Clinical outcomes in end stage renal disease on dialysis and severe coronary artery disease: A real-world study.

BACKGROUND: The optimal management of patients with end-stage renal disease (ESRD) on dialysis with severe coronary artery disease (CAD) has not been determined. METHODS: Between 2013 and 2017, all patients with ESRD on dialysis who had left main (LM) disease, triple vessel disease (TVD) and/or severe CAD for consideration of coronary artery bypass graft (CABG) were included. Patients were divided into 3 groups based on final treatment modality: CABG, percutaneous coronary intervention (PCI), optimal medical therapy (OMT). Outcome measures include in-hospital, 180-day, 1-year and overall mortality and major adverse cardiac events (MACE). RESULTS: In total, 418 patients were included (CABG 11.0%, PCI 65.6%, OMT 23.4%). Overall, 1-year mortality and MACE rates were 27.5% and 55.0% respectively. Patients who underwent CABG were significantly younger, more likely to have LM disease and have no prior heart failure. In this non-randomized setting, treatment modality did not impact on 1-year mortality, although the CABG group had significantly lower 1-year MACE rates (CABG 32.6%, PCI 57.3%, OMT 59.2%; CABG vs. OMT p < 0.01, CABG vs. PCI p < 0.001). Independent predictors of overall mortality include STEMI presentation (HR 2.31, 95% CI 1.38-3.86), prior heart failure (HR 1.84, 95% CI 1.22-2.75), LM disease (HR 1.71, 95% CI 1.26-2.31), NSTE-ACS presentation (HR 1.40, 95% CI 1.03-1.91) and increased age (HR 1.02, 95% CI 1.01-1.04). CONCLUSION: Treatment decisions for patients with severe CAD with ESRD on dialysis are complex. Understanding independent predictors of mortality and MACE in specific treatment subgroups may provide valuable insights into the selection of optimal treatment options.

Impact of Neospora caninum Infection on the Bioenergetics and Transcriptome of Cerebrovascular Endothelial Cells.

In this work, the effects of the protozoan Neospora caninum on the bioenergetics, chemical composition, and elemental content of human brain microvascular endothelial cells (hBMECs) were investigated. We showed that N. caninum can impair cell mitochondrial (Mt) function and causes an arrest in host cell cycling at S and G2 phases. These adverse effects were also associated with altered expression of genes involved in Mt energy metabolism, suggesting Mt dysfunction caused by N. caninum infection. Fourier Transform Infrared (FTIR) spectroscopy analysis of hBMECs revealed alterations in the FTIR bands as a function of infection, where infected cells showed alterations in the absorption bands of lipid (2924 cm-1), amide I protein (1649 cm-1), amide II protein (1537 cm-1), nucleic acids and carbohydrates (1092 cm-1, 1047 cm-1, and 939 cm-1). By using quantitative synchrotron radiation X-ray fluorescence (µSR-XRF) imaging and quantification of the trace elements Zn, Cu and Fe, we detected an increase in the levels of Zn and Cu from 3 to 24 h post infection (hpi) in infected cells compared to control cells, but there were no changes in the level of Fe. We also used Affymetrix array technology to investigate the global alteration in gene expression of hBMECs and rat brain microvascular endothelial cells (rBMVECs) in response to N. caninum infection at 24 hpi. The result of transcriptome profiling identified differentially expressed genes involved mainly in immune response, lipid metabolism and apoptosis. These data further our understanding of the molecular events that shape the interaction between N. caninum and blood-brain-barrier endothelial cells.

Metallome of cerebrovascular endothelial cells infected with Toxoplasma gondii using µ-XRF imaging and inductively coupled plasma mass spectrometry.

