David W Pond

Peter Ward

Importance of diatoms for Oithona in Antarctic waters

Keywords: Oithona, Diatoms, Nutrition, Fatty acids, Pentafluorobenzyl esters Diatoms, Oithona, nutrition, fatty acids, Pentafluorobenzyl esters

Importance of diatoms for Oithona in Antarctic waters

D W Pond, Peter Ward

INTRODUCTION

The importance of smaller species of zooplankton and particularly Oithona similis has been increasingly realized [START_REF] Paffenhöfer | On the ecology of marine cyclopoid copepods (Crustacea, Copepoda)[END_REF][START_REF] Gallienne | Is Oithona the most important copepod in the worlds oceans[END_REF][START_REF] Dubischar | High summer abundances of small pelagic copepods at the Antarctic Polar Front -implications for ecosystem dynamics[END_REF]. Oithona similis is globally distributed, is often the dominant component of plankton communities and has been demonstrated to be an important link between microbial food webs and higher trophic levels [START_REF] Nielsen | Role of cyclopoid copepods Oithona spp. in the North Sea plankton communities[END_REF]. O. similis is also likely to benefit from increasing sea temperatures, particularly in high latitudes, where reduced ice cover is predicted to increase the prevalence of microbial, recycling based ecosystems [START_REF] Hansen | Impact of changing ice cover on pelagic productivity and food web structure in Disko Bay, West Greenland: a dynamic model approach[END_REF].

Studies of the diet of Oithona are not numerous, but this genus is thought to prefer motile prey and in particular protozoans, although diatoms and flagellates are also ingested in smaller amounts [START_REF] Atkinson | Omnivory and feeding selectivity in five copepod species during spring in the Bellingshausen Sea, Antarctica[END_REF][START_REF] Lonsdale | Predation by Oithona spp. on protozoopankton in the Ross Sea, Antarctica[END_REF][START_REF] Castellani | Temperature affects respiration rate of Oithona similis[END_REF], 2008). Initially it was suggested that Oithona recycles detrital aggregates [START_REF] Lampitt | Diet and respiration of the small planktonic marine copepod Oithona nana[END_REF] and faecal pellets (coprophagy, [START_REF] González | The possible role of the cyclopoid copepod Oithona in retarding vertical flux of zooplankton faecal material[END_REF] and this was supported by numerous direct and indirect observations [START_REF] Smetacek | Zooplankton standing stock, copepod faecal pellets and particulate detritus in Kiel Bight[END_REF][START_REF] Lampitt | What happens to zooplankton faecal pellets? Implications for material flux[END_REF][START_REF] Kiørboe | Predator and prey perception in copepods due to hydromechanical signals[END_REF], Gonzalez et al. 2000, Wexels Riser et al. 2001[START_REF] Svensen | Is the sedimentation of copepod faecal pellets determined by cyclopoids? Evidence from enclosed ecosystems[END_REF].

However, more recent experimental evidence has established that Oithona does not readily ingest faecal pellets [START_REF] Reigstad | Fate of copepod faecal pellets and the role of Oithona spp[END_REF]) and protozoans have now been identified as key players in this process [START_REF] Poulsen | Degradation of copepod faecal pellets: key role of protozooplankton[END_REF].

Lipids are often used to study food web interactions with specific fatty acids diagnostic of major microplankton taxa and can be traced through food webs [START_REF] Stevens | Copepod omnivory in the North Water Polynya (Baffin Bay) during autumn: spatial patterns in lipid composition[END_REF][START_REF] Stowasser | Using fatty acid analysis to elucidate the feeding habits of Southern Ocean mesopelagic fish[END_REF]. 16:1(n-7), 16:4(n-1) and 20:5(n-3) are often used as biomarkers for diatoms; 22:6(n-3) and 18:4(n-3) for flagellates; and 15:0, iso15:0, anteiso15:0, 17:0 and iso17:0 for bacteria [START_REF] Stevens | Copepod omnivory in the North Water Polynya (Baffin Bay) during autumn: spatial patterns in lipid composition[END_REF]). Although fatty acid biomarkers in many species of marine zooplankton have been well studied, analyses of Oithona have been limited, possibly owing to the small size of these copepods and the need for considerable numbers of individuals per analysis. [START_REF] Kattner | Fatty acid and alcohol composition of the small polar copepods, Oithona and Oncaea: indication on feeding modes[END_REF] provided initial fatty acid biomarker analyses of O. similis and other small copepod species from Antarctic waters. More recently, [START_REF] Lischka | Seasonal lipid dynamics of the copepods Pseudocalanus minutes (Calanoida) and Oithona similis (Cyclopodia) in the Arctic Kongsfjorden (Svalbard)[END_REF] provided a more detailed seasonal analysis of this species from Arctic waters. Both these studies showed that O. similis has an omnivorous diet with a prevalence of biomarkers characteristic of motile prey. Additionally, these studies also provided lipid biomarker evidence for the ingestion of detrital aggregates and faecal pellets by O. similis. Here we utilise fatty acid biomarker analysis to compare the diet of O. similis with its larger, but less well studied congener, Oithona frigida. Collectively, both species are among the numerically dominant zooplankton in Antarctic waters, the former being widely distributed (Metz 1996, Ward and[START_REF] Ward | Oithona similis in a high latitude ecosystem: abundance distribution and temperature limitation of fecundity rates in a sac spawning copepod[END_REF] whereas the latter appears particularly abundant near the Polar Front (Dubischaar et al. 2002). We collected Oithona from the Scotia Sea in the Atlantic sector of the Southern Ocean. The Scotia Sea is bounded by the Scotia Ridge where primary productivity is generally high and surface chlorophyll a levels can exceed 20 µg L -1 . [START_REF] Atkinson | South Georgia, Antarctica: a productive, cold water, pelagic ecosystem[END_REF]). These intense blooms can persist for months and are typically dominated by large diatoms [START_REF] Korb | Magnitude and maintenance of the phytoplankton bloom at South Georgia: a naturally iron-replete environment[END_REF]. By contrast, primary production in the central Scotia Sea is much lower, with surface chlorophyll a concentrations typically not exceeding 0.5 µgL -1 and where flagellates and microzooplankon comprise a substantial proportion of the microplankton standing stock [START_REF] Korb | Primary production across the Scotia Sea in relation to the physiochemical environment[END_REF].

We sampled Oithona from a number of sites across the Scotia Sea characterised by distinctly different production and microplankton communities with the aim of understanding how the food environment of Oithona was linked to both their nutritional condition and abundance. Given the small size of both species (prosome length of 500 µm and 700 µm for O. similis and O. frigida respectively), fatty acid biomarkers were analysed using a highly sensitive technique, involving the preparation of pentafluorobenzyl ester of the fatty acids, which were then detected by electron capture detector (ECD). This method is described in detail.

METHOD

Sample collection

Samples were collected from six stations during cruise JR177 of the RRS James Clark Ross to the Scotia Sea sector of the Southern Ocean during January and February 2007 (Fig. 1). At each station and immediately prior to zooplankton net hauls, vertical profiles of temperature and fluorescence were determined using a SeaBird 911+CTD equipped with a 12 position carousel water sampler and 10 L Niskin bottles (Table I). Seawater was collected from the fluorescence maxima and 3.2 L aliquots were filtered onto 47 mm ashed GF/F filters and samples stored in chloroform:methanol (2:1 v/v) at -80ºC until analysis. All zooplankton samples were collected using vertical net hauls from 400 m to the surface and conducted at dawn (approx. 04:00 local time). Nets were hauled at ~0.22 ms -1 . Zooplankton samples for the determination of abundances of the various developmental stages of the target copepod species were collected using paired 53µm mesh bongo nets and preserved in buffered formaldehyde (4 % w/v). These samples were analysed in the UK using a binocular microscope at 60X magnification. Counts were made of all copepodid stages of O. similis and O. frigida (CI-CVI) and nauplii of O. similis. Nauplii of O. frigida were extremely rare and not counted. Also counted were the total numbers of egg sacs of O. similis in each haul, both those attached to females and detached and floating free in the sample and eggs female -1 determined. Egg sacs of O. frigida, which are easily distinguishable from those of O similis [START_REF] Ward | Oithona similis in a high latitude ecosystem: abundance distribution and temperature limitation of fecundity rates in a sac spawning copepod[END_REF] were extremely rare and not counted.

Additional net hauls were made for the collection of animals for biochemical analyses. For carbon and nitrogen determinations of female copepods from selected stations, samples of 400-500 O. similis and 100-340 O. frigida, depending on availability, were sorted into a glass cavity well which enabled a final check of the sample identity and contaminating material, i.e. non target copepod species and microplankton material was removed. Purified samples of Oithona were then pipetted onto ashed GF/F filters (25 mm) and subjected to gentle vacuum to remove excess water. For fatty acid analysis, replicates containing 20 copepods were sorted onboard before preservation in 500 µl of chloroform:methanol (2:1 v/v) at -80ºC.

Microplankton species composition was determined on samples collected from 20 m depth and preserved in 1-2% Lugol solution in 250 ml brown glass bottles. In the laboratory, 50 ml subsamples were settled for 20-24 hours in Hydro-Bios setting chambers (Duncan and Associates, UK) and cell enumeration followed [START_REF] Hasle | The inverted microscope method[END_REF]. Microplankton carbon (µgC L - 1 ) was estimated from cell measurements following [START_REF] Poulton | Phytoplankton community composition around the Crozet Plateau, with emphasis on diatoms and phaeocystis[END_REF]. No attempt was made to enumerate cell sizes of < 5 µm since these counts are unreliable using inverted microscopy [START_REF] Poulton | Phytoplankton community composition around the Crozet Plateau, with emphasis on diatoms and phaeocystis[END_REF]. For full details of cell counting and biomass estimate procedures, see [START_REF] Korb | Summer microplankton community structure across the Scotia Sea: implications for biological carbon export[END_REF].

Biochemical analysis

Carbon and nitrogen analyses were conducted on both species of Oithona from selected sites, where both the numbers of copepods and time permitted the collection of the substantial numbers of animals required for these analyses. Carbon and nitrogen analyses of female copepods were performed on samples dried for 24 hours at room temperature in a vacuum dessicator before analysis in a CE-440 elemental analyser (Exeter Analytical Inc).

Given the small size of Oithona, fatty acids were determined using a highly sensitive technique that involves converting the fatty acids to Pentafluorobenzyl (PFB) esters and detecting the fluoride with an Electron Capture Detector (ECD). After the addition of an internal fatty acid standard (23:0), the sample was phase separated using 0.88 % (w/v) KCl and total lipid extracted following [START_REF] Folch | A simple method for the isolation and purification of total lipid[END_REF]. Total lipid was initially saponified using 100 µL 1M KOH in ethanol (5:95 v/v) then acidified using 0.6M HCl to produce free fatty acids. After the addition of water (250 µL), free fatty acids were extracted using 2 x 250 µL diethylether. After drying under a stream of nitrogen, free fatty acids were converted to PFB esters by reacting with 140 µL of acetonitrile:diisopropylamine/ PFB bromide (1000:10:1 v/v/v) at 60 ºC for 30 minutes. PFB esters of the fatty acids were purified using thin layer chromatography (TLC, [START_REF] Pond | Vertebrate nutrition at deep sea hydrothermal vents: Fatty acid and stable isotope evidence[END_REF]) and analyzed using a Trace 2000 GC (Thermo) equipped with a Restek Stabilwax column (30 m x 0.32 mm) and an electron capture detector (ECD). Hydrogen was used as the carrier gas and nitrogen as the ionizing gas for the ECD. The output from the ECD (mol %) was converted to weight % to be consistent with most other studies of marine zooplankton. Fatty acids were identified by comparison with a standard mixture (Marinol) and by GC-MS following [START_REF] Pond | Stable carbon isotope composition of fatty acids in hydrothermal vent mussels containing methanotrophic and thiotrophic bacterial endosymbionts[END_REF].

