Distinguishing Ewing sarcoma and osteomyelitis using FTIR spectroscopy

Abstract The differential diagnosis of Ewing sarcoma and osteomyelitis can be challenging and can lead to delays in treatment with possibly devastating results. In this retrospective, small-cohort study we demonstrate, that the Fourier Transformed Infrared (FTIR) spectra of osteomyelitis bone tissue can be differentiated from Ewing sarcoma and normal bone tissue sampled outside tumour area. Significant differences in osteomyelitis samples can be seen in lipid and protein composition. Supervised learning using a quadratic discriminant analysis classifier was able to differentiate the osteomyelitis samples with high accuracy. FTIR spectroscopy, alongside routine radiological and histopathological methods, may offer an additional tool for the differential diagnosis of osteomyelitis and ES

Increased cellular senescence and vascular rarefaction exacerbate the progression of kidney fibrosis in aged mice following transient ischemic injury.

Recent findings indicate that elderly patients with acute kidney injury (AKI) have an increased incidence of progression to chronic kidney disease (CKD) due to incomplete recovery from an acute insult. In the current study, a co-morbid model of AKI was developed to better mimic the patient population and to investigate whether age exacerbates the fibrosis and inflammation that develop in the sequelae of progressive kidney disease following acute injury. Young (8-10 weeks) and aged (46-49 weeks) C57BL/6 mice were subjected to 30 min bilateral renal ischemia-reperfusion (I/R) to induce AKI. The aged animals have greater mortality and prolonged elevation of plasma creatinine correlating with less tubular epithelial cell proliferation compared to the young. Six weeks post-reperfusion, interstitial fibrosis is greater in aged kidneys based on picrosirius red staining and immunolocalization of cellular fibronectin, collagen III and collagen IV. Aged kidneys 6 weeks post-reperfusion also express higher levels of p53 and p21 compared to the young, correlating with greater increases in senescence associated (SA) β-galactosidase, a known marker of cellular senescence. A higher influx of F4/80(+) macrophages and CD4(+) T lymphocytes is measured and is accompanied by increases in mRNA of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). Importantly, microvascular density is significantly less, correlating with an increase in nitro-tyrosine, a marker of oxidative stress. Collectively, these data demonstrate that prolonged acute injury in the aged animals results in an accelerated progression of kidney disease in a chronic state

Discrete areas of leukocyte infiltrates surrounding the vasculature are only present in aged animals 6 weeks post-reperfusion.

<p>Representative H&E micrograph (A) showing a blood vessel surrounded by leukocytes. It is not the same vessel depicted in subsequent figures. Representative micrographs of serial sections indicate that CD4⁺ T lymphocytes (B) and CD19⁺ B lymphocytes (C) are the predominant immune cells in the area surrounding the blood vessel. Some CD8⁺ T lymphocytes also are present in lower numbers (D); however, F4/80⁺ macrophages do not co-localize with these leukocytes (E). *denotes the lumen of the blood vessel. Hatch marks indicate peripheral margin of the vascular sheath of denoted blood vessel. Bar = 100 microns.</p

Primary antibodies.

<p>Primary antibodies.</p

Extracellular matrix proteins are greater in aged kidneys compared to young at 6 weeks post-reperfusion.

<p>Representative micrographs of cellular fibronectin (A) and collagen III (B) immunostained sections of the outer medulla of young and aged kidneys. Quantitation of pixels per field indicates significantly increased matrix deposition in the aged kidneys 6 weeks post-reperfusion. (n = 4–5/group). There are equivalent low levels in young and age matched normals. *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. Bar = 100 microns.</p

Inflammation in the cortex and outer medulla 6 weeks post-reperfusion is greater in aged kidneys.

<p>Representative micrographs of F4/80 (A) and CD4 (B) immunostained sections of the outer medulla of young and aged kidneys. Quantitation of pixels per field indicate significantly increased numbers of cells in the aged kidneys 6 weeks post-reperfusion (n = 4–5/group). There are equivalent low levels in young and age matched normals. *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. Bar = 100 microns. C. Gene expression of MCP-1 and TNF-α indicate increased mRNA levels in the aged kidneys 6 weeks post-reperfusion (n = 7 for young and 3 for aged mice). *p≤0.05 indicates a significant difference between young and aged ischemia.</p

Microvascular loss and oxidative stress are greater in the aged kidney 6 weeks post-reperfusion.

<p>A. Representative micrographs of CD31-immunostained outer medulla from young and aged mice at 6 weeks post-reperfusion. The enlarged view (area indicated by box) highlights CD31⁺ endothelial cells lining the vasculature. Quantitation of CD31 immunostaining is shown in the graph. There is no difference between young and aged normal; however, vascular loss is greater in the aged kidney 6 weeks post-reperfusion. *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. B. Oxidative stress measured by nitro-tyrosine staining is greater in the outer medulla of aged kidneys 6 weeks post-reperfusion as compared to the young. Images were recorded with identical exposure settings. Bar = 100 microns.</p

Aged mice have greater mortality and prolonged duration of AKI.

<p>A. Percent survival is greater in young mice 6 weeks post-reperfusion (n = 14–15 at day 0). Plasma creatinine (B) and BUN (C) levels were measured at indicated times post-reperfusion (n = 4–10/group) with corresponding shams that are equivalent between young and aged regardless of timepoint. *p≤0.05 indicates a significant difference from the respective sham. **p≤0.05 indicates a significant difference from the young I/R at the same timepoint.</p