In this study, we measured the levels of elements in human brain microvascular endothelial cells (ECs) infected with T. gondii. ECs were infected with tachyzoites of the RH strain, and at 6, 24, and 48 hours post infection (hpi), the intracellular concentrations of elements were determined using a synchrotron-microfocus X-ray fluorescence microscopy (µ-XRF) system. This method enabled the quantification of the concentrations of Zn and Ca in infected and uninfected (control) ECs at sub-micron spatial resolution. T. gondii-hosting ECs contained less Zn than uninfected cells only at 48 hpi (p < 0.01). The level of Ca was not significantly different between infected and control cells (p > 0.05). Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis revealed infection-specific metallome profiles characterized by significant increases in the intracellular levels of Zn, Fe, Mn and Cu at 48 hpi (p < 0.01), and significant reductions in the extracellular concentrations of Co, Cu, Mo, V, and Ag at 24 hpi (p < 0.05) compared with control cells. Zn constituted the largest part (74%) of the total metal composition (metallome) of the parasite. Gene expression analysis showed infection-specific upregulation in the expression of five genes, MT1JP, MT1M, MT1E, MT1F, and MT1X, belonging to the metallothionein gene family. These results point to a possible correlation between T. gondii infection and increased expression of MT1 isoforms and altered intracellular levels of elements, especially Zn and Fe. Taken together, a combined µ-XRF and ICP-MS approach is promising for studies of the role of elements in mediating host-parasite interaction.

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization.

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.

Automated multimodal spectral histopathology for quantitative diagnosis of residual tumour during basal cell carcinoma surgery.

Multimodal spectral histopathology (MSH), an optical technique combining tissue auto-fluorescence (AF) imaging and Raman micro-spectroscopy (RMS), was previously proposed for detection of residual basal cell carcinoma (BCC) at the surface of surgically-resected skin tissue. Here we report the development of a fully-automated prototype instrument based on MSH designed to be used in the clinic and operated by a non-specialist spectroscopy user. The algorithms for the AF image processing and Raman spectroscopy classification had been first optimised on a manually-operated laboratory instrument and then validated on the automated prototype using skin samples from independent patients. We present results on a range of skin samples excised during Mohs micrographic surgery, and demonstrate consistent diagnosis obtained in repeat test measurement, in agreement with the reference histopathology diagnosis. We also show that the prototype instrument can be operated by clinical users (a skin surgeon and a core medical trainee, after only 1-8 hours of training) to obtain consistent results in agreement with histopathology. The development of the new automated prototype and demonstration of inter-instrument transferability of the diagnosis models are important steps on the clinical translation path: it allows the testing of the MSH technology in a relevant clinical environment in order to evaluate its performance on a sufficiently large number of patients.

Impact of Chronic Kidney Insufficiency on Cardiovascular Outcomes in Patients that Undergo Coronary Revascularization: A Historical Review.

Chronic kidney disease (CKD) is associated with poorer short and long-term cardiovascular morbidity and mortality. Even after the commencement of haemodialysis in end stage renal failure patients, mortality exceeds 20% in the first year1. More than 50% of these deaths are contributed by cardiovascular diseases (CVD), of which 20% are caused by acute myocardial infarction2. Consequent to these findings, the degree and impact of coronary revascularization on CKD patients represents a clinical challenge, especially in the setting of advanced stages of CKD.

Tissue diagnosis using power-sharing multifocal Raman micro-spectroscopy and auto-fluorescence imaging.

We describe a multifocal Raman micro-spectroscopy detection method based on a digital micromirror device, which allows for simultaneous "power-sharing" acquisition of Raman spectra from ad hoc sampling points. As the locations of the points can be rapidly updated in real-time via software control of a liquid-crystal spatial light modulator (LC-SLM), this technique is compatible with automated adaptive- and selective-sampling Raman spectroscopy techniques, the latter of which has previously been demonstrated for fast diagnosis of skin cancer tissue resections. We describe the performance of this instrument and show examples of multiplexed measurements on a range of test samples. Following this, we show the feasibility of reducing measurement time for power-shared multifocal Raman measurements combined with confocal auto-fluorescence imaging to provide guided diagnosis of tumours in human skin samples.

Raman spectroscopy for medical diagnostics--From in-vitro biofluid assays to in-vivo cancer detection.