Condition factor

Condition factor (CF), involving mass of carbon or nitrogen per unit volume has previously been used as a measure of a copepods nutritional status [START_REF] Campbell | Growth and development rates of the copepod Calanus finmarchicus reared in the laboratory[END_REF]).

Here we use total fatty acid as a measure of nutritional condition in O. similis. Insufficient data was available to perform a similar analysis on O. frigida.

CF = TfaL -3

where Tfa = total fatty acid (µg) and L = prosome length (µm).

Statistical Analysis

Linear regressions were used to determine the relationship between food availability (estimated carbon biomass) and abundances of the different developmental stages of Oithona.

Linear regression was also used to investigate the relationship between condition of O. similis and an 'index' of fatty acid composition. This index was derived from a principal component analysis (PCA, see below) of the Oithona fatty acid data. The first principal component (PC1) is effectively a fatty acid biomarker spectrum ranging from diatom dominated to microzooplankton + bacteria + detritus dominated signatures. To investigate differences in the fatty acid composition between the different developmental stages and species of Oithona, Principal component analysis (PCA) was conducted on the percentage contributions of fatty acids for each sample. Variables included in the analyses were either correlated with the first two principal components, or were included on the basis that they are recognised biomarkers. [START_REF] Meglen | Examining large databases: a chemometric approach using principal component analysis[END_REF]. Principal components were generated from a correlation matrix of the percentage contributions of fatty acids for each species, stage and site. Graphical representation of scores derived from the PCA analysis indicate relationships among samples. All statistical analyses were conducted using MINITAB 15 statistical software.

RESULTS

The environment

Values of sea temperature, integrated from the surface to 100m, exhibited a general latitudinal gradient with lowest values of 0.85 °C at station Su 5 and increasing to 2.97 °C at Su 9 (Fig. 1, Table I). Integrated chlorophyll a values ranged from 0.21 µg L -1 at Su 5 to 1.46 µg L -1 at Su 9. Integrated sea temperature and chlorophyll a values were correlated (Chl a = -0.466 + 0.545 sea temp., P = 0.030, F = 10.93).

Microplankton

Percent composition of the major taxonomic groups of microplankton was determined from estimates of microplankton biomass (µgC L -1 ) and striking differences were evident between stations (Fig. 2a). Microplankton composition varied between Su 11, which was dominated by dinoflagellates (87 %) to Su 9, which was dominated by diatoms (92%, Fig. 2a). Proportions of ciliates were most abundant at Su 6 comprising 6 % of total microplankton. Since cell sizes of <5 µm were not counted, most naked flagellates were not included in the counts and only low levels were detected (Fig. 2b). Dinoflagellates Fatty acid composition of microplankton at the sites was consistent with the microscopic analysis with the highest proportions of the dinoflagellate biomarkers 18:4(n-3), 18:5(n-3) and 22:6(n-3) at Su 11, whilst diatom markers 16:1(n-7), 16:4(n-1) and 20:5(n-3) were most abundant at the diatom dominated sites (Su 7-Su 9, Fig 2a, Table II).

Oithona abundance

Depth integrated abundances of O. similis copepodids and nauplii a exhibited a similar pattern between sites with highest abundances of all developmental stages present at Su 7 (Fig 3 a,b). Abundances of both developmental stages of O. similis were positively correlated with food availability (estimated carbon biomass) at the sites (O. similis, copepodids = 13 + 8.85 POC, p =0.001, F = 15.87; nauplii = 208 + 7.68 POC, P =0.001, F = 17.29). For O. similis, eggs female -1 was also correlated with food availability (eggs female - 1 = 11.0 + 0.0852 POC, P = 0.026, F = 5.87). O. frigida was comparatively scarce with copepodids comprising mostly adult females. Younger stages were either rare, or absent from the net hauls (Fig. 3d). In contrast with O. similis, no relationship was found between the abundance of O. frigida copepodids and POC (O. frigida copepodids = 25.14 -0.0230 POC, P = 0.795, F = 0.07).

Oithona biochemical composition

Maximum levels of carbon in female O. similis (1.62 µg copepod -1 , Su 8) were almost 3 times lower than the maximum levels found in female O. frigida (4.33 µg copepod -1 , Su 9, Fig. 4). Similar comparison of the levels of nitrogen indicated that O. frigida only contained 1.4 times more nitrogen than O. similis (Fig 4). This was reflected in differences in the overall mean C:N atom ratios which were 5.6 and 10.9 for O. similis and O. frigida respectively With a few exceptions, the levels of total fatty acids in the two species and different developmental stages of Oithona tended to be quite similar between the six sites and were not correlated with food availability (POC, Fig. 5). Levels of total fatty acids in female O. similis were lower than those found in CV's (with the exception of Su 9) and tended to be more variable (Fig. 5). Less data are available for O. frigida, which reflects their low abundance at the sites, but it is clear that their levels of total fatty acids are typically 2-3 times higher than those in O. similis (Fig 5). III. Overall, the mean fatty acid compositions of the different developmental stages and species were similar although O. frigida did contain higher levels of 16:1(n-7, 18:1(n-7) and 16:4(n-1) compared with O. similis (Table III). Highest proportions of 18:1(n-9) were found for CV O. similis (15.6%, Table III). Principal component analysis (PCA) of the fatty acid comprising 41 samples of Oithona indicated a broad separation of O. frigida and O. similis with the first two principal components accounting for 57% or the variability within the dataset. The score plot indicated that the diatoms biomarker fatty acids 16:1(n-7), 18:1(n-7) and 16:4(n-1) were key variables distinguishing O. frigida from O. similis for which the bacterial biomarkers 15:0, iso15:0, anteiso15:0, 17:0 and iso17:0 and the flagellate marker 22:6(n-3) were characteristic (Fig. 6a,b). Three samples of O. frigida were grouped with the cluster for O. similis and it is notable that two of these samples contained both low levels of total fatty acid and diatom biomarkers.

Since the identification of patterns between development stage and sites is not immediately apparent in Fig 6, site specific PCA's were conducted and all showed a similar pattern. The best example of this is provided by Su 8, for which most data are available (Fig.

Fatty acid biomarker ratios

Ratios of diatom to flagellate biomarker fatty acids, i.e 20:5(n-3)/22:6(n-3) and 16:1(n-7)/18:4(n-3) can be a good indicator of diet in marine zooplankton (Fig. 8,9). Low ratios on both the x and y axes indicate diatom dominance in the diet, whilst higher values indicate that protozoans are more important in the diet. Comparison of these ratios in the microplankton and Oithona at each site is presented in Fig. 8. Values for Oithona are between ratios determined for the flagellate and diatom dominated sites which is consistent with a mixed diet, albeit with a dominance of flagellates and protozoans (Fig. 8). A more detailed plot of the diatom/flagellate biomarker ratios in Oithona grouped copepods according to site rather than species or stage (Fig. 9). This suggests that although there are clear differences in the fatty acid profiles of the two species, the microplanktonic food environment is also a key determinant of the fatty acid composition of these species (Fig. 9).

Condition factor

To investigate if diet and therefore fatty acid composition of O. similis was related to nutritional condition we compared the scores from PC1 of Fig. 6a with a measure of condition, i.e. the concentration of total fatty acid in each unit volume of copepod. Condition factor for each copepod was determined from body volume estimates derived from prosome lengths following Campbell et al. (2001, Table IV). Regression analysis indicated a significant relationship between fatty acid composition and fatty acid content in the O. similis samples. (Fig. 10, CF = 3.99 -0.561 PC1, p = 0.000, F = 29.91). Insufficient data was available to perform a similar analysis on O. frigida.

DISCUSSION

The life history traits of Oithona spp. from high latitudes differ from those of most calanoids from similar environments, in that they actively feed and reproduce throughout the year, albeit at lower rates in winter compared to the warmer months. They also exhibit extended female longevity and comparatively low respiration and ingestion rates [START_REF] Paffenhöfer | On the ecology of marine cyclopoid copepods (Crustacea, Copepoda)[END_REF][START_REF] Castellani | Temperature affects respiration rate of Oithona similis[END_REF]. Oithonidae are clearly a very successful group exhibiting considerable dietary plasticity, being capable of carnivory [START_REF] Lampitt | Carnivorous feeding by a small marine copepod[END_REF], omnivory including a wide range of microplankton taxa but particularly protozoans [START_REF] Atkinson | Subantarctic copepods in an oceanic, low chlorophyll environment: ciliate predation, food selectivity and impact on prey populations[END_REF][START_REF] Lonsdale | Predation by Oithona spp. on protozoopankton in the Ross Sea, Antarctica[END_REF] and also coprophagy [START_REF] González | The possible role of the cyclopoid copepod Oithona in retarding vertical flux of zooplankton faecal material[END_REF].

Food available to Oithona in the Scotia Sea varied considerably between stations with microplankton species composition, ranging from diatom dominated to dinoflagellate dominated communities and this reflected the oceanographic conditions, nutrient supply and food web structure at each site [START_REF] Korb | Summer microplankton community structure across the Scotia Sea: implications for biological carbon export[END_REF]. [START_REF] Korb | Summer microplankton community structure across the Scotia Sea: implications for biological carbon export[END_REF] has previously catagorised stations Su 5 and Su 6 as a high nutrient low chlorophyll region (HNLC) with low primary production and a dominance of heterotrophic dinoflagellates. The region to the south west of South Georgia (Su 7) supported moderate production with a shift towards diatom dominated communities, whilst the north west of South Georgia (Su 8, Su 9) is a region that supports large blooms of sustained primary production again dominated by diatoms [START_REF] Korb | Summer microplankton community structure across the Scotia Sea: implications for biological carbon export[END_REF]. Su 11, which was not reported on by [START_REF] Korb | Summer microplankton community structure across the Scotia Sea: implications for biological carbon export[END_REF] was located on the South Georgia Shelf and was in a post bloom situation supporting low microplankton biomass and being dominated by heterotrophic dinoflagellates. At each station in the current study, the fatty acid composition of both species of Oithona reflected that of the microplankton in their environment. These findings are consistent with the view that Oithona ingests motile prey, but also highlights the importance of diatoms in the diet of both species. The catholic nature of their diet has also been remarked upon by [START_REF] Hopkins | Food web of an Antarctic midwater ecosystem[END_REF][START_REF] Hopkins | Midwater food web in Mc Murdo Sound, Ross Sea, Antarctica[END_REF] and [START_REF] Hopkins | Midwater food web in the vicinity of a marginal ice zone in the western Weddell Sea Deep[END_REF]. The fatty acid composition of O. frigida with comparatively high levels of 16:1(n-7), 18:1(n-7) and 16:4(n-1) indicates a greater contribution of diatoms in its diet compared with O. similis. 18:1(n-7) is not generally considered a biomarker for diatoms but most zooplankton are able to elongate this fatty acid from the diatom derived 16:1(n-7) [START_REF] Hirche | Limnocalanus macrurus in the Kara Sea (Arctic Ocean): an opportunistic copepod as evident from distribution and lipid patterns[END_REF]. The elevated levels of diatom biomarkers in O. frigida are consistent with the anatomy of this species. O. frigida is larger than O. similis with a prosome length of approximately 700 µm compared with 500 µm for O. similis. Oithona frigida also possesses substantial and robust maxillipeds relative to those of O.similis, and it is entirely feasible that these adaptations facilitate the ingestion and processing of diatoms, since the frustules of this group can be extremely robust and require considerable force to break [START_REF] Hamm | Architecture and material properties of diatom shells provide effective mechanical protection[END_REF][START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]). Enlarged maxillipeds are also typical of carnivory, a feeding mode that some species of Oithona are known to adopt [START_REF] Lampitt | Carnivorous feeding by a small marine copepod[END_REF]. However the fatty acid data for O. frigida in the current and other studies [START_REF] Kattner | Fatty acid and alcohol composition of the small polar copepods, Oithona and Oncaea: indication on feeding modes[END_REF][START_REF] Lischka | Seasonal lipid dynamics of the copepods Pseudocalanus minutes (Calanoida) and Oithona similis (Cyclopodia) in the Arctic Kongsfjorden (Svalbard)[END_REF] do not indicate that carnivory is important. It should be noted that the current research was conducted during summer and that no information is currently available regarding the feeding habits of O. frigida during winter, a season when the abundance of its microplanktonic prey is low. Further feeding studies, including stomach content analyses over a seasonal cycle could help to resolve this issue.

