The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal differentiation, they can be used to generate a model of endochondral ossification, but this limitation must be kept in mind when using them in cartilage tissue engineering application. The two major areas of musculoskeletal medicine are trauma care and degenerative disorders. Degenerative joint diseases account for half of all chronic conditions in people aged over 65. In young adults, it is foremost traffic accidents that produce a tremendous demand for restorative medical attention. It is estimated that traffic injuries worldwide result in more than 30 million severe or disabling injuries, with associated costs of 500 billion US dollars annually . The newBone marrow-derived mesenchymal stem cells (MSCs) possess a number of abilities, making them interesting candidate cells for tissue engineering applications . They haex vivo expanded articular chondrocytes into cartilaginous defects of the knee joint was one of the first successful applications of tissue engineering [In 1994, the transplantation of ineering . Introduineering . A dedifineering , but theineering .Another example for the potential use of tissue engineering in musculoskeletal medicine is the management of large bone defects which still pose a considerable challenge in musculoskeletal surgery. Other than autologous bone grafting, either in a local fashion or through bypass grafting or bone transport, there is no other effective method for management of diaphyseal defects . It is a3) and the glucocorticoid dexamethasone (Dex) [Cartilage is routinely transformed into bone in in the process of endochondral ossification, during long bone growth and in fracture healing. Here, chondrocytes undergo a terminal step of differentiation, increasing in size and finally undergoing cell death by apoptosis, leaving behind a suitable matrix for bone ingrowth, vascularization and mineralization. This process was initially described in growth plate chondrocytes  and the ne (Dex) .in vivo and in vitro on cartilage and chondrocytes, their effect on this tissue is not yet fully understood, and their role in induction or prevention of endochondral ossification remains ambiguous. GCs signal largely through the GC receptor, a member of the nuclear receptor family that translocates into the nucleus upon ligand binding and acts as transcription factor [in vitro [Though glucocorticoids (GCs) are frequently used n factor , but then factor . Also Dein vitro . Extensiin vitro  as well in vitro .3-Receptor alpha (TRα) and T3-Receptor beta (TRβ), modifying transcriptional activity on the genome [In vitro, thyroid hormones have been frequently used to induce hypertrophy in fetal and growth plate chondrocytes in various culture systems [3 on adult articular cartilage, but it’s possible role in the development of OA has been suggested previously [Triiodothyronine, the deiodinated and thereby activated form of the thyroid hormone, might well be the most important growth hormone. It affects virtually every cell in the organism by binding to its intranuclear receptors Te genome . Its role genome . Congenie genome . In vitr systems –28. Veryeviously .3 and Dex on primary articular chondrocytes and differentiated chondrogenic MSCs, using serum-free induction and the alginate bead three-dimensional (3D) cell culture model. The study was performed on material of a large animal model (bovine), so the results can be used for later establishment of in vivo models for cartilage degradation or bone tissue engineering constructs with some biomechanical validity. In addition to cell survival and matrix production, the experiments were designed to monitor phenotypic stability and furthermore to detect signs of terminal chondrocyte differentiation as a preliminary phase of endochondral ossification and mineralization. We hypothesized that both hormones will induce terminal differentiation in adult articular chondrocytes and chondrocyte-like cells. Furthermore, we expected articular chondrocytes and chondrogenically differentiated MSCs to be similar in their response to the hormonal stimuli.In this study, we investigated the effects of the two hormones TChondrocytes were isolated from articular cartilage of bovine fetlock joints from three calves (two to six month of age) . Cartila2 culture flasks  in control medium consisting of DMEM low glucose (1g/L) with 1% PenStrep, 25 mM HEPES Buffer (Gibco), 3.7 mg/ml NaHCO3, and 10% FCS. After being cultured for four days, non-adherent cells were removed by changing the culture medium. In the following, the medium was changed every two to three days. Primary cultures were considered passage 0 (P0). Upon subconfluence, the cells were detached by using 0.25% trypsin (Gibco) and split in three for the next passage. Cells of passage P2 to P3 were encapsuled in the alginate beads and used for experiments.MSCs were isolated from full bone marrow aspirate of two calves (four and six month of age) as described in the literature . Bone ma6 cells/ml, and expressed through a 22-gauge needle in a dropwise fashion into a 102 mM calcium chloride (CaCl2) solution. After instantaneous gelation, the beads were allowed to polymerize for no more than 10 minutes, washed and cultured in 6-well plates, coated with 3% agarose, approximately 30 beads per well in 6 ml of medium [Alginate is a natural carbohydrate polymer that is derived from brown algae. The monomers gelate upon contact with a divalent ion such as calcium, forming an elastic hydrogel. Removal of calcium through a chelator unlinks the monomers, thereby dissolving the hydrogel and releasing the cells without digestion or disruption . It is ff medium .5 MSCs were resuspended in expansion medium and were spun down (10 min @ 500g) in a 15-ml conical tube. Pellets received standard chondrogenic medium as described above, with TGF-β1 [2 with loosened caps for 20 days, changing the medium three times per week.For differentiation, alginate beads with encapsulated MSCs of passage 2 to 3 were grown in a standard chondrogenic medium, consisting of DMEM, supplemented with 1 µg/ml insulin, 1ng/ml transferrin and 1 ng/ml sodium selenite (ITS+1), 60 µg/ml ascorbic acid, 100 nM dexamethasone  and 10 ng/ml transforming growth factor beta-1  for 3 weeks. Alginate beads without TGF-β1 were cultured as controls to evaluate the effect of the three dimensional culture on the cells. MSCs in monolayer, receiving standard expansion medium, were used as a negative control. Though the standard protocol for chondrogenic differentiation is the pellet culture system , alginath TGF-β1 . Pellets-7 M) triiodo-L-thyronine sodium salt (T3) in 10mM sodium hydroxide  or 1 µM Dex, respectively. Hormone concentrations were adapted from related studies in the literature to ensure comparability. Pilot experiments with lower concentrations of the hormones showed no significant effect of differences in concentration (data not shown).Alginate beads with cells were kept in agarose-coated 6-well tissue culture plastic dishes  in a cell culture incubator at standard conditions . The chondrocyte medium was composed of high glucose DMEM, supplemented with 1% Pen/Strep, 0.3% Gentamycin, 25mM HEPES Buffer, 60 µg/ml L-ascorbic acid 2-phosphate and 10% FCS. For hormonal treatment, serum was replaced by 1 mg/ml bovine serum albumin, 1 µg/ml insulin, 1ng/ml transferrin and 1 ng/ml sodium selenite, and the medium was also supplemented with 100nM  was added to the medium of some beads on day 35. The samples were then cultured for a further 7 days and harvested.Quantification of (DNA) was carried out using the Hoechst 33258 fluorescent dye as described elsewhere  with somFor hydroxyproline (HYP) detection, 250 µl of the digested sample were mixed with an equal volume of 12N HCl (Fluka) and hydrolyzed at 110°C for 12h. Samples were neutralized adding 300 µl of 10N NaOH (Fluka), cleared using a charcoal resin mix, pH was checked with indicator paper and titrated to neutrality with HCl. A standard curve was prepared from a stock solution of trans-4-hydroxy-L-proline (Fluka), and 250 µl of all samples and standards was transferred into a fresh 2 ml tube. All samples and standards were mixed with 250 µl of saturated NaCl. Oxidation of HYP was reached by addition of 500 µl of a pH 6-buffered Chloramine T solution, containing 120 mmol/l citric acid, 440 mmol/l sodium acetate, 425 mmol/l sodium hydroxide, 105 mmol/l acetic acid, 33 mmol/l Chloramine T trihydrate and 30% v/v 2-Methoxyethanol in water, mixing and leaving 20 minutes at room temperature. Color formation occurred after addition of 500 µl of a color solution, containing 11.25% w/v 4-(Dimethylamino)benzaldehyde, 2 mol/l perchloric acid and 50% v/v 1-Propanol in water, and incubating at 65°C for 15 minutes . Absorba3Citrate (Sigma), 3 mM Na 2EDTA (Sigma), 18.75 mM NaCl (Fluka) and 0.05% Tween 20 (Sigma). Samples were diluted in the same buffer to match the linear range of the standard curve. Dye was added to samples and standards in a 96-well culture plate , after all samples and standards were mixed with Guanidine Hydrochloride solution to a final concentration of 240 mM. Absorbance was measured immediately at 535nm and 595nm in the Bio Assay Reader. Using the absorbance ratio, total amounts of GAG were calculated from the linear range of the standard curve. All measurements were done in duplicates.Total sulfated proteoglycans (GAGs) were quantified as a marker of matrix synthesis in the alginate beads, using the dimethylmethylene blue assay as described elsewhere. The dye is known to give a positive reaction with alginate, which is constant over a broad range of values, and the problem can be circumvented by some modifications of the assay . Samples3Citrate.2H2O (Sigma). Cells were collected by centrifugation (10 minutes at 700 G). The pellet was resuspended in a 10mM Tris-HCl buffered (pH 7.4) solution containing 0.1% Triton-X and extracted at 4°C under agitation for exactly 60 minutes. Samples were then centrifuged 10 minutes at 1000g to remove cells and cell debris, and the supernatant was transferred into a fresh tube. Samples were stored frozen at -20°C until analysis. Activity of ALP protein was measured by adding 50 µl of a substrate solution, containing 25 mg/ml of 