Raman spectroscopy is an optical technique based on inelastic scattering of light by vibrating molecules and can provide chemical fingerprints of cells, tissues or biofluids. The high chemical specificity, minimal or lack of sample preparation and the ability to use advanced optical technologies in the visible or near-infrared spectral range (lasers, microscopes, fibre-optics) have recently led to an increase in medical diagnostic applications of Raman spectroscopy. The key hypothesis underpinning this field is that molecular changes in cells, tissues or biofluids, that are either the cause or the effect of diseases, can be detected and quantified by Raman spectroscopy. Furthermore, multivariate calibration and classification models based on Raman spectra can be developed on large "training" datasets and used subsequently on samples from new patients to obtain quantitative and objective diagnosis. Historically, spontaneous Raman spectroscopy has been known as a low signal technique requiring relatively long acquisition times. Nevertheless, new strategies have been developed recently to overcome these issues: non-linear optical effects and metallic nanoparticles can be used to enhance the Raman signals, optimised fibre-optic Raman probes can be used for real-time in-vivo single-point measurements, while multimodal integration with other optical techniques can guide the Raman measurements to increase the acquisition speed and spatial accuracy of diagnosis. These recent efforts have advanced Raman spectroscopy to the point where the diagnostic accuracy and speed are compatible with clinical use. This paper reviews the main Raman spectroscopy techniques used in medical diagnostics and provides an overview of various applications.

Optimization of multimodal spectral imaging for assessment of resection margins during Mohs micrographic surgery for basal cell carcinoma.

Multimodal spectral imaging (MSI) based on auto-fluorescence imaging and Raman micro-spectroscopy was used to detect basal cell carcinoma (BCC) in tissue specimens excised during Mohs micrographic surgery. In this study, the MSI algorithm was optimized to maximize the diagnosis accuracy while minimizing the number of Raman spectra: the segmentation of the auto-fluorescence images was optimized according to the type of BCC, sampling points for Raman spectroscopy were generated based on auto-fluorescence intensity variance and segment area, additional Raman spectra were acquired when performance of the segmentation algorithm was sub-optimal. The results indicate that accurate diagnosis can be achieved with a sampling density of ~2,000 Raman spectra/cm(2), based on sampling points generated by the MSI algorithms. The key benefit of MSI is that diagnosis of BCC is obtained based on intrinsic chemical contrast of the tissue, within time scales similar to frozen-section histopathology, but without requiring laborious sample preparation and subjective interpretation of stained frozen-sections.

Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman spectroscopy.

Label-free imaging using Raman micro-spectroscopy (RMS) was used to characterize the spatio-temporal molecular changes of T. gondii tachyzoites and their host cell microenvironment. Raman spectral maps were recorded from isolated T. gondii tachyzoites and T. gondii-infected human retinal cells at 6 h, 24 h and 48 h post-infection. Principal component analysis (PCA) of the Raman spectra of paraformaldehyde-fixed infected cells indicated a significant increase in the amount of lipids and proteins in the T. gondii tachyzoites as the infection progresses within host cells. These results were confirmed by experiments carried out on live T. gondii-infected cells and were correlated with an increase in the concentration of proteins and lipids required for the replication of this intracellular pathogen. These findings demonstrate the potential of RMS to characterize time- and spatially-dependent molecular interactions between intracellular pathogens and the host cells. Such information may be useful for discovery of pharmacological targets or screening compounds with potential neuro-protective activity for eminent effects of changes in brain infection control practices.

Susceptibility to experimental infection of the invertebrate locusts (Schistocerca gregaria) with the apicomplexan parasite Neospora caninum.

Neuropathogenesis is a feature of Neospora caninum infection. In order to explore this in the absence of acquired host immunity to the parasite, we have tested infection in locusts (Schistocerca gregaria). We show for the first time that locusts are permissive to intra-hemocoel infection with N. caninum tachyzoites. This was characterized by alteration in body weight, fecal output, hemoparasitemia, and sickness-related behavior. Infected locusts exhibited progressive signs of sickness leading to mortality. Also, N. caninum showed neuropathogenic affinity, induced histological changes in the brain and was able to replicate in the brain of infected locusts. Fatty acid (FA) profiling analysis of the brains by gas chromatography and multi-variate prediction models discriminated with high accuracy (98%) between the FA profiles of the infected and control locusts. DNA microarray gene expression profiling distinguished infected from control S. gregaria brain tissues on the basis of distinct differentially-expressed genes. These data indicate that locusts are permissible to infection with N. caninum and that the parasite retains its tropism for neural tissues in the invertebrate host. Locusts may facilitate preclinical testing of interventional strategies to inhibit the growth of N. caninum tachyzoites. Further studies on how N. caninum brings about changes in locust brain tissue are now warranted.