Oithona contrasts with most species of marine zooplankton since the levels of the flagellate-protozoan marker 22:6(n-3) generally exceed that of 20:5(n-3), a biomarker characteristic of diatoms (Lischka and Hagen 2007 and this study). This undoubtedly reflects the high levels of 22:6(n-3) in the diet of Oithona given their preference for motile prey which contain comparatively high levels of this fatty acid. However, since 22:6(n-3) serves critical neurological functions in many organisms, high levels of this fatty acid in Oithona could potentially be related to energy efficient transfer of nerve impulses which either facilitates a fast escape response and/or the capture of prey particles [START_REF] Scott | Species Differences, Origins and Functions of Fatty Alcohols and Fatty Acids in the Wax Esters and Phospholipids of Calanus hyperboreus, Calanus glacialis and Calanus finmarchicus from Arctic Waters[END_REF][START_REF] Waggett | Escape reaction performance of myelinated and nonmyelinated calanoid copepods[END_REF][START_REF] Kiørboe | Mechanisms and feasibility of prey capture in ambush-feeding zooplankton[END_REF].

Ratios of fatty acid biomarkers are often used to study diets of marine plankton. [START_REF] Schmidt | Protozoans as food sources for Antarctic Krill, Euphausia superba: complementary insights from stomach content, fatty acids and stable isotopes[END_REF] plotted the ratios of 20:5(n-3)/22:6(n-3) and 16:1(n-7)/18:4(n-3) to 3) and 16:1(n-7) are biomarkers for diatoms whilst 22:6(n-3) and 18:4(n-3) are biomarkers for protozoans and flagellates. Previous studies on marine zooplankton have also indicated a relationship between fatty acid profiles and the levels of total lipid, an index of condition [START_REF] Schmidt | Protozoans as food sources for Antarctic Krill, Euphausia superba: complementary insights from stomach content, fatty acids and stable isotopes[END_REF]). Here we have adopted a slightly different approach by plotting the data from the overall PCA analysis (PC1) which is effectively an index of fatty acid composition, and the levels of total fatty acids in the same samples. Given that the loadings on PC1 are effectively a spectrum of diatom dominated (-loading) to flagellate and protozoan dominated (+ loading) fatty acid profiles, it can be concluded that those copepods inhabiting environments where diatoms are abundant are in the best condition. Initially this seems to be at odds with the view that Oithona preferentially ingest flagellates and protozoans [START_REF] Atkinson | Subantarctic copepods in an oceanic, low chlorophyll environment: ciliate predation, food selectivity and impact on prey populations[END_REF]. However, environments where diatoms are abundant are generally hotspots of secondary production with elevated abundances of all types of microplankton taxa, particularly protozoans, [START_REF] Verity | Growth rates of natural tintinnid populations in Narragansett Bay[END_REF][START_REF] Brussard | Impact of phytoplankton bloom magnitude on a pelagic microbial food web[END_REF]Boyd et al. 2000) and are also favourable environments for zooplankton growth and development [START_REF] Pond | Diatom fatty acid biomarkers indicate recent growth rates in Antarctic krill[END_REF]Ward et al. 2007). It is equally possible that the Oithona population inhabiting the Southern Ocean have a greater preference for diatoms than those in other oceanic regions, especially given the abundance and importance of diatoms in the Southern Ocean food web [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]).

Given the close relationship between food availability and the population of O. similis, this species is clearly able to rapidly exploit increases in food availability. This finding is consistent with [START_REF] Ward | Oithona similis in a high latitude ecosystem: abundance distribution and temperature limitation of fecundity rates in a sac spawning copepod[END_REF] who investigated the effect of chlorophyll a concentrations, a proxy for food availability and sea temperature on the abundances of O. similis in the Scotia Sea. Abundances of all developmental stages of O. similis were correlated with both temperature and chlorophyll a concentrations [START_REF] Ward | Oithona similis in a high latitude ecosystem: abundance distribution and temperature limitation of fecundity rates in a sac spawning copepod[END_REF].

These findings contrast with the situation for O. frigida for which no similar relationship has been established and whose lifecycle remains largely undescribed. C:N atomic ratios of O. similis and O. frigida were within the range reported for other species of marine copepod [START_REF] Båmstedt | Chemical composition and energy content[END_REF]), although O. frigida with a mean C:N value of 10.9 contained proportionally more carbon that O. similis with a C:N value of 5.6. A higher C:N ratio in female O. frigida compared with O. similis could be attributable to lower fecundity rates in the former during the current study. Eggs of marine copepods are typically rich in lipid and the production of eggs can deplete maternal lipid reserves [START_REF] Mayor | Egg production and associated losses of carbon, nitrogen and fatty acids from maternal biomass in Calanus finmarchicus before the spring bloom[END_REF] The potential role of O. similis in recycling detrital and faecal material in the world's oceans has been debated for nearly two decades, with much direct and indirect evidence providing support for this hypothesis [START_REF] González | The possible role of the cyclopoid copepod Oithona in retarding vertical flux of zooplankton faecal material[END_REF][START_REF] Kiørboe | Predator and prey perception in copepods due to hydromechanical signals[END_REF][START_REF] Svensen | Is the sedimentation of copepod faecal pellets determined by cyclopoids? Evidence from enclosed ecosystems[END_REF]. More recent evidence has established that although copepods do ingest and recycle faecal pellets, the dominant organisms responsible for the degradation and retention of this material in surface waters are protozoans, particularly heterotrophic dinoflagellates [START_REF] Poulsen | Degradation of copepod faecal pellets: key role of protozooplankton[END_REF]. In the current study, key fatty acids distinguishing O. similis from O. frigida were the bacterial biomarkers 15:0, i15:0, a15:0, 17:0 and i17:0. Since both detrital material and faecal pellets are rapidly colonised by bacteria [START_REF] Hansen | Bacteria associated with a marine planktonic copepod in culture. II. Degradation of fecal pellets produced on a diatom, a nanoflagellate or a dinoflagellate diet[END_REF], bacterial markers in O. similis are most likely derived indirectly from the ingestion of heterotrophic dinoflagellates. O. similis readily ingest dinoflagellates and these were major components of the microplankton community at the more southerly and northerly sites.

The protozoan and bacterial biomarkers in O. similis are consistent with this species being integral to recycling food webs, either directly by ingestion of faecal-detrital material or indirectly, by the ingestion of coprophageous protozoans [START_REF] Poulsen | Degradation of copepod faecal pellets: key role of protozooplankton[END_REF].

Whilst the biomarker composition and morphological characteristics of O. frigida suggests this copepod is well adapted to ingesting diatoms, the nutritional condition of O. similis was also linked to the levels of diatoms in its diet and indicates the greater importance of diatoms for Oithona in Antarctic waters, compared to other oceanic regions.

Fig. 4

Carbon and nitrogen contents of Oithona at selected sites (n=1). (Mean C:N atom ratios for; Oithona similis = 5.6, Oithona frigida 10.9).

Fig. 5

Total fatty acid contents of Oithona sp. at the six study sites.

Fig. 8

Relationship between the ratios of diatom fatty acid biomarkers (20:5(n-3) and 16:1(n-7)) and those for flagellates (22:6(n-3) and 18:4(n-3)) for the microplankton and Oithona sp. fatty acids at the six stations.

Fig. 9

Ratios of diatom to flagellate fatty acid biomarkers in Oithona sp.

  comprised mostly 20-40 µm naked dinoflagellates, whilst diatoms were more diverse and dominated by Thalassiothrix antarctica, Eucampia anatarctica, Odontella sp, Chaetoceros sp. and Coscinodiscus sp. Ciliates were mostly Strombidium sp. See Korb et al. (2010) for full details on microplankton species composition. Total amounts of microplankton varied substantially between sites, ranging from 116 µg C L -1 at Su 9 to 8 µg C L -1 at Su 6 (Fig 2b). Levels of microplankton carbon were highest at the three sites where the microplankton community was dominated by diatoms (Fig 2b).

  Mean fatty acid compositions for CV and female O. similis and female O. frigida are given in Table

  7a,b). Again it is apparent that diatom biomarkers separate O. frigida from O. similis for which bacterial and flagellates biomarkers are key variables with the first two principal components accounting for 80.2% of the variability within the dataset (Fig 7a, b). Notably, CV and female O. similis are separated by PC2 and this separation is driven by the higher amounts of the storage reserve fatty acids 18:1(n-9) and 20:1(n-9) in the CV's (Fig 7b).

  importance of diatoms and flagellates in the diet of Antarctic krill. 20:5(n-

  Fig. 1.Locations of the six sampling sites. Major oceanographic boundaries indicated are APF, Antarctic Polar Front; SACCF, Southern Antarctic Circumpolar Current Front; SB, Southern Boundary.

Fig. 2 .

 2 Fig. 2.(a) Microplankton percent composition calculated from estimates of carbon biomass derived from microscopy and (b) estimated carbon biomass (µg L -1 ) at the six study sites.

Fig. 3

 3 Fig. 3Abundances of different developmental stages of Oithona. Abundances are values integrated over 400 m water depth. Note that although nets hauls were conducted from 400 m, 95 to 98% of all developmental stages occupied the upper 100 m, Ward unpublished).

Fig. 6

 6 Fig. 6 Principal component analysis of fatty acid composition (percentage data) for female and CV Oithona similis and female Oithona frigida (a) Score plot. Number denotes station followed by code for species. os v = O. similis CV, os f = O. similis female, of f = O. frigida female (b) loading plot indicating the importance of each fatty acid for determining the scores for the different species and maturity stages plotted in Fig 6a.

Fig. 7

 7 Fig. 7Principal component analysis of fatty acid data for Oithona similis female and CV and female Oithona frigida at station Su 8.

