metabolism (glucose-responsive) versus differentiation into storage cells and sucrose-starch converting potential (sucrose-responsive). In chapter 5 is also discussed how these mechanisms might affect sink strength and thus the capacity to enhance the flow of photosynthate towards the cells of the two different types of growing sink organs and its importance for growth and/or storage.