Towards intra-operative diagnosis of tumours during breast conserving surgery by selective-sampling Raman micro-spectroscopy.

Breast-conserving surgery (BCS) is increasingly employed for the treatment of early stage breast cancer. One of the key challenges in BCS is to ensure complete removal of the tumour while conserving as much healthy tissue as possible. In this study we have investigated the potential of Raman micro-spectroscopy (RMS) for automated intra-operative evaluation of tumour excision. First, a multivariate classification model based on Raman spectra of normal and malignant breast tissue samples was built and achieved diagnosis of mammary ductal carcinoma (DC) with 95.6% sensitivity and 96.2% specificity (5-fold cross-validation). The tumour regions were discriminated from the healthy tissue structures based on increased concentration of nucleic acids and reduced concentration of collagen and fat. The multivariate classification model was then applied to sections from fresh tissue of new patients to produce diagnosis images for DC. The diagnosis images obtained by raster scanning RMS were in agreement with the conventional histopathology diagnosis but were limited to long data acquisition times (typically 10,000 spectra mm(-2), which is equivalent to ~5 h mm(-2)). Selective-sampling based on integrated auto-fluorescence imaging and Raman spectroscopy was used to reduce the number of Raman spectra to ~20 spectra mm(-2), which is equivalent to an acquisition time of ~15 min for 5 × 5 mm(2) tissue samples. This study suggests that selective-sampling Raman microscopy has the potential to provide a rapid and objective intra-operative method to detect mammary carcinoma in tissue and assess resection margins.

Metabolic footprinting of extracellular metabolites of brain endothelium infected with Neospora caninum in vitro.

BACKGROUND: The survival of the intracellular protozoan parasite Neospora caninum depends on its ability to adapt to changing metabolic conditions of the host cell. Thus, defining cellular and metabolic changes in affected target tissues may aid in delineating pathogenetic mechanism. We undertook this study to assess the metabolic response of human brain microvascular endothelial cells (HBMECs) to N. caninum infection in vitro. METHODS: HBMECs were exposed to N. caninum infection and the cytotoxic effects of infection were analyzed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromidin (MTT) assay and lactate dehydrogenase (LDH) release assay. Metabolic footprinting of the extracellular metabolites of parasite-infected and non-infected culture supernatant was determined by using targeted (Randox RX Imola clinical chemistry analyser) and unbiased RS (Raman microspectroscopy) approaches. RESULTS: The MTT assay did not reveal any cytotoxic effect of N. caninum challenge on host cell viability. Measurement of LDH activity showed that N. caninum significantly induced loss of cell membrane integrity in a time-dependent and dose-dependent manner compared to control cells. Targeted biochemical analysis revealed that beta hydroxybutyrate, pyruvate, ATP, total protein, non-esterified fatty acids, and triglycerides are significantly different in infected cells compared to controls. RS-based footprinting with principal component analysis (PCA) were able to correctly distinguish extracellular metabolites obtained from infected and control cultures, and revealed infection-related spectral signatures at 865 cm-1, 984 cm-1, 1046 cm-1, and 1420 cm-1, which are attributed to variations in the content of lipids and nucleic acids in infected cultures. CONCLUSIONS: The changing pattern of extracellular metabolites suggests that HBMECs are target of metabolic alterations in N. caninum infection, which seem to reflect the changing metabolic state of infected cells and constitute a level of information exchange that host and parasite use to coordinate activities.

Diagnosis of tumors during tissue-conserving surgery with integrated autofluorescence and Raman scattering microscopy.