Fig. 10

 10 Fig. 10Relationship between the condition factor (CF) Oithona similis. (where CF= WL-3, W = mass fatty acid copepod-1, L = prosome length) with an index of fatty acid composition (i.e. first principal component, PC1 of the O. similis fatty acid analysis, Fig.6a).

Table I .

 I Integrated temperature and chlorophyll a values for the six stations in the Scotia Sea. Data are integrated mean values generated from determinations of chlorophyll a and temperature from 10 m depth increments from 100 m to the surface).

	Station	Date	Temperature	Chlorophyll a
			(°C)	(µg L -1 )
	Su 5	19/01/08	0.85	0.21
	Su 6 Su 7 Su 8 Su 9 Su 11	22/01/08 25/01/08 01/02/08 04/02/08 09/02/08 F o 1.78 2.54 2.94 2.97 2.24 r	0.17 1.10 0.94 1.46 0.58
			P
			e
			e r
				R
				e
				v i e
				w

Table II .

 II Fatty acid composition of microplankton sampled from the six stations in the Scotia Sea. Data presented as percent composition (n = 1). ). Sat = saturated fatty acids, Mono = monounsaturated fatty acid, Diene = diunsaturated fatty acids, PUFA = polyunsaturated fatty acids, i.e. those containing 3 or more double bonds.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table III :

 III Average fatty acid composition (%) of Oithona similis and Oithona frigida from the six stations. (n = number of samples, each containing 20 copepods. Mean values, SE = standard error). Sat = saturated fatty acids, Mono = monounsaturated fatty acid, Diene = diunsaturated fatty acids, PUFA = polyunsaturated fatty acids, i.e. those containing 3 or more double bonds.

		O. similis		O. similis		O. frigida
		(female) (SE)	(CV)	(SE)	(female)	(SE)
		n=15		n=16		n=10
	10:0	0.7	0.2	1.2	0.2	2.8	0.5
	12:0	5.7	0.9	5.9	0.8	3.5	1.2
	13:0 14:0 i15:0 a15:0 15:0	0.3 6.9 0.7 0.5 2.0 F 0.0 0.4 0.0 0.0 0.1 o r 0.3 6.5 0.5 0.4 1.7	0.0 0.4 0.0 0.0 0.1	0.5 4.7 0.5 0.3 1.4	0.1 0.6 0.1 0.1 0.1
	16:0 16:1(n-9) 16:1(n-7) i17:0 a17:0 16:2(n-4) 17:0	15.1 1.7 3.7 1.0 0.7 0.5 1.2	0.5 0.2 0.2 0.1 0.1 0.1 0.1	12.7 1.4 3.8 P 0.9 e 0.6 0.4 1.0 e 0.6 0.2 0.3 0.1 0.1 0.0 0.1 r	10.0 1.2 6.1 0.5 1.1 0.9 0.7	0.5 0.2 0.4 0.1 0.2 0.2 0.1
	16:3(n-4) 16:4(n-1) 18:0 18:1(n-9) 18:1(n-7) 18:1(n-5) 18:2(n-6) 18:3(n-6) 18:3(n-3) 18:4(n-3)	0.4 0.3 5.2 9.7 2.0 1.8 2.1 0.5 0.7 2.4	0.0 0.1 0.5 0.9 0.1 0.1 0.1 0.0 0.1 0.2	0.4 0.4 4.2 15.6 1.8 1.7 2.2 0.6 0.8 3.0	0.1 0.1 0.4 R 1.8 e 0.9 1.6 3.2 11.4 0.1 5.3 0.1 0.9 0.1 3.2 0.1 0.9 v i e 0.1 1.0 0.2 3.8 w 0.1 0.3 0.5 1.0 0.7 0.1 0.2 0.1 0.1 0.3
	20:0	0.3	0.0	0.2	0.0	0.3	0.1
	20:1(n-9)	1.2	0.2	1.5	0.2	1.2	0.2
	20:4(n-6)	0.3	0.1	0.3	0.0	0.5	0.0
	20:4(n-3)	1.4	0.1	2.0	0.2	2.5	0.3
	20:5(n-3) 11.9	0.7	10.6	0.5	11.3	0.5
	22:1(n-9)	0.2	0.0	0.2	0.0	0.3	0.0
	22:5(n-3)	1.9	0.2	2.5	0.2	4.3	0.5
	22:6(n-3) 17.0	1.1	14.6	0.7	13.5	0.6
	Sat	40.3		36.2		29.4
	Mono	20.3		26.0		26.4
	Diene	2.6		2.6		4.1
	PUFA	36.9		35.2		40.2

Table IV .

 IV Mean prosome length for CV and female O. similis (n=30).

		CV O. similis	Female O. similis
		Mean	(SE)	Mean	(SE)
	Su 5	0.49	0.005	0.51	0.007
	Su 6	0.50	0.005	0.53	0.008
	Su 7	0.44	0.005	0.53	0.008
	Su 8	0.48	0.005	0.53	0.005
	Su 9	0.47	0.004	0.52	0.005
	Su 11	0.48	0.004	0.54	0.009

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

Journal of Plankton Research

ACKNOWLEDGEMENTS

We are indebted to the officers and crew of the RRS James Clark Ross for their support while at sea. We also thank Paul Geissler for conducting the carbon and nitrogen analyses and Alex Poulton for the microscopic analysis of the microplankton communities. This work was funded as a component to the Ecosystems program of the British Antarctic Survey.

F o r P e e r R e v i e w


Magdalena Katharina Hertweck

Felix Erdfelder

Karl-Anton Kreuzer
email: karl-anton.kreuzer@uni-koeln.de

CD44 in hematological neoplasias

Keywords: CD44, hematological malignancies, CD44 antibody, hyaluronan

come

Introduction

The class I transmembrane glycoprotein CD44 has first been described in 1980 as a surface molecule on T-lymphocytes, cortical thymocytes, and granulocytes [START_REF] Dalchau | Monoclonal antibody to a human leukocytespecific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat[END_REF]. It plays an important role in cell proliferation, migration, survival, and apoptosis. Due to its various functions in physiological as well as pathological processes, CD44 has been given several names as for example phagocytic glycoprotein 1 (Pgp-1), Hermes antigen, and extracellular matrix receptor type III (ECM-III) [START_REF] Ponta | The CD44 protein family[END_REF]. In this review we convey structural understanding of the CD44 protein and outline the physiological function on molecular and cellular level in order to provide a broad background for comprehension of CD44 engagement in hematological neoplasias and potential strategies for diagnosis and therapy of these malignancies.

Genomic organization and splice pattern

The highly conserved gene of the CD44 transmembrane protein family is located on the short arm of chromosome 11p13 in humans and on chromosome 2 in mice. It spans approximately 50 to 60 kb of genomic DNA and codes for about 360 amino acids in 19 exons in humans, and 20 exons in mice. It comprises two kinds of exons, constant and variable ones. Former encode the extracellular globular part (exon 1-5), a short stem as connection to the cell membrane (exon 16 and 17) and the transmembrane domain (exon 18). Exon 19 and 20 are subject to alternative splicing creating either a short or more often a long cytoplasmic tail [START_REF] Screaton | Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons[END_REF].

The exons 6 to 15 are variable (v1-10), enlarging the stem on its distal site and forming several distinct CD44 isoforms, referred to as CD44 variants (CD44v1-10) (Fig. 1). Different combinations of the variable exons create a CD44 repertoire of several dozen isoforms.

CD44 is a mainly acidic charged molecule with a t 1/2 turnover of approximately 8 hours [rev.

by [START_REF] Ponta | The CD44 protein family[END_REF][START_REF] Günthert | CD44: a multitude of isoforms with diverse functions[END_REF][START_REF] Naor | Involvement of CD44, a molecule with a thousand faces, in cancer dissemination[END_REF].

The splicing process can be influenced by several factors including cytokine and growth factor (GF) stimulation: Without detectable changes in total CD44 m-RNA levels an upregulation of CD44v3, v5, v6, v7, v8 and v9 is obtained by treatment with 12-Otetradecanoyl phorbol-13-acetate (TPA), insulin-like growth factor-1 (IGF-1), and plateletderived growth factor (PDGF) [START_REF] Fichter | Expression of CD44 isoforms in neuroblastoma cells is regulated by PI 3-kinase and protein kinase C[END_REF]. IL-1 upregulates CD44 standard and induces appearance of v3-and v6-containing splice variants, most likely mediated by Egr-1 [START_REF] Fitzgerald | Characterization of CD44 induction by IL-1: a critical role for Egr-1[END_REF]. Further Ras [START_REF] König | Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator[END_REF],

OPN [START_REF] Khan | Enhanced cell surface CD44 variant (v6, v9) expression by osteopontin in breast cancer epithelial cells facilitates tumor cell migration: novel post-transcriptional, post-translational regulation[END_REF], and poly(rC)-binding protein 1 (PCBP1) involvement [START_REF] Zhang | PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells[END_REF], as well as DNA damage result in a changed splicing pattern of CD44 [START_REF] Yan | Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing[END_REF].

Protein structure

The CD44 protein core has a molecular weight of only 37 kDa but post-translational modifications and insertion of variable exons enlarge it up to 200 kDa. Lacking all variable exons, CD44 standard (CD44s) is the smallest isoforms with a protein product size of approximately 80 kDa. Six cysteine residues creating disulfide chains form the globular shape of the distal extracellular region that serves as a docking site for several elements of the extracellular matrix (ECM). The stem contains possible proteolytic cleavage sites on its carboxyl terminal end. The whole extracellular domain is subject to post-translational modifications: N-linked glycosylations, mostly seen in the globular and variable region, and O-linked glycosylations as well as the attachment of glycosaminoglycans usually found in the stem. The hydrophobic transmembrane domain embodies a cysteine residue that appears to play a role in CD44 oligomerization. The cytoplasmic tail embodies 6 putative serine phosphorylation sites and provides a connection to the cytoskeleton and to the tyrosine kinase family [rev. by [START_REF] Ponta | The CD44 protein family[END_REF][START_REF] Günthert | CD44: a multitude of isoforms with diverse functions[END_REF][START_REF] Naor | Involvement of CD44, a molecule with a thousand faces, in cancer dissemination[END_REF][START_REF] Naor | CD44: structure, function, and association with the malignant process[END_REF][START_REF] Ponta | CD44: from adhesion molecules to signalling regulators[END_REF]. Recent experiments assume that the juxtamembrane region of the cytoplasmic tail runs parallel to the negatively charged inner membrane because of its mostly basic residues. Contrarily the distal part, containing basically acidic residues, would project into the cytoplasm (Fig. 2) [START_REF] Mori | Structural basis for CD44 recognition by ERM proteins[END_REF].

CD44 exists membrane bound and soluble. The latter can be generated by proteolytic cleavage of e.g. membrane-type 1 matrix metalloproteinase (MT1-MMP) [START_REF] Kajita | Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration[END_REF]. This shedding of CD44 from the membrane can be triggered e.g. by oncostatin M (OSM), transforming growth factor beta 1 (TGF-beta 1) [START_REF] Cichy | Cytokines regulate the affinity of soluble CD44 for hyaluronan[END_REF], and EGF [START_REF] Murai | Epidermal growth factor-regulated activation of Rac GTPase enhances CD44 cleavage by metalloproteinase disintegrin ADAM10[END_REF].