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma) in a 1M diethanolamine-buffered 0.5mM MgCl2 solution (pH 9.8), to 100 µl of sample in 250 µl of alkaline buffer containing 1.5 M 2-amino-2-methylpropanol (pH 10.3) and 100 µl of water, and incubating at 37°C for exactly 15 minutes. The reaction was stopped by addition of 500 µl of 0.1M NaOH. The amount of p-nitrophenol released was measured spectrophotometically at 405 nm, and amounts were calculated from a standard curve, prepared from a stock solution of p-nitrophenol (Sigma) in the same buffer [Alkaline phosphatase (ALP) can be considered a marker of osteogenesis. Cells were released from alginate beads as described above with slight modifications. Three samples of three beads each were dissolved in a sterile dissolution buffer consisting of 150 mM NaCl (Fluka) and 55 mM Na e buffer .2 to prevent the beads from dissolving. Beads were soaked in Cryocompound (Leica) and sectioned on a cryotome to sections of 8 µm and mounted on SuperFrost Plus slides. Sections were kept frozen until stained.Methanol fixed alginate beads or pellets were transferred to a 30% sucrose solution overnight, containing 0.1 M CaCl2, pH adjusted to 1.0) for 30 minutes. Slides were washed in 0.1 M CaCl2, dehydrated and mounted with DPX water-free mountant (Sigma).Alcian blue interacts with alginate but lowering the pH of the dye solution to 1.0 suppresses background staining of alginate and enables a selective staining of sulfated PGs only . Slides 2 solution, bound to cause considerable background staining. The slides were rehydrated and stained in a filtered AR-solution , blot dried, and dipped in acetone [Though described in the literature ,43 aliza acetone , then deT method, using 18S rRNA as endogenous control and the day 0 control-group as calibrator [Three times ten beads per group and time point were dissolved in the sterile dissolution buffer described above and cells were collected by centrifugation (10 minutes at 700g). Total RNA was then extracted from the samples using TRI Reagent with poliacrylic carrier (Molecular Research Center), and RNA was purified using the Sigma GenElute Mammalian Total RNA Miniprep Kit (Sigma), according to manufacturer’s directions. Isolated mRNA was transcribed to complementary DNA (cDNA), using Applied Biosystems reverse transcription kit and a reaction of 25°C for 10 minutes, 48°C for 30 minutes, 95°C for five minutes on a thermo cycler (Eppendorf). The primers and probes (MicroSynth) used for the detection of collagen types I, II and X mRNA were custom designed and the sequences are listed in librator .+: p<0.05; + +: p < 0.01; + + + p < 0.001; + + + +: p < 0.0001.Raw data from measurement apparatus  was exported to CSV files, and imported into Microsoft Excel 2007 . Data management, calculations from standard curves and dCt-Calculations for RT-PCR, as well as averaging of duplicate or triplicate measurements of the same sample was completed using Microsoft Excel 2010. Calculated and averaged data was then transferred to Graph Pad Prism 5.04  for statistical analysis. Statistics were completed using multifactor ANOVA, and p-values were calculated with Bonferroni post-hoc tests from the ANOVA, and are stated in the text and graphs. P < 0.05 was assumed “significant difference”. All analysis was completed with GraphPad Prism 5.04 for Windows, and all graphs were prepared with the same software. The authors received help with the statistical analysis by other persons as mentioned below (see acknowledgments). Unless stated otherwise, within the graphs, statistically significant difference between time points are indicated in asterisks as follows: *: p<0.05; **: p < 0.01; *** p < 0.001; ****: p < 0.0001. Statistically significant differences between treatment groups are indicated in crosses as follows: To compare primary chondrocytes to chondrogenically differentiated MSCs, the stem cells were differentiated in the alginate beads. During the differentiation, the cells acquired the properties of chondrocytes, including production of the cartilage-specific Col II and the production of a hyaline like extracellular matrix, composed of collagen and proteoglycans. Successful differentiation was therefore assumed with the expression of Col II mRNA and an increase in proteoglycan production.3 and Dex could influence chondrocyte differentiation towards hypertrophy and endochondral ossification. For this, the beads with chondrogenically differentiated MSCs, or serum precultured chondrocytes, were cultured in serum free medium containing either dexamethasone or T3 for five weeksMSCs in alginate beads proliferated only little, and at day 21, DNA content was increased by app. 30%, while in pellet culture the total DNA content decreased by 20% during the differentiation period see . This deNo differences in DNA content were found between the chondrocyte treatment groups at any time point after “day 0”, which indicates the start of the hormonal treatment. In the MSCs, as described above, DNA content increased significantly during differentiation then dropped back to the levels of the initial seeding density within the first 7 days of hormonal treatment. No differences in DNA content were seen due to the hormonal treatment see .The mRNA levels of Col I, II and X over 21 days of MSC chondrogenesis are shown in 3 had an up-regulating or stabilizing effect on mRNA expression of Col I and Col II. Col II was not significantly modulated by Dex, but after five weeks of culture, the T3 treated group showed the highest Col II mRNA, even surpassing the amounts in freshly isolated chondrocytes . Col I, foremost a marker of de-differentiation in the articular chondrocytes, was significantly decreased by Dex, and no up regulation occurred during the whole observation period. Control and T3 group, in contrast, showed a steady state to slight increase, starting after two weeks of culture. With regard to Col I, Dex had a very similar decreasing effect on the chondrogenically differentiated MSCs as on adult chondrocytes . T3 had no effect significantly different than from the control group, and by day 35, Col I mRNA levels in T3 and control group were not significantly different than on day 0. For Col II, however, T3 significantly decreased mRNA expression as compared to control , while the levels remained mostly stable under control treatment or with Dex supplementation, thereby showing a reversed effect on the hormones when compared to adult chondrocytes , to then drop again when treated with control medium or Dex, with the drop quickest in the control group . Correlating to the increase in col X mRNA, ALP activity also went up in the T3-treated chondrocyte group .Col X mRNA expression and ALP protein activity were monitored over the culture period as markers of hypertrophy . Collage3 over the culture period of five weeks, as compared to control or over time. After the aforementioned increase during the differentiation, the levels of Col X mRNA remained stable, with a slight but non-significant tendency to decrease during culture with T3. In the MSCs, Dex had a significant impact, and increased Col X mRNA production significantly over time  and against control group . After five weeks, Col X mRNA levels were increased by two orders of magnitude compared to start of induction.The chondrogenically differentiated MSCs showed no change in Col X mRNA expression in response to TIn contrast to articular chondrocytes, freshly isolated MSCs showed no measurable ALP protein activity. Expansion in monolayer did not change this. After encapsulation and chondrogenic differentiation, ALP activity became measurable and rose significantly to levels comparable to those of serum-cultured articular chondrocytes .GAG content of freshly prepared chondrocyte beads after cell isolation and matrix digestion was low as expected and increased during serum preculture. Thereafter, only with Dex did the production increase further. The MSCs displayed a similar pattern as the articular chondrocytes. As stated above, the total amount of GAG had already increased during the chondrogenic differentiation period see , comparaAlcian Blue staining of cryosectioned alginate beads for glycosaminoglycans was completed to confirm findings of biochemical analysis. After five weeks of culture, cells demonstrate a typical chondrogenic phenotype with a darker stained ring of pericellular matrix around each cell. In histology, no difference was seen between the Dex and T3 group, and both showed intense uptake of alcian blue .3 group in the chondrocytes seems to show an increase over control and Dex after 14 and 35 days, an effect that was not seen in the MSCs , none of the changes seen were statistically significant.To determine whether the changes in the mRNA expression actually lead to differences in collagen production levels, total hydroxyproline per DNA was measured in the samples. The TMSCs see . During MSCs see , but in The alizarin red staining of samples to investigate calcification led to a degree of background staining due to the calcium present to gelate the alginate. In our hands, this background staining with alizarin red was present on freshly prepared and cultured beads alike see .Despite the backgroup staining observed, differences could be detected. For MSC beads, an increase in the AR staining intensity was observed from day -21 and day 0. The Dex-treated beads then showed a much more intense staining, and at higher magnification, the Dex-treated cells showed a pericellular matrix and dark red calcium depositions around the cells, which was not seen at this intensity in the other samples . The sam3 and Dex, on chondrocytes and chondrogenically differentiated MSCs in different settings, aiming to determine if and how these hormones would influence cell proliferation, matrix production and the expression of Col I, Col II and Col X mRNA as markers for phenotypic stability. We had hypothesized that the hormones would induce Collagen X expression, ALP protein activity and increased calcification as markers of terminal differentiation. We also expected articular chondrocytes and chondrogenically differentiated MSCs to be similar in their response to the hormonal stimuli.In this study, we investigated the effects of two hormones, T6 cells / ml of alginate, a concentration that is likely to be close to the cell density in human adult knee cartilage [We have used full thickness cartilage from bovine fetlock joints as a source of articular chondrocytes, and encapsuled the cells into alginate beads. The cells were seeded at a concentration of app. 4*10artilage . Experimartilage , but is Most of the cells acquired in the digestion process were fully differentiated into the articular chondrocyte phenotype, as seen with Col II mRNA being expressed in high amounts and very little Col I mRNA being expressed. Unexpectedly there was considerably high expression of Col X mRNA in the freshly isolated chondrocytes. In young animals, ossification is still occurring in the lower level of articular cartilage where it is directly adjacent to bone. Though care was taken during harvest not to scrape into the mineralizing layer, it is possible cells were taken from the calcifying zone that were expressing both Col X mRNA and ALP activity . A rapidThe cells readily responded to serum as a medium supplement inducing proliferation and matrix production. In the presence of serum, the cells doubled in number within two weeks, and produced both collagen and proteoglycan as the typical markers of adult chondrocytes. However, we also saw the previously described trend towards a dedifferentiation  by way oin vitro conditions can also induce features of fibrocartilage (Col I) as well as features typical of hypertrophy (Col X and ALP) [This did not hold true for MSCs: Their count decreased significantly when medium was changed from differentiation medium (with TGF-β1 and Dex) to the serum-free medium. The most common and best described method of chondrogenic differentiation of MSCs is that of pellet culture under the influence of TGF-β, resulting in differentiation towards a chondrocyte-like phenotype, characterized by upregulation of cartilage-specific mRNA and cartilage ECM molecules such as collagens and proteoglycans . Apart fand ALP) ,53. The and ALP) . The expand ALP) . The matWe have treated the entrapped cells with two different agents in a chemically defined medium to investigate the effect of the hormones in regards to cell proliferation, matrix production and collagen expression pattern. Our main issues were phenotypic stability as well as signs of terminal differentiation of the chondrocytes.3 on chondrocytes of different origin, mostly growth plate and fetal, and in many different culture systems and media conditions. While human articular chondrocytes subjected to T3 in monolayer and serum containing medium showed enhanced proliferation [3 or T4 on adult bovine cartilage cell proliferation or GAG synthesis in high-density cultures in serum-free medium [3-treated chondrocytes vs. control in means of DNA and GAG content, and in line with other studies performed in serum-free medium [3.Review of the literature shows many studies using Tferation , earliere medium . This ise medium . The cone medium . Obvious3 generally had an upregulating effect on all types of Col mRNA, including Col X mRNA. While the receptor and pathways of T3 induced effects in growth plate chondrocytes could be recently identified [3, it seems likely that these pathways, remnants of the growth plate cartilage, are still active in the “permanent” articular chondrocytes as well [3 was found to be stabilizing dedifferentiated human chondrocytes of various sources and inhibit hypertrophy-inducing effects of other agents [3 and show enhanced ALP activity and an increase in Col X production when stimulated in monolayer and serum culture [As markers for chondrocyte hypertrophy we have measured Col X mRNA levels, ALP protein activity in the beads and evaluated calcium incorporation histologically. Collagen Type X is considered a marker of chondrocyte hypertrophy and has been frequently used as such in many publications. Along with its appearance in the growth plate, Col X mRNA is also expressed by chondrocytes in osteoarthritic joints . Over thentified , this ha as well . In other agents . Adult p culture .3 in the absence of Dex also showed a tendency to induce terminal differentiation in the articular chondrocytes. The increase in Col X mRNA was accompanied by a rise in ALP activity over the control group. Apoptosis is closely associated with terminal chondrocyte differentiation [3 in the absence of Dex is sufficient to promote Collagen X expression in the articular chondrocytes, but does not necessarily induce the full features of the hypertrophic chondrocyte. The differential effect of T3 on collagen type II and col X gene expression in chondrocytes and chondrogenically induced MSCs would suggest that they are not at the same developmental stage. For both collagens, addition of T3 lead to an increased expression in chondrocytes compared to control, which was not seen in chondrogenically induced MSCs.In our study, Tntiation , but no ntiation . We also3 on chondrogenically differentiated MSCs are very sparse, but evidence suggests that removal of TGF-β, reduction of Dex and addition of T3 should induce hypertrophy in the chondrogenically differentiated MSCs at least in pellet culture [3, promoting MSC osteogenic differentiation at 10 pM [3 had little effect at all on the MSCs, the only significant difference was a marked and steady decrease in Col II mRNA, while Col I and X mRNA levels remained stable. Indeed, if T3 is suitable to promote a more osteogenic phenotype in MSCs, this would be in line with the some of the evidence available so far. The dose used in our experiments was identical for chondrocytes and MSCs, and therefore 100-fold higher than the dose used before to induce hypertrophy in pellet cultures – on the other hand, the use of Dex in those experiments might also be a key factor, as discussed further below.The facts currently known about an effect of T culture . Here isat 10 pM . We had It is striking that especially the differentiated MSCs of the control group in serum-free ITS-supplemented medium do seem to remain in their chondrocyte-like phenotype over five weeks, with no significant increase of ALP protein activity and no increase in Col X mRNA expression. Alginate as the chosen scaffold material might also have a certain influence to this fact. Alginate binds divalent calcium ions, producing a positive charge within the gel, a feature that seems beneficent to chondrogenesis, as surface chemistries promoting a rounded cell morphology in general seem to do . On the 3, the effect of Dex on the growth plate is a matter of ongoing research, but without doubt, glucocorticoids such as Dex have a major role in the development of the growing skeleton, both through systemic [The second hormone investigated in this study was dexamethasone. As with Tsystemic  as well systemic . Besides3-treated beads was observed. While some studies describe a decreased cell survival or increase of apoptosis in chondrocytes submitted to Dex or other steroids in vitro [in vivo [Looking at cell survival, no significant differences between the Dex-treated alginate beads versus control or Tin vitro ,70 and i[in vivo , others [in vivo . We have3 treated group. Such an increase in GAG production was seen by others previously [Dex is capable of regulating both degrading and matrix-building components of the ECM in a dose-dependent manner. In the current literature, numerous, partly conflicting results have been reported ,73. We feviously . We posteviously  downreguApart from its previously debated apoptotic effect, Dex is also under debate as an inductor of chondrocyte terminal differentiation. Addition of Dex, to monolayer human chondrocytes in serum supplemented culture increased hypertrophy and apoptosis , but notin vitro induction of chondrogenesis, the differentiating cells also exhibited Col X as a marker of hypertrophy. The phenotype of MSCs in cartilage repair can therefore be so unstable that differentiation will continue along the endochondral ossification pathway, especially with Dex present in the medium [Phenotypic stability is a major issue with MSCs in tissue engineering, especially when aiming for a cartilage repair graft. For this reason, differentiated MSCs must be committed to their cartilage-like gene expression pattern, also in the absence of chondrogenic factors or in the presence of osteogenic factors, a feature that seems to be created even through a short TGF-β exposure . The abse medium . While ue medium .3 as an inductor of chondrocyte hypertrophy in postnatal immature articular chondrocytes. We have also shown a contrary effect of Dex to contribute to the ongoing discussion of the use of this hormone in cartilage tissue engineering. We also want to suggest the idea that even those chondrocytes deemed “permanent” may not be so, but rather be “slow transient” in their response to stimuli, a fact that must be considered when using these cells in tissue engineering applications.In summary, chondrocytes are capable of more than merely synthesizing cartilage matrix. Their terminal differentiation is an important process in long bone growth and secondary fracture healing. It is also suspected that it plays a role in onset of degenerative OA, yet the factors that influence it are poorly understood. Also, chondrocytes from various sources show different behavior towards their response to any stimuli. Without defined conditions, a comparison of completed studies is without benefit. Currently, too little work is completed on primary adult chondrocytes to lead to later clinical applications. In this study we have taken a step towards probing TThe use of MSCs in bone and cartilage tissue engineering is well established, but phenotypic stability is a major issue, especially when aiming for a cartilage repair graft. It remains under debate whether or not the phenotype of the chondrocyte-like cell after differentiation is stable enough to be used in cartilage repair. When MSCs are indeed moving on towards terminal differentiation after chondrogenic differentiation, they can be used to generate a model of endochondral ossification. We can contribute with our results, recommending the use of alginate beads rather than micromass pellet cultures as a scaffold, and the use of Dex to trigger Col X mRNA expression in these cells.