Tissue-conserving surgery is used increasingly in cancer treatment. However, one of the main challenges in this type of surgery is the detection of tumor margins. Histopathology based on tissue sectioning and staining has been the gold standard for cancer diagnosis for more than a century. However, its use during tissue-conserving surgery is limited by time-consuming tissue preparation steps (1-2 h) and the diagnostic variability inherent in subjective image interpretation. Here, we demonstrate an integrated optical technique based on tissue autofluorescence imaging (high sensitivity and high speed but low specificity) and Raman scattering (high sensitivity and high specificity but low speed) that can overcome these limitations. Automated segmentation of autofluorescence images was used to select and prioritize the sampling points for Raman spectroscopy, which then was used to establish the diagnosis based on a spectral classification model (100% sensitivity, 92% specificity per spectrum). This automated sampling strategy allowed objective diagnosis of basal cell carcinoma in skin tissue samples excised during Mohs micrographic surgery faster than frozen section histopathology, and one or two orders of magnitude faster than previous techniques based on infrared or Raman microscopy. We also show that this technique can diagnose the presence or absence of tumors in unsectioned tissue layers, thus eliminating the need for tissue sectioning. This study demonstrates the potential of this technique to provide a rapid and objective intraoperative method to spare healthy tissue and reduce unnecessary surgery by determining whether tumor cells have been removed.

Label-free molecular analysis of live Neospora caninum tachyzoites in host cells by selective scanning Raman micro-spectroscopy.

A selective scanning method was used to measure spatially resolved Raman spectra of live Neospora caninum tachyzoites colonizing human brain microvascular-endothelial cells. The technique allowed the detection of nucleic acids, lipids and proteins linked to the parasites and their cellular micro-environment at ∼10× shorter acquisition time compared to raster scanning.

Molecular and crystal deformation of cellulose: uniform strain or uniform stress?

The molecular and crystal deformations of a range of lyocell cellulose fibres, produced using different drawing conditions, are reported. The fibres are spun using increasing draw ratios to both increase the molecular and crystal orientation and, consequently, mechanical stiffness. Raman spectroscopy and X-ray diffraction are used to follow molecular and crystal deformation, respectively. It is shown that these techniques are complementary, and that both must be used for semicrystalline cellulose fibres if a full picture of their micromechanics is to be obtained. By following the shift in the 1095 cm(-1) Raman band with respect to external tensile deformation of the fibres we show that we can build up a picture of the microstructure. Using theoretical predictions of the relationship between the Raman band shift rates with respect to strain and stress and the modulus of the fibres we show that the fibres have properties that suggest a hybrid series-series aggregate structure. By using X-ray diffraction we show that the crystal modulus of the fibres appears to change with increasing draw ratio. Furthermore the crystal modulus of the fibres appears to vary systematically with the crystallinity of the sample. Other relationships between the predicted fibre modulus and the experimental values and between the Raman band shift rates and modulus suggest that the assumption of a uniform stress microstructure prior to the measurement of crystal modulus may be an incorrect one. A more realistic structure is proposed for semicrystalline regenerated cellulose fibres, wherein crystals and amorphous regions are both in series and in parallel with each other.

Influence of domain orientation on the mechanical properties of regenerated cellulose fibers.

The determination of the crystal orientation of regenerated cellulose fibers produced under different drawing regimes is presented. Orientation is determined by using wide-angle X-ray diffraction from a synchrotron source and by measuring the azimuthal width of equatorial reflections. The orientation parameter theta is then determined to compare fiber samples. By using a 500 nm beam size, clear differences between the crystal orientations of the skin and the core of the fibers are reported for a range of differently processed fibers for the first time. These results are shown to have implications for the mechanical properties of regenerated cellulose fibers. By applying tensile deformation to fiber bundles it is shown that the most misoriented samples undergo rapid decreases in the orientation parameter, which is an indication of crystal reorientation. However, the more highly oriented fibers undergo little reorientation. An average shear modulus for these fibers is determined by placing the data on a master curve and fitting with a model equation. By using another model for the fibers of low orientation and the shear modulus from the master curve analysis, it is shown that the deformation of less oriented fibers is dominated by shear between crystals, whereas the more oriented filaments are likely to undergo more significant chain deformation. By using a new model for fibers of low orientation, a parameter ksigma is introduced that gives the proportion of the fiber stress that is due to crystal shear. Systematic differences between this parameter for fibers of increasing initial orientation are reported. Moreover it is shown that the fibers of initially lower average orientation are governed by uniform strain, in agreement with the new model, whereas more highly oriented fibers deform under uniform stress. Furthermore, the model that we propose for misoriented domains and the use of a new factor dictating the proportion of shear stress may have general applications in materials engineering.