Expression pattern

CD44s is almost ubiquitously expressed whereas physiological CD44v expression is restricted to a subset of tissues. Despite CD44s expression on e.g. connective tissues, blood vessels, and muscle [START_REF] Mackay | Expression and modulation of CD44 variant isoforms in humans[END_REF], highest expression is observed on hematopoietic cells. Therefore CD44s is also referred to as hematopoietic CD44 (CD44H) [START_REF] Naor | CD44: structure, function, and association with the malignant process[END_REF]. Basically epithelial cells express CD44 variants, CD44v9 being the dominant isoform. It is expressed in most stratified squamous epithelia showing particular high expression levels in oesophagus, skin, and tonsil. Additionally CD44v6 and CD44v4 are expressed in epithelial cells, although in smaller amounts. The longest isoform containing all variable exons is expressed in keratinocytes.

Within the individual tissues generative cells display the highest amount of CD44v [START_REF] Ponta | CD44: from adhesion molecules to signalling regulators[END_REF][START_REF] Mackay | Expression and modulation of CD44 variant isoforms in humans[END_REF].

CD44v expression is transiently upregulated on activated T cells and cells of the innate immune system [START_REF] Naor | CD44: structure, function, and association with the malignant process[END_REF]. The expression pattern of the different variants of CD44 varies during lineage commitment, e.g. CD44v6 is upregulated in monopoiesis and downregulated in granulopoiesis [START_REF] Legras | A strong expression of CD44-6v correlates with shorter survival of patients with acute myeloid leukemia[END_REF].

CD44v6 is found to confer metastatic behavior to non-metastatic tumor cells [START_REF] Günthert | A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells[END_REF] and plays an important role in various tumor entities concerning pathophysiology and prognosis. As reviewed by Heider et al. a high frequency of positive cells up to hundred percent is found in squamous cell carcinomas of head and neck, oesophagus, lung, skin, and cervix as well as in metastases of these tumors. In adenocarcinoma, high levels of CD44v6 are observed in breast cancer cells and even higher levels in cells of metastatic lesions. In cells of Barrett's, lung, gastric, pancreatic, colon, endometrium, and prostatic cancer, more varying levels are reported, displaying a more moderate expression rate. Also in thyroid carcinoma and basal cell carcinoma CD44v6 is frequently expressed [START_REF] Heider | CD44v6: a target for antibody-based cancer therapy[END_REF].

Additionally CD44 has been proposed to identify tumor initiating cells, also referred to as cancer stem cells, in a multitude of different tumor entities as e.g. breast cancer [e.g. [START_REF] Kai | Breast cancer stem cells[END_REF],

colon cancer [e.g. [START_REF] Todaro | Colon cancer stem cells: promise of targeted therapy[END_REF], pancreatic cancer [e.g. [START_REF] Lee | Pancreatic cancer stem cells[END_REF], and liver cancer [e.g. [START_REF] Zou | Liver cancer stem cells as an important target in liver cancer therapies[END_REF] and thereby plays an important role in tumor pathogenesis, prognosis and therapy strategies.

Ligands and protein interactions

Protein interactions of CD44 are divided into three groups according to the compartment the interaction takes place. Extracellular, CD44 mediates outside-in signaling and the connection to ECM and surrounding cells. The cytoplasmic tail initiates intracellular signaling cascades and creates the connection to the cytoskeleton, thereby influencing cell shape and migration.

The membrane region is characterized by recruitment of proteins to the membrane and interaction with membrane spanning receptors.

Extracellular region: CD44 binds to several components of the ECM: fibronectin, collagen type I, and type IV, laminin, osteopontin (OPN), and hyaluronan (HA), its principal ligand.

Thereby HA fulfills a passive 'linking' function between CD44 expressing cells as well as signal transduction [START_REF] Ponta | The CD44 protein family[END_REF][START_REF] Ponta | CD44: from adhesion molecules to signalling regulators[END_REF]. The binding activity of HA to CD44 in general displays a great variability dependent on CD44 surface expression and clustering [START_REF] Perschl | Role of CD44 cytoplasmic domain in hyaluronan binding[END_REF], insertion of variant exons in the CD44 molecule [START_REF] Fichter | Expression of CD44 isoforms in neuroblastoma cells is regulated by PI 3-kinase and protein kinase C[END_REF], and post-translational modifications of CD44 [among others [START_REF] Cichy | Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells[END_REF][START_REF] Katoh | Glycosylation of CD44 negatively regulates its recognition of hyaluronan[END_REF] and can be regulated by cytokines. CD44/HA signaling itself can be affected by the molecular weight of HA e.g. during inflammation: While low molecular weight HA (LMW-HA) stimulates cell growth, high molecular weight HA (HMW-HA) inhibits proliferation [START_REF] Puré | Rheostatic signaling by CD44 and hyaluronan[END_REF]. Furthermore, the localization of the CD44/HA interaction at the cell surface has influence on the mitotic spindle axis formation.

Here, binding of HA to CD44 at the apical membrane results in parallel orientation of the spindle, while ligation at the basal membrane leads to increased spindle axis rotation [START_REF] Fujiwara | Hyaluronan-CD44 pathway regulates orientation of mitotic spindle in normal epithelial cells[END_REF].

In hematopoietic stem cells (HSC), even though there is a high CD44 expression on CD34+ hematopoietic cells, spontaneous binding activity to HA is low. Binding affinity can be induced by stem cell factor (SCF), GM-CSF, and IL-3 without affecting the amount of total CD44 expressed on the cell surface. This effect might be due to conformational changes of CD44. Thereby pre-existing 'inactive' CD44 with low HA binding affinity would be activated by interaction of cytoplasmic tail and cytoskeleton [START_REF] Legras | CD44-mediated adhesiveness of human hematopoietic progenitors to hyaluronan is modulated by cytokines[END_REF]. Also monocytic CD44 can be transferred into a high (e.g. by TNF-alpha) or low (e.g. by IL-4) HA-affinity state by cytokines via post-translational modifications as N-linked glycosylation or chondroitin sulfate modification [START_REF] Levesque | TNFalpha and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44[END_REF][START_REF] Brown | Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14(+) peripheral blood monocytes[END_REF]. Similarly T cell binding to HA is regulated by cytokines (e.g. IL-2, IL-8, TNF) activating CD44 [rev. [START_REF] Cichy | Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells[END_REF]. Additionally antigen-activation of T cells enhances the affinity of their surface CD44 to HA [START_REF] Degrendele | CD44 activation and associated primary adhesion is inducible via T cell receptor stimulation[END_REF].

CD44 plays a pivotal role in cell-cell interaction by interaction with integrins. Cooperation of CD44 with alpha4beta1 integrin, also referred to as very late antigen-4 (VLA-4) mediates adhesion of hematological progenitor cells to the bone marrow [START_REF] Verfaillie | Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrin alpha 4 beta 1 and the CD44 adhesion receptor[END_REF] and plays an important role in the process of leukocyte extravasation [START_REF] Nandi | Bimolecular complex between rolling and firm adhesion receptors required for cell arrest; CD44 association with VLA-4 in T cell extravasation[END_REF]. CD44v ligation by OPN induces integrin activation via Src-signaling leading to increased survival of cells [START_REF] Lee | Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPN-CD44V interaction enhances survival in gastrointestinal cancer cells[END_REF]. Furthermore, crosslinking of CD44 results in higher expression levels of lymphocyte function-associated antigen-1 (LFA-1) and VLA-4 which are responsible for enhanced transendothelial migration of tumor cells [START_REF] Wang | CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)[END_REF].

Transmembrane region: Additionally to transmembrane proteins and components of the ECM, several soluble molecules are reported to bind to CD44 as e.g. members of the galectin family and several GFs (see table 1 and2) and trigger intracellular signaling cascades. Hereby CD44 recruits GFs to the cell membrane and mediates GF interactions with their respective receptor tyrosine kinase. By this means CD44 functions as co-receptor for a wide range of receptor tyrosine kinases as members of the ErbB family, c-Met, VEGFR-2, and BMP receptor type II (see table 3) [rev. [START_REF] Ponta | CD44: from adhesion molecules to signalling regulators[END_REF]. Further CD44 signaling activates transcription and translocation of MMPs to the cell surface. Thereby CD44 enhances invasiveness of tumor cells either by anchoring or increased secretion of proteolytic active MMPs (see table 4) [START_REF] Torre | Reduction of hyaluronan-CD44mediated growth, migration, and cisplatin resistance in head and neck cancer due to inhibition of Rho kinase and PI-3 kinase signaling[END_REF][START_REF] Baronas-Lowell | Differential modulation of human melanoma cell metalloproteinase expression by alpha2beta1 integrin and CD44 triple-helical ligands derived from type IV collagen[END_REF][START_REF] Yu | Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion[END_REF].

The cytoplasmic tail cooperates with intracellular molecules as members of the Src tyrosine kinase family including c-Src, Lyn, Fyn, and Lck [START_REF] Lefebvre | CD44 interacts directly with Lck in a zinc-dependent manner[END_REF][START_REF] Bourguignon | CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration[END_REF][START_REF] Wong | CD45 downregulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells[END_REF][START_REF] Ilangumaran | Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes[END_REF], Moesin-Ezrin-Radixin-Like Protein Merlin [START_REF] Mcclatchey | Merlin and ERM proteins: unappreciated roles in cancer development?[END_REF], T-cell lymphoma invasion and metastasis-inducing protein 1 and 2 (Tiam1/2) [START_REF] Terawaki | The PHCCEx domain of Tiam1/2 is a novel protein-and membrane-binding module[END_REF], intracellular OPN [START_REF] Wang | Osteopontin: role in immune regulation and stress responses[END_REF], as well as members of the Smad protein family [START_REF] Mori | Structural basis for CD44 recognition by ERM proteins[END_REF][START_REF] Tanikawa | Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway[END_REF][START_REF] Peterson | CD44 modulates Smad1 activation in the BMP-7 signaling pathway[END_REF].

Hereby c-Src signaling stimulates microRNA-mediated down-regulation of tumor suppressor gene transcription [START_REF] Bourguignon | Hyaluronan-CD44 interaction promotes c-Src-mediated twist signaling, MicroRNA-10b expression and RhoA/RhoC upregulation leading to Rho-kinase-associated Cytoskeleton Activation and Breast Tumor Cell Invasion[END_REF].

Intracellular region: The connection to the cytoskeleton is provided by ankyrin and the ezrinradixin-moesin (ERM) protein family functioning as cross-linkers to the actin filaments. The ERM proteins have a well conserved FERM domain in common that binds to CD44, and possess a carboxyl terminal tail linking to F-actin [START_REF] Bretscher | ERM proteins and merlin: integrators at the cell cortex[END_REF][START_REF] Mcclatchey | Merlin and ERM proteins: unappreciated roles in cancer development?[END_REF]. Affinity is regulated by phosphorylation of either the ERM protein family or the cytoplasmic tail of CD44, latter mediated by protein kinase C (PKC). Likewise ankyrin activity which mediates contact to the cytoskeleton component spectrin depends on its phosphorylation status. Thereby it modulates HA-dependent cell adhesion and motility. [START_REF] Ponta | CD44: from adhesion molecules to signalling regulators[END_REF][START_REF] Mori | Structural basis for CD44 recognition by ERM proteins[END_REF]. Ankyrin binding is further modulated by RhoA and Rac1, members of the Rho-GTPase family [START_REF] Turley | Signaling properties of hyaluronan receptors[END_REF]. Activation of both, RhoA and Rac1, is mediated by CD44 itself: CD44/MMP-9 activates TGF-beta [START_REF] Yu | Transforming growth factor-beta facilitates breast carcinoma metastasis by promoting tumor cell survival[END_REF] which is shown to regulate RhoA activity, rearrangement of the cytoskeleton, and adhesion [START_REF] Tian | TGF-beta1-mediated inhibition of HK-2 cell migration[END_REF].