Burkholderia pseudomallei causes the disease melioidosis, which often presents as a serious suppurative infection that is typically fatal without intensive treatment and is a significant emerging infectious disease in Southeast Asia. Despite intensive research there is still much that remains unknown about melioidosis pathogenesis. New animal models of melioidosis are needed to examine novel aspects of pathogenesis as well as for the evaluation of novel therapeutics. The objective of the work presented here was to develop a subacute to chronic caprine model of melioidosis and to characterize the progression of disease with respect to clinical presentation, hematology, clinical microbiology, thoracic radiography, and gross and microscopic pathology. Disease was produced in all animals following an intratracheal aerosol of 104 CFU delivered, with variable clinical manifestations indicative of subacute and chronic disease. Bronchointerstitial pneumonia was apparent microscopically by day 2 and radiographically and grossly apparent by day 7 post infection (PI). Early lesions of bronchopneumonia soon progressed to more severe bronchointerstitial pneumonia with pyogranuloma formation. Extrapulmonary dissemination appeared to be a function of pyogranuloma invasion of pulmonary vasculature, which peaked around day 7 PI. Histopathology indicated that leukocytoclastic vasculitis was the central step in dissemination of B. pseudomallei from the lungs as well as in the establishment of new lesions. While higher doses of organism in goats can produce acute fatal disease, the dose investigated and resulting disease had many similarities to human melioidosis and may warrant further development to provide a model for the study of both natural and bioterrorism associated disease.Infection with  Burkholderia pseudomallei, remains an emerging infectious disease of global importance Despite its discovery nearly a century ago rd leading infectious cause of death, behind human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and tuberculosis In northeast Thailand, melioidosis is currently the 3Mus spp., despite the very wide host range of animals affected by melioidosis B. pseudomallei to humans Mice have been the predominant host used for studying the pathogenesis and potential therapeutic interventions for melioidosis, and the availability of a large variety of inbred strains, immune function mutants, and immunologic reagents make murine models attractive for the study of this disease Goats are naturally affected by melioidosis and offer several potential advantages for use as an animal model of melioidosis, including a large body size allowing clinical monitoring and therapeutic intervention in a manner relevant to humans. Natural disease has been reported to occur in goats from Northern Australia et al. (1982) used a goat isolate of B. pseudomallei passaged twice through hamsters prior to intraperitoneal (IP) or subcutaneous (SC) inoculation of goats with either 6.5×107 or 6.5×109 colony forming units (CFU) et al. (1988) used sheep, goat, or bird isolates of B. pseudomallei to infect goats SC with doses ranging from 90 CFU to 5×105 CFU B. pseudomallei isolates, as suggested by Thomas et al. Two previous experimental models of caprine melioidosis have been published. Narita B. pseudomallei and melioidosis has changed considerably. Significant advances have been made in the study of melioidosis, but there is still a great deal about B. pseudomallei infection that remains unknown. A greater understanding of pathogenesis and immune function can be achieved through comparative studies of different animal models and human disease. In pursuit of these goals, the objective of this study was to create a non-fulminant, subacute to chronic caprine model of aerosol-transmitted melioidosis. Such disease was readily produced, allowing for the description of the clinical, hematologic, microbiologic, radiographic, and pathologic features of caprine disease, adding a novel and useful model for the study of melioidosis.In the 20 years since description of these original caprine models, which primarily focused on disease in goats and the potential impact on human health from raw milk consumption B. pseudomallei (Bp 4176/MSHR 511), isolated from an outbreak at an Australian goat farm 4 CFU/ml. The bacterial suspension used for infection was backtitrated in duplicate on MH agar plates incubated at 37°C in air.et al. B. pseudomallei. Nasal swabs, organ homogenates, and abscess material were also cultured on MH-gentamicin plates at 37°C in air and read at 96 h, 48 h, and 48 h, respectively.Quantitative blood culture was performed using a pour plate technique modified from Simpson B. pseudomallei were performed in a biosafety level-3 facility.All experiments using 4 CFU in 5 ml of PBS was selected based on pilot studies, which investigated a dose range of 104–108 (data not shown). Nebulization time was approximately 10–15 min. Total dose of bacteria delivered to the lungs was calculated as 10% of the bacteria placed in the nebulizer, a figure that was derived from pilot studies in which two goats were euthanized immediately after exposure and lung tissues samples representing all lung fields were homogenized and plated to determine CFU delivered per gram of lung tissue.Bacteria were delivered as an intratracheal aerosol. The bacterial suspension was aerosolized using a LC Sprint® nebulizer (mass mean diameter 3.5 µm) and Vios® air compressor . The nebulizer was modified so that all exhaled air passed through a HEPA filter. The modified nebulizer was then connected to a cuffed endotracheal tube (size 10) for intratracheal delivery of the aerosol. A target dose of 1×10This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and every effort was made to minimize suffering. The protocol was approved by the Animal Care and Use Committee of Colorado State University . Group size was determined subjectively based on pilot studies as the study was designed to be descriptive (vs. quantitative) in nature.ad libitum access to water and were fed a complete pelleted feed twice daily. Goats were acclimatized to the facility for approximately one week prior to infection. Infection was performed (Day 0) under intravenous general anesthesia using xylazine hydrochloride (premedication) and ketamine hydrochloride (induction/maintenance). Endotracheal intubation and nebulization was performed with the goats in sternal recumbency.Twelve yearling Nubian-cross goats were obtained through private sale for use in this experiment. Goats weighed between 35 to 50 kg. There were seven males and five females. Animals were housed in an ABSL-3 facility for the duration of the experiment. They had Beginning on Day -4, the goats’ attitude, appetite, and temperature were monitored daily for the duration of the experiment. Normal rectal temperature was defined as 38°C to 40°C. Four pre-infection complete blood counts (CBCs)  were performed on each goat  to establish baseline hematologic values. Post-infection CBCs were performed on Days 1–7, 9, 11, 13, 16, 19, and 21.Nasal swabs and blood cultures were collected on the same schedule as the complete blood counts, except that only one pre-infection sample was collected. For nasal swabs, both nostrils were sampled with a Dacron® fiber tipped plastic applicator swab  and immediately streaked onto a MH-gentamicin agar plate. For blood culture, the collection site over the jugular vein was aseptically prepared with povidone iodine and ethyl alcohol, and 5 ml of venous blood was collected into a heparinized syringe.Three-view thoracic radiographs  were taken under xylazine sedation for all goats prior to infection and on days 7, 14, and 21. Radiographs of the extirpated lungs were also taken at the time of necropsy. Initial studies showed little to no radiographic change in the lungs on day 2; therefore, this necropsy time point was not aligned with radiography. Radiographs were taken using a MinXRay® 100HF and Agfa® Computed Radiography (CR) 43×35 CR MD 4.0 General Cassettes. During use within the ABSL-3, cassettes were triple sealed in 6 mil polyethelene bags . The cassettes were processed with a CR 85-X digitizer and the images were processed with NX software. All radiographs were read by AJM.Humane euthanasia of three goats with intravenous pentobarbital was planned for days 2, 7, 14, and 21. One goat that was going to be euthanized on Day 2 died during recovery from anesthesia. Necropsies were performed on all goats. Based on organ involvement detected in pilot studies, the following tissues were collected into 10% neutral buffered formalin for histology: mandibular lymph node, retropharyngeal lymph node, tracheobronchial lymph node, mediastinal lymph node, lungs, heart, spleen, liver, kidney, adrenal, thyroid, brain, mesenteric lymph node, and testis or ovary. Samples were routinely embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for microscopic evaluation.B. pseudomallei. In the absence of macroscopic abscesses, samples of the following organs were aseptically collected to calculate the organ burden (CFU/g): lung , spleen, liver, kidney, retropharyngeal lymph node, tracheobronchial lymph node, and mesenteric lymph node. Tenbroeck homogenizers were used to homogenize 0.5 g of each tissue sample in 2 ml PBS +20% glycerol (limit of detection 50 CFU/g). Neat urine was also collected and cultured from each goat at the time of necropsy. The homogenate or urine (50 µL) was plated in duplicate on MH-gentamicin agar, and the remaining sample was frozen at −80°C. If the colony count was above the limit of quantitation (>300 colonies/plate), the frozen homogenate was thawed and serial log dilutions were plated in duplicate.In organs with macroscopic abscesses, the abscesses were counted or estimated as a percentage of the total organ volume. Representative abscesses were cultured on MH-gentamicin agar to confirm positive growth of 5 CFU. The delivered dose to individual goats was approximately one log lower and is reported in Infection was performed on four separate days. The backtitration of the nebulized bacterial suspension ranged from 1.07–2.33×10The median rectal temperature for all goats prior to infection was 38.9°C. Ten of 11 goats developed fever by day 1 (median temperature 40.7°C), with the remaining goat developing a fever by day 2 . Fevers were variable, but peak temperatures were frequently seen on day 6  or day 8  after the day 2 peak. Representative temperatures are shown in Other than fever, the most frequent clinical sign recognized was cough, which was observed in seven of 11 goats. Cough was most frequently observed early after infection, days 2–5. Nasal discharge was only observed in Goat 16, which developed on day 11 and persisted until the time of euthanasia on day 16.3 cells/µL (range 3.5–23×103 cells/µL). This relatively high value was attributed to an epinephrine response, which is often seen in young, excited goats. The median pre-infection granulocyte and monocyte counts for all goats over all independent samples were 5.7×103 cells/µL (range 2.9–15.7×103 cells/µL) and 1.0×103 cells/µL (range 0.4–3.0×103 cells/µL). While some granulocyte and monocyte counts were elevated, no goat maintained a