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We report a case of differentiation syndrome in a patient receiving the IDH1 inhibitor ivosidenib, with skin biopsy showing isocitrate dehydrogenase (IDH) R132H-mutated leukemia cutis. A 72-year-old man with IDH1-mutated acute myeloid leukemia (AML), status-post allogeneic cell transplantation, on ivosidenib for six months, was admitted for culture-negative neutropenic fever, pink and purpuric plaques and patches on the legs, abdomen and back, edema, hypotension, and shortness of breath. Skin biopsy revealed an infiltrate of atypical, immature, myeloperoxidase-positive mononuclear cells compatible with leukemia cutis or Sweet syndrome. Although dermal edema and interstitial neutrophilic infiltrate with karyorrhexis characteristic of Sweet syndrome were not seen, the atypical cells lacked expression of CD117 and CD34, which were expressed in the original leukemia. Additional immunohistochemical staining of suspected blasts was strongly positive for IDH1 R132H, suggesting a diagnosis of leukemia cutis. As the immunophenotype of blasts in skin infiltrates can significantly differ from the immunophenotype seen in blood and bone marrow, this case demonstrates that mutation-specific antibodies such as anti-IDH1 R132H may be useful to help distinguish malignant from non-malignant infiltrates in the skin. Furthermore, differentiation syndrome may demonstrate histologic features of leukemia cutis on skin biopsy. This article is protected by copyright. All rights reserved.

Identification of melanoma or worrisome moles is often taught as an important part of routine skin checks. We sought to evaluate the efficacy of gamified education vs. traditional ABCDEs education on melanoma identification and self-confidence in identifying worrisome moles. We report that in our cohort (n = 271), participants randomized to the gamified intervention were more likely to correctly identify melanoma and non-melanoma skin lesions than those randomized to the ABCDE control cohort (74.2% vs 63.5% correct, P

The aim of this study was to record growth-related changes in collagen network organization and proteoglycan distribution in intermittently peak-loaded and continuously lower-level-loaded articular cartilage. Cartilage from the proximal phalangeal bone of the equine metacarpophalangeal joint at birth, at 5, 11 and 18 months, and at 6-10 years of age was collected from two sites. Site 1, at the joint margin, is unloaded at slow gaits but is subjected to high-intensity loading during athletic activity; site 2 is a continuously but less intensively loaded site in the centre of the joint. The degree of collagen parallelism was determined with quantitative polarized light microscopy and the parallelism index for collagen fibrils was computed from the cartilage surface to the osteochondral junction. Concurrent changes in the proteoglycan distribution were quantified with digital densitometry. We found that the parallelism index increased significantly with age (up to 90%). At birth, site 2 exhibited a more organized collagen network than site 1. In adult horses this situation was reversed. The superficial and intermediate zones exhibited the greatest reorganization of collagen. Site 1 had a higher proteoglycan content than site 2 at birth but here too the situation was reversed in adult horses. We conclude that large changes in joint loading during growth and maturation in the period from birth to adulthood profoundly affect the architecture of the collagen network in equine cartilage. In addition, the distribution and content of proteoglycans are modified significantly by altered joint use. Intermittent peak-loading with shear seems to induce higher collagen parallelism and a lower proteoglycan content in cartilage than more constant weight-bearing. Therefore, we hypothesize that the formation of mature articular cartilage with a highly parallel collagen network and relatively low proteoglycan content in the peak-loaded area of a joint is needed to withstand intermittent stress and shear, whereas a constantly weight-bearing joint area benefits from lower collagen parallelism and a higher proteoglycan content.