Further Rac1 activation proceeds via HA-ligation to CD44, leading to actin cytoskeleton and cell reorientation [START_REF] Oliferenko | Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth[END_REF]. Besides direct interactions, CD44 influences filament organization by co-activation of several tyrosine kinases resulting in downstream phosphorylation of cytoskeleton proteins [START_REF] Bourguignon | CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration[END_REF] and F-actin rearrangement [START_REF] Wong | CD45 downregulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells[END_REF].

Intracellular signaling cascades

Complex intracellular signaling cascades, including the PI3K/Akt and the Ras/ERK pathway, mediate CD44-regulated adhesion, migration, proliferation, survival, apoptosis, and differentiation via three distinct mechanisms:

(i) Activation and association of intracellular molecules to the cytoplasmic tail of CD44 and downstream phosphorylation. Hereby e.g. complex formation with Lyn leads to subsequent regulation of Akt phosphorylation, modulation of the actin-binding protein cofilin, and enhanced migration as observed in colon carcinoma [START_REF] Subramaniam | CD44 regulates cell migration in human colon cancer cells via Lyn kinase and AKT phosphorylation[END_REF]. Further CD44/HA-activated Rho-kinase increases cell motility by phosphorylation of myosin phosphatase and consecutive cytoskeletal activation, and confers ECM degrading properties by triggering MMP-9 and MMP-2 secretion.

Additionally PI3K signaling is induced and increases cell proliferation and survival [START_REF] Torre | Reduction of hyaluronan-CD44mediated growth, migration, and cisplatin resistance in head and neck cancer due to inhibition of Rho kinase and PI-3 kinase signaling[END_REF]. The ERK1/2 pathway can be stimulated e.g. by CD44/CD74 complex activation, which leads in turn to Scr association with the cytoplasmic tail of CD44 and subsequent ERK1/2 phosphorylation [START_REF] Shi | CD44 is the signaling component of the macrophage migration inhibitory factor-CD74 receptor complex[END_REF]. Furthermore association to the ERM protein family mediates not only cell motility as described above but also cell death and survival as seen in Jurkat cells where Fas-mediated apoptosis can be modulated by CD44s induced downstream ezrin/actin interactions [START_REF] Mielgo | The CD44 standard/ezrin complex regulates Fas-mediated apoptosis in Jurkat cells[END_REF].

(ii)Cleavage and subsequently translocation of the cytoplasmic tail of CD44 into the nucleus followed by transcription of several target genes inclusive the CD44 gene itself [START_REF] Okamoto | Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway[END_REF].

(iii) Downstream signaling of the CD44-activated receptor tyrosine kinases.

The epidermal growth factor receptor (EGF-R) for instance can be activated by CD44 colocalization and trigger the Akt as well as the ERK pathway. HMW-HA induced CD44/EGF-R colocalization results in protein kinase C (PKC) phosphorylation by activated EGF-R, Akt and Rac-1 activation, focal adhesion kinase (FAK)-mediated upregulation of MMP-2 secretion, and enhanced cell motility [START_REF] Kim | CD44-epidermal growth factor receptor interaction mediates hyaluronic acid-promoted cell motility by activating protein kinase C signaling involving Akt, Rac1, Phox, reactive oxygen species, focal adhesion kinase, and MMP-2[END_REF]. Further TGF-beta1 stimulated CD44/EGF-R complex formation is followed by ERK1/2 signaling and fibroblast differentiation [START_REF] Simpson | Aging fibroblasts resist phenotypic maturation because of impaired hyaluronan-dependent CD44/epidermal growth factor receptor signaling[END_REF].

Physiological function in cells of hematopoietic origin

CD44 is involved in a wide range of important and very different functions: Cell proliferation, wound healing, angiogenesis, migration, homing, hematopoiesis, differentiation, immune response, and cell survival. Many of these functions are regulated by CD44-mediated cytokine production and secretion (table 5) and have been reviewed extensively [among others [START_REF] Ponta | The CD44 protein family[END_REF][START_REF] Ponta | CD44: from adhesion molecules to signalling regulators[END_REF][START_REF] Puré | A crucial role for CD44 in inflammation[END_REF]]. Here we focus on the role of CD44 during hematopoiesis and in mature blood cells.

CD44 plays two pivotal roles in early hematopoiesis: (i) mediation of the interaction of the progenitor cells with their respective niche in the bone marrow (BM), (ii) stimulation of cell proliferation and differentiation by regulation of local cytokine secretion [START_REF] Legras | CD44-mediated adhesiveness of human hematopoietic progenitors to hyaluronan is modulated by cytokines[END_REF][START_REF] Verfaillie | Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrin alpha 4 beta 1 and the CD44 adhesion receptor[END_REF][START_REF] Lapidot | How do stem cells find their way home?[END_REF][START_REF] Khaldoyanidi | CD44 variantspecific antibodies trigger hemopoiesis by selective release of cytokines from bone marrow macrophages[END_REF].

During fetal erythropoiesis CD44 expression on erythroid progenitor cells as well as on fetal hepatoblasts declines during maturation in order to prepare cells to leave the liver, suggesting a hematopoiesis dependent expression of CD44 [START_REF] Ohata | Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells[END_REF]. Likewise in adult erythropoiesis, decreasing CD44 expression is found. Therefore the authors claim CD44 to be a better marker for discrimination between erythroblasts at different stages of development than CD71 [START_REF] Chen | Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis[END_REF].

Similarly during B cell maturation in the bone marrow, CD44 displays a very organized expression pattern characterized by two waves of downregulation: After expression of CD44 on uncommitted CD34 positive progenitor cells, there is a loss of CD44 at the very early stage of B cell development. Thereafter CD44 expression increases, followed by a second wave of down-regulation before B cells regain CD44 when entering the periphery [START_REF] Vaskova | CD44 and CD27 delineate B-precursor stages with different recombination status and with an uneven distribution in nonmalignant and malignant hematopoiesis[END_REF]. A possible reason for this expression pattern might be the proliferation regulating effect of CD44. CD44 expression occurs at the same time as RAG-1 expression and thereby immunoglobulin chain rearrangement is down-regulated, and proliferation is increased.

During subsequent immunoglobulin light chain recombination CD44 expression decreases again.

During T cell maturation CD44 mediates homing of progenitor cells from the BM to the thymus where T cell differentiation and selection takes place. Furthermore intrathymic expansion of immature T cells through the different zones is regulated by CD44v6. Hereby CD44v6 cross-linking promotes proliferation and reduces apoptosis in early thymocytes [START_REF] Rajasagi | CD44 promotes progenitor homing into the thymus and T cell maturation[END_REF].

Clustering of CD44 and Lck leads to F-actin rearrangement via PI3K activation and regulates spreading of mature T cells [START_REF] Wong | CD45 downregulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells[END_REF].

Regulation of apoptosis plays an important role in mature T cells, especially in context of activation induced cell death (AICD). CD44 reduces Fas-mediated apoptosis in T helper 1 cells (Th1 cells) and increases effector cell survival. Thereby CD44 contributes to memory Th1 cell generation [START_REF] Baaten | CD44 regulates survival and memory development in Th1 cells[END_REF]. At the same time CD44 is able to promote AICD in T cells by at least two distinct mechanisms: (i) up-regulation of Fas ligand surface expression on peripheral T cells [START_REF] Nakano | Engagement of CD44 upregulates Fas ligand expression on T cells leading to activation-induced cell death[END_REF], (ii) Fas independent induction of apoptosis in activated T cells via HA [START_REF] Ruffell | Hyaluronan induces cell death in activated T cells through CD44[END_REF].

Extravasation of mature lymphocytes depends on at least three different CD44/protein interactions: (i) CD44 mediates lymphocyte rolling and adhesion to endothelial HA. Affinity is regulated by conformational shifts of latter. This results in low and high binding activity of cells to HA. Hereby the change between these two states is essential for unimpaired rolling [START_REF] Ogino | Two-state conformations in the hyaluronanbinding domain regulate CD44 adhesiveness under flow condition[END_REF]. (ii) Binding of CD44v to different members of the selectin family mediates rolling via selectin-dependent tethering [START_REF] Napier | Selectin ligand expression regulates the initial vascular interactions of colon carcinoma cells: the roles of CD44v and alternative sialofucosylated selectin ligands[END_REF]. (iii) Thus firm adhesion, essential for subsequent squeezing through the endothelial cells, leads to CD44/VLA-4-mediated transendothelial migration [START_REF] Nandi | Bimolecular complex between rolling and firm adhesion receptors required for cell arrest; CD44 association with VLA-4 in T cell extravasation[END_REF].

In macrophages CD44 regulates migration behavior and cytokine production. CD44-/macrophages show decreased response to chemoattractants, implied by impaired migration [START_REF] Zhu | Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin[END_REF]. Ligation of CD44v6 and v7 on monocytes trigger the release of GM-CSF and IL-6, which thus leads to increased proliferation of myeloid and lymphoid progenitor cells [START_REF] Khaldoyanidi | CD44 variantspecific antibodies trigger hemopoiesis by selective release of cytokines from bone marrow macrophages[END_REF]. In cooperation with OPN, CD44 inhibits the expression of IL-10 an anti-inflammatory cytokine, promoting a cytotoxic immune response [START_REF] Ashkar | Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity[END_REF]. In cooperation with HA, CD44 displays protective effect against septic response [START_REF] Muto | Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS[END_REF]. On the other hand, CD44v7 ligation in mononuclear cells in an inflammational setting is accompanied with apoptosis induction [START_REF] Hoffmann | CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria[END_REF].

Taken together these findings indicate a crucial role of CD44 not only in stimulating the immune response but also in limiting an excessive reaction. Expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, cytokine responsive gene-2 (CXCL-10), monocyte chemoattractant protein-1 (MCP-1), as well as IL-8 can be downregulated by blocking of CD44 by antibodies in macrophages [START_REF] Mckee | Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44[END_REF].

Further cell polarization, migration, and migration speed in neutrophils are dependent on CD44. CD44-/-neutrophils display impaired migration concerning direction and speed and reduced activation of RhoA [START_REF] Alstergren | Polarization and directed migration of murine neutrophils is dependent on cell surface expression of CD44[END_REF]. Additionally CD44 ligation triggers neutrophil apoptosis in vitro [START_REF] Takazoe | CD44-mediated neutrophil apoptosis in the rat[END_REF]. Similarly Langerhans and dendritic cell migration is mediated at least in parts by CD44, since antibodies against CD44 block OPN-induced cell migration to lymphatic organs in mice [START_REF] Weiss | Osteopontin is involved in the initiation of cutaneous contact hypersensitivity by inducing Langerhans and dendritic cell migration to lymph nodes[END_REF]. Also the proliferation of mast cells is regulated by CD44 [START_REF] Takano | Involvement of CD44 in mast cell proliferation during terminal differentiation[END_REF].