granulocyte or monocyte count above the reference range (1.2–7.2×103 cells/µL and 0.0–1.0×103 cells/µL respectively) for all samples. Variations were attributed to a cortisol response from handling. Pre-infection red cell parameters were generally unremarkable.Pre-infection hematology revealed a marked lymphocytosis in nearly all goats. The median pre-infection lymphocyte count for all goats over all independent samples was 12.8×103 cells/µL and peaked on day 2 at 16.7×103 cells/µL (n = 11). A second peak of 14.9×103 cells/µL was seen on day 9 (n = 6). Representative changes in granulocyte counts can be seen in The only cell types to show a marked response post-infection were granulocytes. On day 1 the median granulocyte count increased to 14.2×10B. pseudomallei for 3 of 11 goats on day 1. This was very light growth, with three or fewer colonies per plate. At all other time points, positive nasal swabs were found only for goat 16 on days 5, 11, 13, and 16; growth was light for this goat on days 5 and 11 (less than 20 colonies) and heavy on days 13 and 16 (dense mat of colonies).Nasal swabs were positive for B. pseudomallei for all goats except for goat 16, which was positive on day 13, with 1 CFU/5 ml, and on day 16 with 19 CFU/ml.Quantitative blood culture was negative for There were no significant findings on pre-infection thoracic radiographs, except for goat 27, which had a moderate bronchointerstitial pattern; signs of respiratory disease were not evident in this goat prior to infection. Pulmonary disease was radiographically evident in all goats by day 7 (n = 9), which ranged in severity from a mild to moderate bronchointerstitial infiltrates in the caudal lung lobes  to severMild focal consolidation in the cranioventral and caudodorsal lung fields was noted in the two goats euthanized on day 2. Retropharyngeal and tracheobronchial lymph nodes were moderately enlarged. No other lesions were noted in any other organs.By day 7 pulmonary abscessation was present in each of the three goats examined. Abscesses were typically 5 mm or smaller and had a creamy to tan center containing variable amounts of thick purulent material. The parenchyma immediately surrounding the abscess was consolidated, appearing as a rim of dark red tissue around the tan center. This configuration was most easily observed in abscesses just beneath the visceral pleura . There wOn day 14, pulmonary abscesses remained consistent in appearance and distribution, but varied widely in number (range 12 to 80). The abscesses were larger than at day 7, most were 5–10 mm in diameter, with isolated nodules as large as 20 mm. Retropharyngeal, tracheobronchial, and mediastinal lymphadenomegaly was also consistent with the day 7 findings. Abscessation of the mediastinal lymph node was seen in one goat, which appeared as multiple small (1–2 mm) caseous nodules. This was typical for most lymph node abscesses, which were small in size and multiple in most cases.Extrapulmonary abscessation was evident in all goats euthanized on day 14. Splenic abscesses  were seeGoat 16, assigned to the group of three scheduled for euthanasia on day 21, was the most severely affected animal in the study. On day 16, the goat was acutely moribund and was humanely euthanized. Approximately 50% of the lungs were affected with numerous abscesses of varying sizes up to 25 mm in diameter. Abscesses were seen in the retropharyngeal and tracheobronchial lymph nodes, spleen, and kidneys, which were similar in appearance to the abscesses seen at earlier time points. The left adrenal gland was enlarged and edematous, and when cut longitudinally, it was found to contain a solitary abscess that lacked a distinctive structure with no apparent capsule.Two goats survived with minimal clinically-evident disease until euthanasia on day 21. Pulmonary abscessation with widely disseminated disease was evident in both goats at necropsy. Lung abscesses range from 20 to 45 in number and 10 to 20 mm in diameter. One very large abscess in the cranial lung lobe was adhered the visceral pleura to the rib cage. In both goats, abscesses were seen in the tracheobronchial lymph nodes, spleen (grossly enlarged by >10 abscesses in one goat), and kidney. Abscesses in the mediastinal lymph nodes and one small (2 mm) hepatic abscess were also observed, which were similar in appearance to the small splenic abscesses. Organ involvement for individual goats is summarized in B. pseudomallei. Positive growth was generally restricted to areas of gross lesions, with the exception of day 2 samples and tracheobronchial lymph nodes, both of which often cultured positive in the absence of gross lesions. Multiple organs from goat 16 also cultured positive, with relatively high levels of growth, which appeared to be associated with fulminant septic shock.The bacterial organ burden for all goats is summarized in B. pseudomallei from neat urine with 4.4×104 CFU/ml.Only goat 16 was positive for growth of Early bronchiolar lesions at 2 days PI comprised any combination of the following changes: apical surface deciliation; single cell necrosis of the lining pseudostratified ciliated epithelium; neutrophil transcytosis; and luminal aggregates of mucopurulent to fibrinopurulent exudate . NeutropBy day 7 well-formed abscesses were cuffed by mantles of macrophages (including epithelioid histiocytes) and lymphocytes, which in turn were ensheathed by fibrous connective tissue capsules of varying thickness with a significant histiocytic component. Many of the intervening alveolar spaces were lined by plump cuboidal epithelium indicative of proliferation of type 2 pneumocytes. Interlobular septa were also thickened by edema and inflammatory infiltrates, extending to the pleura. Pleural surfaces were variably expanded by pleocellular exudate, subpleural edema, fibroplasia, and lymphangectasia and lymphangitis. A prominent segmental leukocytoclastic vasculitis with perivascular cuffs of lymphocytes and histiocytes  primarilLesions were less exudative and more chronic on day 14, with multiple coalescing pyogranulomas and suppurative to fibrinopurulent bronchopneumonia. Goat 16, which developed terminal disease on day 16, showed marked pulmonary pathology. Vascular thromboses with perivascular hemorrhages were evident throughout the lungs, which showed fibrin exudation into parenchyma and interlobular septa, prominent peribronchiolar lymphoid hyperplasia, and chronic pleuritis with lymphangectasia and lymphangitis. Lesions on day 21 were more of chronic bronchointerstitial pneumonia with septal fibrosis, multifocal coalescing pyogranulomas, multifocal atelectasis, and pleural fibrosis.Variable tracheitis, with mucous gland hyperplasia, neutrophil transcytosis, and mucosal abscesses , were seMild to severe lymphoid hyperplasia, with occasional edema, was seen in all goats that were euthanized on days 2 to 14. Focal to multifocal pyogranulomas were present in all goats euthanized on days 16 and 21, and all appeared chronic (with thick fibrous capsules) on histopathology.Mild to severe lymphoid hyperplasia was seen at all time points in all goats. The severity of hyperplasia was less in the mandibular lymph node compared to the retropharyngeal lymph node in all cases. Changes in these lymph nodes did not appear to correlate with the severity of hyperplasia in the TBLNs.Histologically, the predominant lesion – regardless of time point – was a minimal to mild random necrosuppurative hepatitis, which was seen in six goats. Hepatic lipidosis was also noted in six goats, but was not associated with significant inflammation as the two lesions were only seen together in two goats.The only findings on days 2 and 7 were congestion, with mild lymphoid hyperplasia additionally noted in one goat. By day 14, capsular changes were present (not necessarily directly associated with abscesses), which varied slightly between individual animals, but included suppurative capsulitis with mesothelial cell hyperplasia and hypertrophy, lymphangectasia and lymphangitis, vasculitis, edema, and fibrosis . VasculiThe apparent histologic age of renal lesions was less associated with the duration of infection than pulmonary or splenic lesions. No significant findings were seen in goats on day 2, nor in one of three goats on days 7 and 14. ‘Early’ lesions were seen in two of three goats on day 7 and one of three goats on days 14 and 21. These lesions ranged from focally hypercellular glomeruli only to glomeruli showing multifocal to global thickening due to hypertrophy of the parietal layer of Bowman’s capsule, mesangial thickening, hyperemia, minimal to mild infiltration of neutrophils, as well as variable tubulointerstitial nephritis and mineralization. The remaining three goats  all had ‘late’ lesions of characteristic linear abscesses extending from the cortex down into the medulla, but not involving the inner medulla or pelvis. Microscopically, the lesions appeared to be centered on the glomeruli and then spread to the tubules, culminating in tubulointerstitial nephritis with protein casts. The lesions were pyogranulomas with a core of degenerate neutrophils surrounded by a mantle of lymphocytes, histiocytes, and epithelioid macrophages . VasculiAcute suppuration/abscessation (without fibrosis/capsule) was noted in two goats on days 7 and 16. This spread by day 7 was the first evidence of dissemination beyond the pulmonary system and directly draining lymph nodes. Lesions were seen at the corticomedullary junction and appeared to originate from blood vessels, which had evidence of local vasculitis .The mesenteric lymph node was typically observed to be edematous with mild to moderate lymphoid hyperplasia regardless of time point.Multifocal suppurative orchitis was noted only in goat 16, which was associated with vasculitis (both arteritis and phlebitis), hemorrhage, testicular degeneration, azospermia, and giant spermatids .B. pseudomallei, which has a very broad host range. Additionally, new animal model systems are also important for the development and testing of novel therapeutics and vaccines.Melioidosis has a diverse clinical presentation in humans. This may be affected by individual characteristics of the infecting strain, dose, and route of infection, which is rarely known in naturally occurring cases. These unknowns make it very difficult to study the pathogenesis of melioidosis despite the large number of individual case reports and several retrospective and prospective studies. Basic pathogenesis research has been largely restricted to murine models, but additional models are necessary for a more complete understanding of the pathogenesis of infection with Patients with melioidosis typically present with protean signs. Since there is no “typical” presentation, the subdivision of cases is highly variable in the human medical literature, with some papers grouping patients by duration of signs (acute vs. chronic), the presence or absence of septic shock/bacteremia, or the primary organ system involved. Pneumonia, with (76%) or without (45%) septic shock, accounts for the largest percentage of clinical presentations of naturally occurring human melioidosis In our goat model, the only consistent clinical sign of infection was fever. The degree