Summary Reasons for performing study: There is ample evidence on topographical heterogeneity of the principal biochemical components of articular cartilage over the surface of the joint and the influence of loading thereon, but no information on depth-related zonal variation in horses. Objectives: To study depth-related zonal variation in proteoglycan (PG) and collagen content in equine articular cartilage. Methods: Two techniques (safranin-O densitometry and Fourier transform infrared spectroscopy) were applied to sections of articular cartilage from the proximal phalangeal bone of the metacarpophalangeal joint of 18-month-old Thoroughbreds that had been raised at pasture from age 0–18 months without (PASTEX) and with (CONDEX) additional exercise. Two sites were investigated: site 1 at the joint margin that is unloaded at rest or at slow gaits, but subjected to high-intensity loading during athletic activity; and site 2, a continuously, but less intensively, loaded site in the centre of the joint. Results: Proteoglycan values increased from the surface to the deep layers of the cartilage, collagen content showed a reverse pattern. PG content was significantly higher at site 2 in both PASTEX and CONDEX animals without an effect of exercise. In the PASTEX animals collagen content was significantly higher at site 1, but in the CONDEX group the situation was reversed, due to a significant exercise effect on site 1, leading to a reduced collagen content. Conclusions: Collagen and PG content gradients agree with findings in other species. The observations on PG levels suggest that the exercise level was not strenuous. The collagen results in the PASTEX group confirmed earlier findings, the lower levels at site 1 in the CONDEX group being possibly due to an advancement of the physiological maturation process of collagen remodelling. Potential relevance: This study confirms earlier observations that even moderate variations in exercise level in early age may have significant effects on the collagen network of articular cartilage.

Summary Reasons for performing study: Subchondral bone provides structural support to overlying articular cartilage and plays an important biomechanical role in osteochondral diseases. Mechanical features of bone correlate strongly with bone mineral density, which is directed by the loading conditions to which the tissue is subjected. Objective: To investigate the influence of physical activity levels on subchondral bone mineral density (sBMD) in foals during early development. Methods: Three groups of foals were subjected to different physical activity levels from birth until age 5 months. A proportion of these foals were subjected to euthanasia at 5 months while remaining foals were subjected to similar physical activity levels for 6 months until euthanasia at 11 months. Osteochondral specimens were collected for measurement of sBMD with peripheral quantitative computed tomography at 2 differently loaded anatomical sites of the proximal phalangeal bone at 1, 2, 3, 4 and 5 mm depth from the osteochondral junction. Results: Growth significantly increased sBMD but by a different amount depending on anatomical location and physical activity level. Significantly higher sBMD was found at the habitually loaded central area in comparison to the intermittently peak loaded marginal site. Exercise increased sBMD throughout the whole depth of analysed tissue, but changes were generally more obvious at a depth of 2 mm. Interestingly, foals subjected to additional sprint training preserved the exercise-induced sBMD increase at the habitually loaded central area during the 6 months of the second phase of the study. Conclusions: Habitual low-intensity loading elicits a greater response in sBMD in quantitative terms than high-intensity low-frequency loading at the sites investigated in this study. Future sBMD may be influenced by means of well-tailored exercise regimens at young age. Potential relevance: Specific physical activity levels during early development may potentially reduce the prevalence of osteochondral injury later in life.

Polarized light microscopy is a traditional method for visualizing the collagen network architecture of articular cartilage. Articular cartilage repair and tissue engineering studies have raised new demands for techniques capable of quantitative characterization of the scar and repair tissues, including properties of the collagen network. Modern polarized light microscopy can be used to measure collagen fibril orientation, parallelism, and birefringence. New commercial instruments are computer controlled and the measurements are easy to perform. However, often the interpretation of results causes difficulties, even errors, because the theoretical aspects of the technique are demanding. The aim of this study was to describe the instrumentation and properties of a modern polarized light microscope, to point out some sources of error in the interpretation of the results, and to recall the theoretical background of the polarized light microscopy.