Hematological Neoplasias

Acute lymphoblastic leukemia

Being the most common malignancy in childhood, acute lymphoblastic leukemia (ALL) displays very young patient pattern and requires well-considered treatment strategies to minimize treatment related morbidity, mortality, and disease relapse. Total CD44 as well as CD44v6 have been identified as prognostic marker, and particular high or low CD44 expression patterns have been reported for several cytogenetic or prognostic subgroups in ALL.

In a small cohort of 16 pediatric patients CD44v6 expression was observed mainly in the medium and high risk group indicating a possible association between CD44v6 expression and unfavorable outcome [START_REF] Magyarosy | Expression of metastasis associated proteins, CD44v6 and NM23-H1, in pediatric acute lymphoblastic leukemia[END_REF]. This is consistent with the finding that high CD44v6 mRNA levels correlate with increased risk of relapse (n=21). Further expression of CD44v6 in ALL cell lines was associated with accelerated engraftment in NOD/SCID mouse transplantation experiments [START_REF] Bendall | Role of CD44 variant exon 6 in acute lymphoblastic leukaemia: association with altered bone marrow localisation and increased tumour burden[END_REF]. Contrarily, in a group of 97 pediatric B precursor ALL patients with neither known adverse nor favorable cytogenetic features no association between CD44v6 expression and prognosis was seen. On the other hand, total CD44 surface expression was identified as an independent predictor of disease relapse in the latter cohort [START_REF] Khan | Expression of CD44, but not CD44v6, predicts relapse in children with B cell progenitor acute lymphoblastic leukemia lacking adverse or favorable genetics[END_REF].

In pediatric ALL, CD44 and CD27 surface expression patterns have been described for different ALL subtypes and prognostic groups. CD44 surface expression in diagnostic bone marrow samples measured by flow cytometry displayed (i) concordance between high CD44 expression and high risk T-ALL, (ii) significant lower CD44 expression in the TEL/AML1 ALL subtype together with high CD27 expression, and (iii) double CD44/CD27 expression pattern typically seen in bcr/abl positive subtypes [START_REF] Vaskova | Transfer of genomics information to flow cytometry: expression of CD27 and CD44 discriminates subtypes of acute lymphoblastic leukemia[END_REF].

Acute myeloid leukemia CD44 signaling plays a pivotal role in acute myeloid leukemia (AML), depicting three different putative points of attack: differentiation arrest, bone marrow niche dependency of leukemia initiating cells (LIC), and acquired therapy resistance.

HA as well as CD44 antibodies can induce reverse of the leukemic differentiation blockage in a dose and time dependent manner, showing the strongest effect in monoblastic AML FAB M5 cells. The CD44 antibodies H90 and A3D8 as well as HA-12, an oligosaccharidic fragment, and HMW-HA enable AML blasts to produce oxidative burst, increase expression of the differentiation specific markers CD11b, CD14, and CD15, and induce cytological changes confirming proceeding differentiation. This in vitro setting indicates that abrogation of the leukemic block might be a therapeutic strategy not only in AML FAB M3 and identified H90 and A3D8 as potential agents [START_REF] Charrad | Ligation of the CD44 adhesion molecule reverses blockage of differentiation in human acute myeloid leukemia[END_REF]. In combination with retinoic acid (RA) both antibodies overcome the maturation deficiency in the KG1a (FAB M0) cell line that is characterized by resistance to most differentiation-inducing agents. This depicts the possibility of synergistic effects of several agents to overcome the leukemic block in AML [START_REF] Charrad | Effects of anti-CD44 monoclonal antibodies on differentiation and apoptosis of human myeloid leukemia cell lines[END_REF]. Molecular mechanisms behind the differentiation process have been identified in parts as e.g. in primary AML FAB M2-5 cells. Here anti-CD44 mAb treatment with HI44a resulted in reduced c-myc transcript expression. Additionally seen increased differentiation and apoptosis was assumed to be responsible for this effect. [START_REF] Song | HI44a, an anti-CD44 monoclonal antibody, induces differentiation and apoptosis of human acute myeloid leukemia cells[END_REF]. Further THP-1 cell differentiation proceeds via autokrine cytokine secretion upon CD44 ligation. Hereby anti-CD44 mAb H3 treatment induced ERK1/2-mediated TNF-alpha and IL-6 secretion.

Subsequent cell differentiation was shown to be dependent on both, TNF-alpha as well as IL-6. [START_REF] Bourcier | CD44-ligation induces, through ERK1/2 pathway, synthesis of cytokines TNFalpha and IL-6 required for differentiation of THP-1 monoblastic leukemia cells[END_REF]. In primary AML FAB M5 cells, CD44-mediated GM-CSF and IL-8 secretion was identified to promote differentiation upon anti-CD44 mAb P245 treatment [START_REF] Delaunay | Contribution of GM-CSF and IL-8 to the CD44-induced differentiation of acute monoblastic leukemia[END_REF]. These findings indicate CD44-mediated stimulation of autokrine cytokine secretion to be a general mechanism for induction of differentiation in AML cells.

IL-8 and GM-CSF also mobilize hematopoietic stem cells (HSC) from BM into peripheral blood [START_REF] Nervi | Cytokines and hematopoietic stem cell mobilization[END_REF]. Differentiation and proliferation of HSC is closely connected to the microenvironment. Therefore they are dependent on finding their specific niche in the BM In DLBCL prognostic value of CD44 is well-documented: In primary nodal DLBCL, CD44s and CD44v6 expression levels are associated to tumor dissemination and survival. In a cohort of 276 patients with DLBCL CD44s as well as CD44v6 expression was correlated to tumor spread. Further CD44s displayed a strong predictive value for tumor related death independent to other parameters of the International Prognostic Index (IPI) [START_REF] Drillenburg | CD44 expression predicts disease outcome in localized large B cell lymphoma[END_REF]. Contrarily in another study of 46 patients with primary nodal DLBCL only CD44v6 expression was significantly correlated to poorer overall survival. While CD44v6 expression was predominantly observed in lymphoma cells, CD44s expression was also seen in nonneoplastic small lymphocytes [111]. Tissue microarray analysis of 90 DLBCL patients revealed correlation of disease stage to CD44v6 expression, and inverse correlation to CD44s expression. In CD44s negative cases CD44v6 expression was associated with poorer overall survival. [START_REF] Tzankov | Prognostic significance of CD44 expression in diffuse large B cell lymphoma of activated and germinal centre B cell-like types: a tissue microarray analysis of 90 cases[END_REF].

Examining 114 cases of mature B cell NHL, including gastric, nongastric extranodal, nodal follicular lymphomas, and nodal DLBCL, a novel translocation t(11;14)(p13;q32) juxtapositioning the regulatory 5' region of CD44 to the IGHS enhancer was indentified. This translocation resulted in the overexpression of a CD44 variant lacking Exon 1 [START_REF] Hu | CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation[END_REF].

Chronic lymphocytic leukemia

CD44v surface levels in chronic lymphocytic leukemia (CLL) are associated with advanced disease, therapy requirement, and lower median survival [114]. Unlike other surface molecules associated with other adhesion molecules like CD11a, CD49d, beta 1-3 integrins, CD54, CD58, and L-selectin, only the expression of CD44 and CD11c is associated with spleenic manifestation of the disease. [115]. Soluble CD44 is related to shorter time of progression free survival [START_REF] Molica | Elevated serum levels of soluble CD44 can identify a subgroup of patients with early B-cell chronic lymphocytic leukemia who are at high risk of disease progression[END_REF], and soluble CD44s as well as soluble CD44v6 correlate with

Multiple myeloma

In human myeloma derived-cells CD44 is shown to be involved in adherence to BM stroma cells (BMSC) and subsequent IL-6 secretion of latter [START_REF] Kim | Cell surface expression and functional significance of adhesion molecules on human myeloma-derived cell lines[END_REF]. Plasma cell lines reveal CD44v3, v6, and v9 expression regulating binding capacity of the plasma cells to stroma cells.

CD44v9-mediated binding triggers IL-6 secretion of BMSC together with subsequent cell growth. Additionally loss of IL-6-dependent proliferation in XG-1 cells coincidences with loss of CD44v9 expression [START_REF] Van Driel | CD44 variant isoforms are involved in plasma cell adhesion to bone marrow stromal cells[END_REF]. In multiple myeloma (MM) cell lines adhesion is dependent on [122]. Further OPN induced proliferation as well as migratory capacity of MM cells is inhibited by anti-CD44v6 antibody [123].

High CD44v9 expression is associated with advanced disease stage, progressive disease, and shorter overall survival but with no other prognostic markers as serum lactic acid dehydrogenase (LDH) or beta2-microglobulin. This indicates CD44v9 to be an independent prognostic marker [START_REF] Stauder | Different CD44 splicing patterns define prognostic subgroups in multiple myeloma[END_REF]. Further acquired CD44v9 expression occurs during disease progression [125]. CD44v6 expression is associated with chromosome 13q14 deletion and advanced disease stages in MM [126].

Hodgkin lymphoma

Resistance to apoptosis in Hodgkin lymphoma (HL) might be conferred by CD44/MIF interaction. CD44 and MIF are secreted in HL cell lines, expressed in primary HL cells, and increased in plasma of HL patient. According to the previous findings of MIF blocking the cytotoxic T lymphocyte (CTL) response and CD44 being essential for MIF/CD74 complex signalling, this interaction has been proposed as a potential strategy of the malignant cells to evade cytotoxic killing [START_REF] Ma | Proteomics analysis of Hodgkin lymphoma: identification of new players involved in the cross-talk between HRS cells and infiltrating lymphocytes[END_REF]. Another hypothesis provided suggests tissue inhibitory of metalloproteinase 1 (TIMP1) in cooperation with CD44 to rescue pre-apoptotic defect B cells in B cell malignancies as HL [128].

The CD44v10 isoform displays prognostic relevance by being correlated to initial BM involvement and risk of relapse in nodular sclerosing HL [129].

Experimental antagonization and drug resistance

For targeting the CD44 function in hematological malignancies, two mechanisms are worth considering: (i) direct targeting of CD44 and its variant isoforms expressed on the malignant cells as a primary therapeutical approach, and (ii) reversing an acquired resistance to primary treatment mediated through altered CD44 expression patterns by antagonizing of CD44 function.

Direct targeting of tumor cells by CD44 can be achieved by antibody based immunotherapy, eventually in context with labeling with radioactive substances or cytotoxic agents.

CD44v6 antibodies have been investigated extensively in squamous cell carcinoma (SCC).

In radioimmunotherapy (RIT) of patients with metastatic and refractory disease, an anti-tumor effect was observed [START_REF] Heider | Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas[END_REF][131][132][133]. In the same setting, the CD44v6 antibody bivatuzumab has been used as vehicle for mertansine, a very strong inhibitor of microtubule assembly.

Treatment related death of one patient resulted in no further clinical testing of the agent [134][135][136]. Bivatuzumab mertansine failure in the clinical setting might be due to the high toxicity of mertansine itself. Being of 100-to 1,000-fold higher cytotoxic potency than other clinically used anticancer drugs such as taxanes or anthracyclines it might have been too potent for this kind of use.