of pyrexia appeared to parallel the increase in granulocyte count over the first seven to nine days, with peaks on day 1 and around day 7. After day 9, temperatures tended to decrease, but this decrease was not necessarily paralleled in the granulocyte count see  and 2. TB. pseudomallei except in the case of goat 16, where increasingly heavy discharge and growth of B. pseudomallei was associated with the progression towards terminal disease. Light positive growth seen on day 1 was interpreted as residual bacteria from intratracheal infection rather than dissemination or expectorated pus. This low rate of nasal shedding suggests that horizontal spread among animals via nasal secretions would be unlikely or insignificant. This appears similar to human disease where there are very few reports of person-to-person transmission Other clinical signs included cough and oculonasal discharge. Nasal swabs, including those from goats with nasal discharge, were typically culture negative for 4–106 CFU/ml were also seen in a small number of goats used in the dose determination portion of this study (data not shown), which is comparable to counts seen in human patients Hematogenous dissemination, the only mechanism consistent with the observed pattern of multiorgan involvement, was not directly confirmed, likely because of the transient nature of bacteremia. Positive blood cultures were only seen in goat 16, which was associated with the progression to terminal disease. The bacteremia observed in goats appears similar to the bacteremia seen in human patients, in which only 50% of patients are culture positive, and of the culture positive patients the median bacteremia is 1.1 CFU/ml 7 CFU produced fulminant pulmonary disease, necessitating euthanasia 48 h PI. Additionally, the use of healthy animals, without any predisposing risk factors for melioidosis, could have limited the development of rapidly progressive disease.Thoracic radiographic findings in human melioidosis are highly variable and the incidence of specific findings also varies considerably among retrospective imaging studies of melioidosis. Radiographic signs in acutely presenting cases often have local, patchy, alveolar infiltrates, or disseminated nodular lesions, which are suggestive of metastatic (hematogenous) spread Organ involvement in melioidosis can be highly variable in both humans and goats. In humans, the lungs are the most commonly affected organ, but virtually any organ can be affected One notable difference in organ involvement and lesion severity observed in goats in this study compared to mice and humans was the rarity of gross hepatic abscesses and only mild severity of microscopic changes within the liver. Previous reports of both natural and experimental melioidosis in goats have had variable findings in regards to liver lesions, with some reporting no lesions B. pseudomallei.In the current study, the lungs were the primary site of infection. Dissemination to extrapulmonary tissues appeared to be predominantly a function of time, with greater numbers of organs involved at the later time points. Dissemination to sites other than lymph nodes draining the pulmonary system was not grossly evident before day 14 with the exception of one small adrenal abscess seen in one goat on day 7. The histologic evidence suggests that the timing of dissemination was a result of the size and extent of the pulmonary pyogranuloma formation. After aerosol delivery, mucopurulent bronchopneumonia rapidly developed and soon progressed to a more severe fibrinopurulent to bronchointerstitial pneumonia with pyogranuloma formation evident radiographically, grossly, and histologically by day 7. Within the lungs, the lesions spread along interlobular septa and subpleural stroma inducing a local leukocytoclastic vasculitis where the pyogranulomas encroached upon and eventually invaded the vessel . The vasB. pseudomallei is the glomerulus, with the earliest detectable lesion being hypercellular glomeruli with neutrophil infiltration. The lesions then suppurate and extend into tubules creating tubulointerstitial disease and ultimately result in the formation of pyogranulomas. The testicle was the only organ with lesions that had evidence of hemorrhage in association with the vasculitis. The significance of this finding is unclear at this time since a testicular lesion was only seen in one goat, but lends support to vasculitis being a central event in pathogenesis.Vasculitis also appears to be the central pathologic step in the establishment and progression of pyogranuloma development in extrapulmonary organs. Vasculitis within an adrenal vessel was noted in association with abscess formation and invasion of the parenchyma. Splenic lesions in the capsule and parenchyma were also seen in association with vasculitis. The predominance of capsular pyogranulomas appears to correlate with the capsulitis observed histologically. The renal lesions suggest that the point of entry for To the authors’ knowledge, vasculitis has not previously been reported as a feature of human disease or in murine models. It has been reported in goats in association with central nervous system lesions B. pseudomallei to more chronic lesions, and even potentially eliminate the organism, makes them a particularly interesting animal model of melioidosis. We have shown that melioidosis can be readily induced in goats following aerosol exposure. As expected, the extent of organ involvement seen was more variable in goats than in murine models, but was comparable to human disease and is likely to be a feature of any model with a truly heterogeneous outbred population. However, it appeared that organ involvement may become more consistent at later time points with more chronic disease in our caprine model.Goats are generally believed to have a natural tendency towards more chronic disease and granulomatous type lesions in melioidosis The larger body size of goats allows for human-relevant clinical monitoring as well as longer-term serial evaluation of disease progression and therapy. We believe the caprine model will be a useful animal model for further investigation of the molecular pathogenesis and host response in melioidosis, as well as evaluation of preventative and therapeutic interventions.


The aim of this study was to compare the effect of 6% hydroxyl ethyl starch solution with 4% gelatin and Ringer’s solutions on the haemodynamic stability of patients after coronary artery bypass graft (CABG) surgery and immediately after discontinuation of cardiopulmonary bypass (CPB).This was a randomised, double-blind clinical trial of 92 patients who were candidates for on-pump CABG. After discontinuation of CPB, all patients were transferred to the intensive care unit (ICU) and divided randomly into three groups. The first group received Ringer’s solution, the second group 4% gelatin, and the third 6% hydroxyl ethyl starch (HES) solution (Voluven). Haemodynamic parameters such as heart rate, mean arterial pressure, systolic blood pressure, diastolic blood pressure, central venous pressure, cardiac output and the presence of arrhythmias were documented.The volume needed for maintaining normal blood pressure and central venous pressure in the range of 10–14 mmHg was less in the HES group than in the other groups. The volume was similar however in the gelatin and Ringer’s groups in the first 24 hours after surgery. Urinary output in the first four and 24 hours after surgery were significantly higher in the HES group than in the other two groups. Mean creatinine levels were significantly lower in the HES group.HES (6%) had a better volume-expanding effect than gelatin (4%) and Ringer’s solutions, and its short-term effects on renal function were also better than gelatin and Ringer’s solutions. Immediately after coronary artery bypass graft (CABG) surgery, patients are haemodynamically unstable and need fluid support.4There is controversy regarding the different types of solutions used after CABG, and various researchers have used materials such as crystalloid solutions or colloids, including albumin and gelatin, or other agents such as hydroxyl ethyl starch solutions. Volume expansion is an important aspect of these solutions, however, side effects, such as inflammatory responses, and effects on endothelial integrity and on organs such as the kidney should also be considered during their administration.4Gelatins are polydispersed polypeptides produced by degradation of bovine collagen. Three types of modified gelatin products are now available: cross-linked or oxypolygelatins (e.g. Gelofundiol®), urea cross-linked (e.g. Haemacel®) and succinylated or modified fluid gelatins (e.g. Gelofusine®). Their molecular weight (MW) ranges from 5 000–50 000 Da, with an average of 30 000–35 000 Da. The various gelatin solutions have comparable volume-expanding powers and all are said to be safe with regard to coagulation and organ function (including kidney function).22:C2 ratio, and solvent. HES solutions with a low MW and a low molar concentration are thought to be safe with regard to coagulation, and increased bleeding tendency no longer appears to be a problem , even when higher doses are given.3Hydroxyl ethyl starch (HES) is a widely used plasma substitute for correcting hypovolaemia in cardiac surgery patients. HES preparations vary with regard to concentration, mean MW, molar concentration, Cet al. have shown that HES and albumin solutions caused mild systemic acidosis in patients undergoing normovolaemic haemodilution after cardiac surgery.7Some authors believe that albumin has a better volume-expanding effect than HES.et al. found better kidney function and less inflammation with the use of HES than with albumin solutions.4Correcting hypovolaemia with HES has been suggested to be associated with an increased risk of acute renal failure, and interest has recently been focused on the influence of HES solutions on renal function.The aim of this study was to compare the effect of 6% hydroxyl ethyl starch solution with 4% gelatin and Ringer’s solutions on haemodynamic stability of patients after CABG surgery and immediately after discontinuation of cardiopulmonary bypass.This was a prospective, randomised, double-blind clinical trial in 92 patients who were candidates for on-pump CABG. The age range of patients was from 40 to 75 years. Exclusion criteria were left ventricular ejection fraction < 40%, right heart failure, emergency patients, pump time > 180 minutes and clamp time > 90 minutes, patients who needed re-operation within the first six hours due to surgical haemorrhage or other reasons, renal failure needing haemodialysis, and those with respiratory failure.All patients received pre-anesthesia medication. Lorazepam  was given the night before the operation and intramuscular morphine (0.1 mg/kg) one hour before induction of anaesthesia in all patients. In the operating room, lidocaine (1%) was used for access to arterial and peripheral vessels and Ringer’s crystal solution was administered in a dose of 5–10 ml/kg. Anaesthesia induction was started with intravenous medazolam sufentanyl and pancranium.After the use of 100% oxygen by mask, patients were intubated with an endotracheal tube and connected