Background Retinal pigment epithelium (RPE) maintains the function of photoreceptors and eyesight and is an important target for gene delivery. Since in some diseases RPE cells proliferate uncontrollably, we investigated the role of cell cycle in non-viral gene delivery into a RPE cell line (D407). Methods D407 (human) cells were transfected with cationic DNA complexes. Cells were synchronized into different phases of the cell cycle and transfected using poly-L-lysine (PLL) or polyethyleneimine (PEI) carriers for 3 h. The effects of different reporters (β-galactosidase, luciferase) or promoters (CMV, SV40, tk, PDE-β) on gene expression were evaluated 43 h later. Cellular uptake of ethidium monoazide/DNA complexes with PLL or PEI was determined by flow cytometry. Fluorescent DNA and the complexes were localized with a confocal microscope. The role of cell cycle in transcription was evaluated by stable luciferase-expressing cells. Results PLL showed lower transfection levels than PEI in synchronized cells and only slight dependence on cell cycle. PEI showed minimal efficiency at G1 phase and maximum level at S phase. All promoters and reporter genes showed dependence on cell cycle. Cellular uptake of polyplexes was highest at S phase (80–90%) and lowest at G1 phase (5–30%). Confocal microscopy showed minor differences of free DNA between groups in the nucleus, where it was largely carrier-bound. Cell cycle effects on luciferase expression were clear in stable cell line Conclusions Transfection by polyplexes in the RPE cell line is influenced by cellular uptake and transcription, and both processes are cell-cycle-dependent. The results have implications in retinal gene therapy. Copyright © 2004 John Wiley & Sons, Ltd.

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.

In Gd-DTPA-enhanced T imaging of articular cartilage, the MRI contrast agent with two negative charges is understood to accumulate in tissue inversely to the negative charge of cartilage glycosaminoglycans (GAGs) of proteoglycans (PGs), and this leads to a decrease in the T relaxation time of tissue relative to the charge in tissue. By assuming a constant relaxivity for Gd-DTPA in cartilage, it has further been hypothesized that the contrast agent concentration in tissue could be estimated from consecutive T measurements in the absence or presence of the contrast agent. The spatial sensitivity of the technique was examined at 9.4 T in normal and PG-depleted bovine patellar cartilage samples. As a reference, spatial PG concentration was assessed with digital densitometry from safranin O-stained cartilage sections. An excellent linear correlation between spatial optical density (OD) of stained GAGs and T with Gd-DTPA was observed in the control and chondroitinase ABC-treated cartilage specimens, and the MR parameter accounted for approximately 80% of the variations in GAG concentration within samples. Further, the MR-resolved Gd-DTPA concentration proved to be an even better estimate for PGs, with an improved correlation. However, the linear relation between MR parameters and PG concentration did not apply in the deep tissue, where MR measurements overestimated the PG content. While the absolute [Gd-DTPA] determination may be prone to error due to uncertainty of relaxivity in cartilage, or to other contributing factors such as variations in tissue permeability, the experimental evidence highlights the sensitivity of this technique to reflect spatial changes in cartilage PG concentration in normal and degenerated tissue.

It has been suggested that orientational changes in the collagen network of articular cartilage account for the depthwise T2 anisotropy of MRI through the magic angle effect. To investigate the relationship between laminar T2 appearance and collagen organization (anisotropy), bovine osteochondral plugs (N = 9) were T2 mapped at 9.4T with cartilage surface normal to the static magnetic field. Collagen fibril arrangement of the same samples was studied with polarized light microscopy, a quantitative technique for probing collagen organization by analyzing its ability to rotate plane polarized light, i.e., birefringence (BF). Depthwise variation of safranin O-stained proteoglycans was monitored with digital densitometry. The spatially varying cartilage T2 followed the architectural arrangement of the collagen fibril network: a linear positive correlation between T2 and the reciprocal of BF was established in each sample, with r = 0.91 +/- 0.02 (mean +/- SEM, N = 9). The current results reveal the close connection between the laminar T2 structure and the collagen architecture in histologic zones.

Inhibition of keratinocyte growth in cell culture and whole skin culture by mast cell mediators