Approaches of using 186 Re-labeled bivatuzumab in early stage breast cancer patients display limited efficiancy because of its higher uptake levels in blood and bone marrow cells than in tumor cells. [137]. This in turn might indicate a promising role in treatment of hematological malignancies. Besides of the therapeutic effect also diagnostic use of CD44v6 antibodies is shown by successful detection of HNSCC lymph node metastases via immuno-PET by 89 Zr-cmAb U36 [139,138].

Using HA, the natural binding partner of CD44, several drugs have been coupled to HA to be transported selectively to transformed cells: HA-But, a hyaluronic acid esterified with butyric acid, showing promising effects on malignant lesions of the liver [140], HA-cross-linked cisplatin displaying superior pharmacokinetics and pharmacodynamics to free cisplatin [START_REF] Li | CD44-targeted microparticles for delivery of cisplatin to peritoneal metastases[END_REF],

HA-containing liposomes carrying mitomycin C [START_REF] Peer | Loading mitomycin C inside long circulating hyaluronan targeted nano-liposomes increases its antitumor activity in three mice tumor models[END_REF], and paclitaxel targeting the malignant cells via CD44/HA interactions in cell lines [143] and in a human ovarian carcinoma nude mouse xenograft model. [START_REF] Auzenne | Hyaluronic acid-paclitaxel: antitumor efficacy against CD44(+) human ovarian carcinoma xenografts[END_REF]. Retroviral gene delivery resulting in enforced CD44s expression and CD44v7-10 knock down experiments suggest CD44 alterations to be a possible target for gene therapy [START_REF] Yang | Stable alterations of CD44 isoform expression in prostate cancer cells decrease invasion and growth and alter ligand binding and chemosensitivity[END_REF].

Multi drug resistance is a major problem in hematological malignancies. One mechanism behind this process is efflux pumps as for example the phosphoglycoprotein (Pgp) MDR1. In murine lymphoma and human leukemic cell lines decreased drug resistance achieved by treatment with HA oligosaccharides (oHA) is shown to be dependent on CD44 as the effect could be blocked by an anti-CD44 antibody. These findings indicate an important role of CD44 in mediating the efflux blocking effect of oHA on the Pgp transporter protein [146].

In DLBCL co-expression of CD44v and HA-mediated motility receptor (RHAMM) leads to unfavorable outcome when treated with cyclophosphamide, doxorubicin, vincristine, and At least two potential mechanisms have been provided in solid tumors as well as in inflammatory disease and might be useful models for treatment of haematological neoplasias: (i) Decrease of treatment resistance and increase of apoptosis in mesothelioma cell lines was achieved by siRNA silencing of CD44 or using CD44-neutralizing antibodies [START_REF] Tajima | Osteopontin-mediated enhanced hyaluronan binding induces multidrug resistance in mesothelioma cells[END_REF]. (ii) Developed for treatment of inflammatory disease PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody, might demonstrate a novel agent also useful in cancer therapy. By binding to CD44 it leads to inhibition of HA binding and thereby causes a loss of CD44 on the cell surface. The putative effect on drug resistance would have to be elucidated further in tumor settings [150].

Furthermore, new Imatinib-resistant CML cell lines display independency to common reported modes of resistance such as mutations of the bcr-abl domain, Lyn, Hck or MDR-1 overexpression. Instead CD44 and Fyn are upregulated in these cells and knock down or inhibition of Fyn results in re-sensitization to Imatinib [START_REF] Grosso | Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors[END_REF]. Because of known CD44/Fyn association [START_REF] Turley | Signaling properties of hyaluronan receptors[END_REF], the role of CD44 in this process has to be elucidated further.

Conclusions and perspective

By interaction with various different, and in some extent functional antagonistic proteins, CD44 seems to take part in a wide range of distinct cell functions. Because of its regulatory activity concerning e.g. cell migration, invasion, survival, and growth, altered CD44 expression might be an important step towards malignant transformation. This underlines the importance of understanding the molecular mechanisms behind the obvious effect of the different CD44 expression pattern in distinct tumor entities.

There have been a lot of previous research approaches concerning CD44 in solid tumors.

Here we focused on hematological neoplasias where CD44 has three main functions: First its role as prognostic marker, second its potential role for diagnosis, and third its role as a promising therapeutic target.

Distinguishing between different prognostic subtypes of one neoplastic disease entity is of great advantage. E.g. this aspect is of eminent importance in ALL because of the very young median age of the patients on the one hand and the very bad prognosis of a disease relapse on the other hand. For preventing secondary malignancies induced by too aggressive treatment regimes a deliberate risk stratification based on the expected tumor prognosis is imperative. Another example gives the observed treatment resistance to several standard therapeutics associated with CD44 expression in a limited group of patients with DLBCL. This provides the possibility of improved individual treatment decisions, which have been a big aim in cancer therapy in the last years.

More and more, CD44 is considered being of diagnostic use. First approaches were made in Burkitt lymphoma, where it was suggested to detect malignant cells on a sub-microscopical level by flow cytometry. In addition CD44 was claimed to be able to distinguish, together with other antibodies, between c-myc-associated lymphoma and DLBCL, which is of great importance concerning treatment decision.

The last function might be the most promising. Several approaches have been made investigating the possible role of CD44 in new therapy strategies of solid tumors. CD44 antibodies as well as HA achieved promising results in therapy and diagnostic. Also first research achievements were made concerning CD44 and gene therapy, opening a wide interesting field for future investigation. The development of CD44 targeted therapeutic concepts in hematopoietic malignancies is far behind compared to treatment of solid tumor entities but latter can be used as model for future approaches in hematological neoplasias.

Furthermore overcoming acquired treatment resistance in hematological malignancies certainly needs to be investigated in the near future.

[ 99 ,

 99 100]. Together with observed eradication of LIC and reduced repopulation in immune deficient mice [101], this might provide a way of antibody-triggered, CD44-and IL-8/GM-CSF-mediated targeting of AML cells via combined effect of reversed differentiation block and dependency of LIC on their bone marrow niche. Co-culturing of M6-AML TF-1 cells with mouse bone marrow-derived MS-5 cells resulted in increased CD34+ cell number and upregulation of CD44s and CD44v10. Anti-CD44 mAb 5F12 treatment displayed reduced adherence of TF-1 cells to the stroma [102]. In acute promyelocytic leukemia (APL) one molecular mechanism behind all-trans RA (ATRA)-resistance is the absence of functional CD44 expression on the cell surface and consequent apoptosis resistance: In the APL cell line NB4 treatment with anti-CD44 antibodie inhibited cell growth and induced apoptosis. The ATRA-resistant subclone NB4-LR1 displayed no CD44 expression due to epigenetic silencing mechanisms, which can be reversed treating the respective line with the DNA methylating inhibitor 5-aza-CdR. A similar effect is seen upon treatment with cyclic AMP and subsequent CD44 ligation by the anti-CD44 specific antibody A3D8, which results in apoptotic cell death [103]. CD44v6 surface expression correlates with shorter overall survival [19]. The t(8;21) translocation in AML is associated with upregulated CD44 both on mRNA and protein level[104]. Aggressive non Hodgkin lymphoma Aggressive non Hodgkin lymphoma (NHL) show a new role of CD44 in diagnostic use, providing a model for minimal residual disease (MRD) detection and for discrimination between c-myc-associated (Burkitt lymphoma) and non-c-myc-associated lymphoma (Diffuse large B cell lymphoma: DLBCL). Further CD44v6 displays strong prognostic potential. Examining 39 cases of childhood Burkitt lymphoma for CD44 surface expression by flow cytometry, deficient expression compared to normal B cells of similar maturity is observed. This characteristic possibly allows detection of tumor cells at a sub-microscopical level [105]. So far two distinct antibody panels for distinguishing between c-myc-rearranged and nonrearranged tumors have been introduced: CD44, CD38, and T cell leukemia 1 (Tcl-1), showing superiority to conventional staining with CD10 and Bcl-2 [106], and combination of CD44 and CD54 [107]. Among others Stauder et al examined a pool of 138 patients, including 76 low and 62 high grade NHL, showing an association between the expression of v6-containing isoforms of CD44 and aggressive NHL. Additionally strong expression levels of CD44v6 were highly significant correlated to a decreased overall survival but not to other prognostic markers as age and Ann Arbor classification determined in both groups. Neither ECOG performance status, extranodal involvement and serum LDH levels in the group of high grade NHL showed any association to CD44v6, indicating CD44v6 to be an independent factor for risk stratification [108, 109].

  extended lymph node involvement, advanced Binet and Rai stage, and chemotherapy requirement [117]. As observed in other entities, CD44 displays an anti-apoptotic effect in CLL cells: In vitro data showed a protective effect of co-culturing CLL-with HK cells, a follicular dendritic cell line, against spontaneous apoptosis by increased levels of the anti-apoptotic Bcl-2 family member Mcl-1. In this setting, blockage of CD44 by antibodies resulted in Mcl-1 downregulation and inhibition of the protective effect through the HK cells, indicating the antiapoptotic mechanism being CD44-dependent [118]. Complex formation of CD44v, VLA-4, and proMMP-9 in CLL but not in normal B cells as well as increased proMMP-9 secretion upon CD44 antibody treatment provide a putative molecular mechanism of regulation of invasiveness. The functional relevance has not been further elucidated [119].

  prednisone [147]. Similarly in MM, treatment success of Dexamethasone was dependent on CD44 with decreased apoptosis rates in cells with high CD44 [148]. Here neutralizing CD44 might reveal new therapeutic strategies in treatment of multidrug resistant haematological malignancies.

  triggered by CD44 ligation in THP-1 monoblastic leukemia cells AML cell line Bourcier et al. [67] GM-CSF CD44 Ligation induces gene transcription and secretion AML FAB M5 blasts Delaunay et al. [68]

Figure 1 .

 1 Figure 1. Exon organization Dark green circles represent the constant exons, light green the variable ones (v1-10). The extracellular part (EC) is encoded by exon 1 to 17 (E1-17), spanning at least 248 amino acids: E1-5 form the amino terminal globular region, E16 and 17 encode the stem (46 amino acids) with possible insertion of v1-v10, whereas v1 is not existent in the human genome. E18 represents the transmembrane region (TM) that consists of 23 hydrophobic amino acids and a cysteine residue. E19 or E 20 encode the intracellular (IC) cytoplasmic tail, creating either a short tailed (3 amino acids) or a more abundant long tailed (72 amino acids) CD44.

Figure 2 .

 2 Figure 2. Protein structure CD44v (left) compared to CD44s (right). Globular extracellular region includes HA binding motifs (yellow), one located inside the so called 'link domain' spanning amino acids 32 to 123, and the other at amino acid positions 150 to 158, and a cleavage site (red). Exon v3 can be modified by heparan sulphate (purple). The cytoplasmic tail embodies a basic amino acid binding site for ERM and Merlin at amino acid 292 to 300 (dark blue), an ankyrin binding site at amino acid 304 to 318 (light blue), and a Lck binding site located at the membrane proximal region.

Table 1 . Extracellular CD44 binding partners

 1

	Ligand	Interaction

Table 2 . Extracellular CD44/Growth factor interactions

 2

	Ligand	Interaction

Table 3 . CD44/receptor tyrosine kinase interactions

 3

	Protein	Interaction

Table 4 . Impact of CD44 signalling on MMPs

 4

	Protein	Interaction

Table 5 . CD44-mediated cytokine expression

 5

	Cytokine