to a mechanical ventilator and central venous pressure (CVP) was introduced in the right internal jugular vein. Maintenance of anaesthesia was achieved with continuous infusion of idazolam, atrocurium and sufentanyl. After infusion of 300 IU/ kg heparin, the patient went on-pump and the activated clotting time (ACT) was above 480 s, mean arterial pressure 60–70 mmHg, haematocrit level was 22–27%, and the temperature was set at 32°C.After discontinuation of cardiopulmonary bypass (CPB) all patients were transferred to the intensive care unit (ICU) and were randomly divided into three groups. The first group received Ringer’s solution, the second gelatin (4%), and the third group hydroxyl ethyl starch solution (HES) (6%) (Voluven) as a volume expander to maintain the CVP between 7 and 14 mmHg. Packed cells were infused where the haemoglobin level was lower than 8 mg/dl and fresh frozen plasma (FFP) was used for continuous bleeding with a normal range of ACT and APTT .2 body surface area, inotrope infusion (dobutamine or epinephrine) was started.Cardiac output was monitored with a NICO instrument and haemodynamic values were monitored continuously. In situations where, after maintaining adequate volume, the mean arterial pressure was below 60 mmHg and cardiac index below 2 l/min/mHaemodynamic parameters such as heart rate, mean arterial pressure, systolic blood pressure, diastolic blood pressure, central venous pressure, cardiac index and the presence of arrhythmias were documented. Other independent variables such as urinary output, serum electrolytes and serum creatinine levels were measured immediately after discontinuation of CPB, before transferring the patient to the ICU, immediately after arriving in ICU, and after two, four, six, 12 and 24 hours in ICU.Study approval was obtained from the ethics committee of our Centre and written informed consent was obtained from the patients. The data were put into spreadsheets and comparison of variables between groups was done using Chi-squared or ANOVA tests.Biometric data were similar in all groups. Mean anaesthesia time, pump time and cross-clamp time were the same in all three groups The volume needed for maintaining normal blood pressure and central venous pressure in the range of 7–14 mmHg was less in the HES group than in the other groups, but similar in the gelatin and Ringer’s groups in the first 24 hours after surgery. Urinary output in the first four and 24 hours after surgery was significantly higher in the HES group than in the other two groups.Mean creatinine levels on the first day post operation were 1.25 ± 0.23 mg/dl in the Ringer’s group, 1.3 ± 0.24 mg/dl in gelatin group and 1.06 ± 0.13 mg/dl in the HES group. On the second day post operation, these values were 1.4 ± 0.25, 1.41 ± 0.26, 1.13 ± 0.16 mg/dl in the Ringer, gelatin and HES group, respectively. Mean creatinine levels were significantly lower in the HES group There were no significant differences between the three groups in the amount of blood, FFP and platelet transfusions in the ICU The main result of this study was that haemodynamic stability could be achieved after CABG surgery with less volume of HES than gelatin and Ringer’s solutions. The kidney function was better in the short term in the HES group than in the other two groups.In our centre, we selected adult patients for CABG. We are aware that enough of a suitable volume expander is needed for haemodynamic stability after CABG, and that some volume expanders have side effects. Patients usually have a systemic inflammatory response after CPB for CABG and the resultant endothelial damage leads to hyperpermeability and interstitial oedema.4Some researchers have shown that HES reduced inflammation and endothelial damage.12et al. reported a lower inflammation rate and better GFR with HES.The most dangerous complication after CABG is kidney damage and some researchers demonstrated kidney damage after the use of HES but found gelatin 4%) to be safer.% to be sIn our study there was better haemodynamic stability with lower volumes of HES. Renal function was good after its use in the first two days after CABG, which indicates that renal function can be maintained after use of 6% HES. Other reporters have shown less renal damage after the use of HES than with gelatin, albumin and Ringer’s solutions.4In our study we used less volumes of HES than Ringer’s solution and gelatin and this produced a better volume-expanding effect with HES than with gelatin and Ringer’s solutions. Better oxygenation and lower serum lactate concentration were shown after the use of HES than with gelatin.There were some limitations to the study. Because of the systemic inflammatory response after CPB, it would have been advisable to compare inflammatory biomarkers in the three groups but this was not done. It has been reported that HES had an effect on the acid–base balance in some studies, but this was not determined in our study.Our study showed that HES (6%) had a better volume-expanding effect than gelatin (4%) and Ringer’s solution, and its short-term effects on renal function were also better than with gelatin and Ringer’s solution.


The determination of altered expression of genes in specific tumor types and their effect upon cellular processes may create insight in tumorigenesis and help to design better treatments. The Flatcoated retriever is a dog breed with an exceptionally high incidence of histiocytic sarcomas. The breed develops two distinct entities of histiocytic neoplasia, a soft tissue form and a visceral form. Gene expression studies of these tumors have value for comparable human diseases such as histiocytic/dendritic cell sarcoma for which knowledge is difficult to accrue due to their rare occurrence. In addition, such studies may help in the search for genetic aberrations underlying the genetic predisposition in this dog breed.Microarray analysis and pathway analyses were performed on fresh-frozen tissues obtained from Flatcoated retrievers with localized, soft tissue histiocytic sarcomas (STHS) and disseminated, visceral histiocytic sarcomas (VHS) and on normal canine spleens from various breeds. Expression differences of nine genes were validated with quantitative real-time PCR (qPCR) analyses.PPBP, SpiC, VCAM1, ENPEP, ITGAD (down-regulated), and GTSF1, Col3a1, CD90 and LUM (up-regulated) in the comparison of both the soft tissue and the visceral form with healthy spleen. DAVID pathway analyses revealed 24 pathways that were significantly involved in the development of HS in general, most of which were involved in the DNA repair and replication process.QPCR analyses identified the significantly altered expression of nine genes; This study identified altered expression of nine genes not yet implicated in histiocytic sarcoma manifestations in the dog nor in comparable human histiocytic/dendritic sarcomas. Exploration of the downside effect of canine inbreeding strategies for the study of similar sarcomas in humans might also lead to the identification of genes related to these rare malignancies in the human. Rare human diseases, such as histiocytic malignancies, might therefore be common in specific dog breeds Fundamental research on rare human diseases is not only hampered by minimal grant supply; lack of sufficient sample numbers is of equal disadvantage. One way to overcome this last catch, is to investigate other species in which a similar disease occurs at a much higher frequency, like in specific dog breeds. In dogs, a downside of selection for breeding purposes is the occurrence of a very large number of breed-specific hereditary diseases (The Flatcoated retriever (FCR) breed has a strongly increased risk for histiocytic sarcoma (HS) development. In the UK, it is likely to account for about 36% of all malignant neoplasms diagnosed in this breed Canine histiocytic malignant disorders were as such first described in the late 1970s In dogs, there are several clinical manifestations of HS. One common form is the localized, soft tissue histiocytic sarcoma (STHS), which manifests itself as a tumor arising in the deeply seated soft tissues of limbs often in association with joints CDKN2A/B, RB1 and PTENMTAP-CDKN2A has recently been identified in this breed RAGE) and the high mobility group box1 protein (HMGB1) potentially have a major effect on the progression of malignant histiocytic disorders Past research using Comparative Genomic Hybridization has already shown various aberrations in HS in the Bernese Mountain Dog, another breed predisposed to histiocytic malignancies, with cases showing numerous shared Copy Number Alterations (CNAs) both gains and losses, throughout the genome. These included deletions of the tumor suppressor genes cDNA microarrays have become powerful tools in the study of gene expression which has enabled improved classification of various naturally occurring cancers The study of spontaneously occurring tumors in the dog, a species which has a genetically stronger relationship to the human than mice http://wetten.overheid.nl.The experimental protocol (ID 2007.III.08.110) was peer-reviewed by the scientific committee of the Department of Animals in Science & Society, Utrecht University, The Netherlands, and approved by the Animal Experiments Committee of the Academic Biomedical Centre, Utrecht, The Netherlands. The Animal Experiments Committee based its decision on 'De Wet op de Dierproeven'  and on the 'Dierproevenbesluit' . Both documents are available online at All tumor samples were confirmed as being spontaneously occurring histiocytic malignancies and were obtained from family-owned FCR with informed owner consent. All tumor material used originated from the Dutch FCR, that had not received radiotherapeutical or cytostatic treatment. Tumors were obtained under sterile conditions, either as part of a routine diagnostic or therapeutic surgical procedure, or immediately following euthanasia. Directly after excision, samples were snap frozen in liquid nitrogen, or alternatively by primary preservation in RNA-later, in both instances followed by storage at minus 70°C. Tumor samples collected adjacent to the site of the frozen or RNA-later preserved samples were fixed in 10% neutral buffered formalin and routinely processed for histological examination.At the time of surgery or necropsy, the evident anatomical location of all tumors was recorded for each individual and categorized as either VHS, if a tumor was present in internal organs (n = 7) or STHS, if the tumor was localized in a limb only without identifiable metastases (n = 6) Histological specimens were classified by a board-certified veterinary pathologist (GCMG) according to the recommendations and classification scheme defined by Affolter and Moore As control tissue, normal spleen from  crossbreed dogs (n  = 6) was used as obtained at postmortem immediately following euthanasia that was not related to neoplastic, endocrine or metabolic diseases.As a common reference pool a multitude of canine organs  were used that had been obtained from healthy crossbreeds (n = 8) euthanized for non-metabolic, non-tumorous lethal conditions.The procedures were approved by the local ethics committee, as required under Dutch legislation (ID 2007.III.08.110).Approximately 