Objective To assess the clinical and histologic effects of an intraarticular application of low-dose (noncytotoxic) liposomal clodronate in established antigen-induced monarthritis (AIA) in rabbits. Methods AIA was monitored by assessments of joint swelling, C-reactive protein levels, and radiographic changes in 17 NZW rabbits for 8 weeks during the course of weekly intraarticular injections of liposomal clodronate (0.145 mg/injection, low dose) or “empty” liposomes. The contralateral knee was injected with liposome buffer alone as the control. End-point analyses included macroscopic joint examination, immuno- and TUNEL staining, Safranin O staining/microspectrophotometry, and tumor necrosis factor α (TNFα) convertase enzyme (TACE) inhibition assay. Results Liposomal clodronate–treated rabbits showed a reduction and delay in joint swelling during the first 3 injections. Expression of matrix-bound (solubilized) TNFα, lining cell hyperplasia, and levels of RAM-11+ macrophages were low in the synovium of the liposomal clodronate treatment group, but the proportion of apoptotic lining cells was not affected. The radiologic score was low at the end of weeks 2 and 4, but at 8 weeks, no difference, compared with controls, was found in pannus formation or in the extent of joint erosion; also, joint swelling was higher than before initiation of treatment. Injections of liposomal clodronate prevented cartilage proteoglycan loss, which was significant in the superficial zone only. TACE activity was not inhibited by clodronate. Conclusion Liposomal clodronate had temporary antiinflammatory and antierosive effects on established AIA in rabbits. Over the long-term, the loss of cartilage proteoglycans was halted. This observed treatment effect may be related to the inhibition of TNFα production and processing in the synovium.

The expression of hyaluronan in human tooth germs was studied by using a biotinylated hyaluronan-binding complex and quantitative digital image analysis. At the cap stage, dental papilla exhibited a moderate staining, while intense reaction was observed in the apical portion of presecretory ameloblasts, stellate reticulum, and in dental basement membrane. When the enamel and dentine matrices started to develop, a strong hyaluronan reaction was evident in the young enamel and the apical portion of secretory ameloblasts. No hyaluronan could be detected in the secretory ameloblasts and enamel matrix of the early (9-wk-old) post-natal stage. It is concluded that hyaluronan may play a transitory role in the early phase of the development of the enamel matrix organization. A very weak signal was observed in the wall of dentin tubules, whereas the rest of the dentine matrix was not stained. The odontoblasts and the pulp were also moderately stained, and these reactions gradually decreased with age, suggesting that hyaluronan may also contribute to the development of dentine matrix and pulp.

The aim of this study was to evaluate the effects of long-term running training on the structural properties of bone. Ten beagle dogs ran according to a strenuous progressive program (up to 40 km/day) for 1 year. At the end of the training program, there was a significant reduction in bone mineral density (up to 9.7%) in the vertebrae of the runner dogs as compared with 10 sedentary control dogs. Polarized light microscopy of the vertebral trabecular bone, however, displayed proportionally higher retardation values of the collagen network of the runner dogs than of the sedentary dogs, suggesting a reorganization in a more parallel manner in the collagen fibrils. The concentration and cross-linking of collagen in the bones remained similar in both groups. No differences were observed in the force to failure of bones of the two groups nor in the histomorphometric analysis of the bones. We suggest that the collagen network in the bones accounted for the maintenance of the strength properties in the bones of the runner dogs despite the loss of mineral density.

To analyze the interrelationships between the structure, composition, and mechanical properties of articular cartilage, canine knee (n = 10) femoral and tibial cartilages were used as experimental tissues. The biomechanical properties, instant shear modulus (IM), and equilibrium shear modulus (EM) of articular cartilage were investigated using an in situ indentation creep technique. The local variations in the concentration of glycosaminoglycans (GAGs) in the cartilage were measured with a microspectrophotometer after safranin-O staining of histological sections. Using a computer-based quantitative polarized light microscopy method, area-specific measurements of the optical path difference were performed to quantitate collagen-related optical retardation (Γ) of cartilage zones. The IM and EM were 131.3 and 51.2% higher (p < 0.001) in the femoral cartilage than in the tibial cartilage, respectively. The mean thickness of the superficial zone and the relative proportion of the superficial zone from the total uncalcified cartilage was 107.1 and 155.3% higher (p < 0.001) at the femoral test points than in the tibial ones, respectively. The mean thickness of the tibial uncalcified cartilage was 21.1% higher (p < 0.001) than the thickness of the femoral cartilage. The GAG concentration of the tibial cartilage was higher (14.8%, p < 0.001) than that of the femoral cartilage, especially in the superficial zone (50.0%, p < 0.05), whereas the Γ of the collagen network in the superficial zone of the femoral cartilage was 64.7% higher (p < 0.001) than in the tibial cartilage. The percent relative thickness and retardation Γ of the superficial zone correlated positively with the indentation stiffness of the canine knee articular cartilage. These observations indicate that cartilage is structurally inhomogenous and layered tissue and the local organization of collagen and GAG concentration of the articular cartilage regulate the biomechanical properties of the tissue. The structure and composition of the superficial articular cartilage significantly affects the indentation response of the canine knee articular cartilage. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 48: 99–107, 1999