30 mg of frozen tumor was transferred to a container with 600 µl of Buffer RLT and was disrupted/lysed and homogenized using a dismembrator  for 45 s at 2200 rpm. Total RNA was isolated and treated with DNase using the RNeasy mini kit  according to the manufacturer's protocol. Quantity and integrity were assessed with the Bioanalyzer Agilent BioAnalyzer-2100  in combination with an RNA 6000 Pico-LabChip. The average RNA integrity number 8.5 (range: 7.2–9.8) was found to be appropriate RNA was labeled twice and hybridized against the common reference RNA on dual channel arrays. RNA concentrations were 0.6 µg/µl at a minimum amount of 3 µg per sample.RNA amplifications and labeling were performed on an automated system  as described Hybridized slides were scanned on an Agilent scanner (G2565BA) at 100% laser power, 30% photomultiplier tube voltage. After automated data extraction using Imagene 8.0 (BioDiscovery), printtip Loess normalization was performed http://www.r-project.org/). In a fixed effect analysis, sample, array and dye effects were modeled. P-values were determined by a permutation F2-test, in which residuals were permutated 5,000 times globally. Genes with P<0.0002 after either family wise error correction (FWER) or determination of false discovery rate (FDR) were considered significantly changed. Genes with log2-fold changes of more than 0.5 or less than −0.5 were then selected to ensure that only robust changes were considered.Analyses were performed to detect common differences in gene expression between the two groups of HS and healthy spleen tissue. Data were analyzed using ANOVA http://www.geneontology.org/) was used to check gene molecular and biological functions of the remaining genes.The Gene Ontology (GO) database (http://david.abcc.ncifcrf.gov/).In general, pathway analysis in dogs has its restraints because pathway identification relies heavily on existing functional annotation, which is still limited for this species. Still, pathway analysis provides an additional way to analyze expression data across species. This may shed light on common pathways important for tumor behavior and on finding new therapeutic targets. To examine whether certain pathways are over- or under-represented in the gene list, all genes significantly differentially expressed between either STHS, VHS and normal spleen, were introduced into DAVID  were used in the microarray experiment , furthermore three additional samples  were added thus creating 3 groups of seven samples for the qPCR experiment. Total RNA from these samples was isolated. After isolation, total RNA was treated with DNase using the RNeasy mini kit  according to the manufacturer’s protocol.Reverse transcription (RT) was performed of all 20 samples in a 80 µl reaction using 2000 ng total RNA, 16 µl iScript Reaction mix and 4 µl iScript Reverse Transcriptase . This includes a mixture of oligo-dT random hexamer primers. The mixture was incubated 5 min. at 25°C, 30 min. at 42°C and followed by 5 min. at 85°C. Minus RT controls were prepared from 500 ng of the same RNA under the same conditions, but without addition of reverse transcriptase.Reference genes were selected as non-regulated reference genes for normalization based on their stable expression in canine tissue ribosomal protein S5 (RPS5), signal recognition particle receptor (SRPR), ribosomal protein L13(RPL13), hypoxanthine phosphoribosyltransferase (HPRT).Using Ensembl, through annotated transcripts, PCR primers were designed using the Perl Primer software (version 2.0.0.7) and primer3 software (version 0.4.0) according to the parameters outlined in the Bio-Rad i-cycler manual. The specificity of each primer pair was confirmed by sequencing its product and also in qPCR by checking the meltcurve and reaction efficiency. GeneNorm was used to establish expression stability Primers for all nine reference genes, including their optimum temperature are listed in For qPCR, the Bio-Rad detection system (Bio-Rad.) with SYBR green fluorophore was used. Reactions were performed in a total volume of 25 µl containing 12.5 µl of 2×SYBR green super mixes (Bio-Rad Laboratories Ltd.), 1 µl of each primer at 400 nM concentration, 0.8 µl of cDNA and 9.7 µl Rnase and Dnase free water as previously described Data analysis was performed with IQ5 Real-Time PCR detection system software (BioRad). Expression levels were normalized using the average relative amount of the reference genes. Log-values of normalized relative expression were used to obtain normal distribution.A Wilcoxon rank sum test was performed to determine the significance of differential gene expression. All results were Bonferroni corrected.Differentiation between STHS and VHS was based on physical examination and radiographic (thorax)/ultrasound examination (thorax/abdomen). All dogs with visceral organ involvement were euthanized followed by immediate autopsy. Histomorphology and immunohistochemical staining for CD18 confirmed the suspected histiocytic origin of tumors studied.The Microarray enabled analysis of the expression of 42,034 features. Since only 21,682 (51% ) were annotated (CanFam 2.0), it is possible that important genes are missed. When comparing VHS and spleen, 4,235 features were significantly differentially expressed. When only looking at 4-foldchanges or larger, 352 features remained. When comparing STHS with spleen, 5,779 features were significantly differentially expressed. In this comparison, when only looking at 4-foldchanges or larger, 437 features remain.Of the total of altered genes, 3,394 features were significantly differentially expressed in both forms of HS versus normal spleen, and 319 features remained when only 4-foldchanges or larger are taking into account.In order to improve knowledge on the genetic basis of HS and to best exploit logistic and financial resources, we chose to examine a selection of the significantly altered genes found in the microarray experiment for confirmation by qPCR. Selection of genes was based on the statistical significance of their differential expression and their potential involvement in tumor development.Since it is not possible to obtain pure samples of sedentary, non-tumorous histiocytes for expression profiling, spleen tissue was chosen as the normal equivalent of HS. HS arises from interstitial DC, and hence emanate from lymphoid domains when these arise from lymphoid organs such as the spleen PPBP, SpiC, VCAM1, ENPEP and ITGAD were downregulated and GTSF1, LUM, Thy1 and Col3a1 were upregulated in both STHS and VHS compared to normal spleen. p-values and Fold Change.As a result from the qPCR experiment, significantly altered expression was confirmed for nine of the ten genes analyzed. Spi-C and VCAM1 gene expression between tumors and healthy spleen must be explained. It is possible that some of these expression differences are based on differences in tissue-origin rather than on actual tumor development. Spi-C plays a critical role in the development of splenic iron homeostasis. It is highly expressed in red pulp macrophages, but not monocytes, dendritic cells or other tissue macrophages. Spi-C is therefore highly expressed in spleen Spi-C is also known to regulate VCAM1 expression VCAM1 in the spleen. ITGAD  is a receptor for VCAM1. In our study, we detected a lowered expression of CD11d in HS as compared to normal spleen.The use of spleen as the healthy equivalent of HS does raise some concern as to how observed differences in CD11d in HS as compared to normal spleen.Staining by immunohistochemistry for the presence of CD11d in both STHS and VHS was found negative in a first study by Moore et al, including16 splenic HS PPBP as was found in this study, has also been reported to play a role in the development of myelomas PPBP an intriguing observation worthy of further pursuit.The down-regulation of LUM has been observed in various types of cancer cells  - with contrasting findings on the relation with type of growth and/or tumor progression or metastasis Expression and overexpression of GTSF1 is already known in the occurrence of mycosis fungoides, which is the most common type of primary cutaneous T-cell lymphoma, in which GTSF1 was proposed as a gene for which expression appears to be restricted to mycosis fungoides tumor stage and that might even serve as diagnostic (bio)marker In humans, an up regulation of ENPEP probably plays a role in regulating growth and differentiation of early B-lineage cells (http://www.wikigenes.org/e/gene/e/13809.html) and down regulation may thus also be involved in HS development.Col3a1 also has been shown to be overexpressed in other types of tumors, such as malignant mesothelioma Finally, Thy1 (CD90) is an important marker of many types of stem cells CD90 has already been identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays CD90 has even been shown to provide a clinical prognostic marker CD90 in HS might herald stem cell characteristics of the type of cancer.Myh11.For technical reasons, no qPCR data could be obtained for P<0.05). Most were involved in the DNA repair and replication process. The biological functions of ten of these pathways, amongst which the P53 signaling pathway was one of the most relevant, are listed in As a result of the pathway analyses in DAVID, 24 pathways were significantly involved in the development of HS  and development of HS in the Flatcoated retriever dog, irrespective of the disseminated or localized form.Our observations provide evidence of an association between altered expression of nine genes , ENSCAFG00000001046 (DESI1) and ENSCAFG00000006138 (LUM) were found to be commonly involved in our study as well as in Hedans study. In our study, eventually two of these genes, SPIC and LUM, were chosen for qPCR confirmation. We were indeed able to identify the significantly altered expression of these two genes.The current study provides the most comprehensive database of genome alterations in histiocytic malignancies to date, revealing genes and signaling pathways not previously associated with this disease. Although mRNA levels do not necessary reflect differences in protein levels, it is very well conceivable that the large difference in mRNA levels of specific genes will result in quantitative differences in protein expression. Lack of verified and specific antibodies for all nine gene products of interest let us to restrain this expression profiles to mRNA levels only. The study of Hedan This is the first study to compare gene expression in HS (both STHS and VHS) and normal spleen using both traditional fold change analysis as well as disease-based pathway analyses using DAVID.PPBP, SpiC, VCAM1, ENPEP, ITGAD (down-regulated), and GTSF1, Col3a1, CD90 and LUM (up-regulated) to be associated with the HS genotype.This study provides evidence for involvement of several genes in HS, irrespective of the form of manifestation, some of which are also related with to other cancers. On the basis of quantitative differences in expression, we consider Extrapolation of this data to human samples may help to further our understanding of the propagation and oncogenesis of histiocytic cells. Eventually, this will contribute to the development of effective therapeutic modalities for both species.