The potentialities of polarization microscopy has been greatly increased by using specific stains for selective enhancement of the optical anisotropy of a macromolecular constituent of cells and tissues. Such stainings have proved to be especially useful in exploring the spatial orientation pattern of the extracellular matrix components. The retardation value, which characterizes quantitatively the degree of submicroscopic orientation, can be measured traditionally with a compensator plate. This technique, however, is time-consuming and greatly dependent on visual judgment. Several attempts have been made to combine digital image analysis and polarization microscopy to improve the measuring technique in unstained structures. In this paper, we summarize theoretical considerations and experimental data to show the advantages and limitations of this methodological approach when using stained and birefringent specimens. The technique we are suggesting is the measurement of the light intensity using a 12 bit cCCD camera attached to a polarized light microscope and digital image analysis system. The theoretical basis is given by the Fresnel equation describing the relationship between light intensity and retardation value. According to this, there is a sin2 function between the light intensity and the retardation value. The same relationship of these two parameters was observed in our experiments on the birefringent extracellular matrix around chondrocytes grown in agarose gel and interterritorial and territorial matrix of canine articular cartilage stained with picrosirius red. Our results suggest that the retardation values can be calculated directly from the light intensity values if the retardation value is lower than lambda/2.

Introduction: The articular cartilage collagen network and proteoglycans are subject to changes in deteriorating joint diseases. In this study, we exposed articular cartilage plugs to cyclic loading and investigated the properties of collagen network and proteoglycans in different zones of the articular cartilage. Methods: Articular cartilage full-depth plugs were exposed in vitro to 4.1 MPa cyclic (0.5 Hz) loading for 1 to 20 hr and investigated using quantitative microscopic methods (i.e., polarized light microscopy, microspectrophotometry, and autoradiography). Results: The loading caused packing or condensation of the tissue. In histological sections, the height of uncalcified articular cartilage decreased by an average of 12.8% (range, 4 to 19.7%). Loading increased the birefringence of collagen in the superficial cartilage (P , 0.05), with thickening of the zone up to 41.4% at 20 hr. The thickness of the intermediate zone increased also (22% at 1 hr and 434% at 20 hr). Concomitantly, the birefringence (P , 0.05) and the thickness of the deep zone decreased (18.5 to 27.8%). Loading for 4 hr increased the 35 S-sulphate incorporation of the cartilage explants by an average of 67% (P , 0.05). The increase was most significant in the deep cartilage. A simultaneous increase was observed in the proteoglycan concentration of the cartilage; the staining intensity with safranin-O increased by 8.8% (P , 0.05). After 8 hr loading, this stimulation decreased; at 20 hr, loading caused a clear inhibitory effect on proteoglycan synthesis in the superficial zone. Discussion: According to these results, the chosen loading regimen increased the thickness and collagen orientation in the superficial zone. In contrast, the thickness and birefringence in the deep cartilage were reduced. The proteoglycan metabolism of chondrocytes was first stimulated deep in the

Background: Inbred C57BL made mice express a high incidence of spontaneous osteoarthritis of the knee joint at the age of 18 months. We used this strain of mice to find out the effects of life-long, moderate running exercise on the health of articular cartilage and the incidence of osteoarthritis. Methods: Male mice (294) were divided into controls and runners. The runners were trained daily between 2 and 18 months of age. The speed was 13.3 m/min and the distance on a flatbelt treadmill was 1,000 m/day. The mice were sacrificed at the ages of 2, 6, 10, 14, and 18 months. The Knee joints were sectioned in frontal direction and the osteoarthritic changes were graded using a conventional light microscope. The reproducibility of the grading method was tested by calculating extended k-coefficient for the results of six researchers. Results: The incidence of osteoarthritis at the age of 18 months increased from 72% in controls to 88% in runners in the medial tibial condyles (P