Characteristic infrared spectroscopic patterns in the protein bands of human breast cancer tissue

Infrared (IR) spectra of normal, hyperplasia, fibroadenoma and carcinoma tissues of human breast obtained from 96 patients have been determined and analyzed statistically. Several spectral differences were detected in the frequency regions of N-H stretching, amide I, II and III bands: (1) the bands in the region 3000-3600cm-1 shifted to lower frequencies for the carcinomatous tissue; (2) the A(3300)/A(3075) absorbance ratio was significantly higher for the fibroadenoma than for the other types of tissues; (3) the frequency of the a-helix amide I band decreased for the malignant tissue, while the corresponding beta -sheet amide I band frequency increased; (4) the A(1657)/A(1635) and A(1553)/A(1540) absorbance ratios were the highest for fibroadenoma and carcinoma tissues; (5) the A(1680)/A(1657) absorbance ratio decreased significantly in the order of normal > hyperplasia > fibroadenoma > carcinoma; (6) the A(1651)/A(1545) absorbance ratio increased slightly for the fibroadenoma and the carcinoma tissues; (7) the bands at 1204 and 1278 cm(-1), assigned to the vibrational modes of the collagen, did not appear in the original spectra as resolved peaks and were distinctly stronger in the deconvoluted spectra of the carcinoma tissue and (8) the A(1657)/A(1204) and A(1657)/A(1278) absorbance ratios, both yielding information on the relative content of collagen, increased in the order of normal < hyperplasia < carcinoma < fibroadenoma. The said differences imply that the information is useful for the diagnosis of breast cancer and malignant breast abnormalities, and may serve as a basis for further studies on conformational changes in tissue proteins during carcinogenesis. (C) 2001 Elsevier Science B.V. All rights reserved.

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.

A novel breast tissue density classification framework

Oliver, A., Freixenet, J., Marti, R., Pont, J., Perez, E., Denton, E. R. E., Zwiggelaar, R. (2008). A novel breast tissue density classification framework. IEEE Transactions on Information Technology in BioMedicine, 12 (1), 55-65

'Adipaging': Aging and obesity share biological hallmarks related to a dysfunctional adipose tissue

The increasing aging of our societies is accompanied by a pandemic of obesity and related cardiometabolic disorders. Progressive dysfunction of the white adipose tissue is increasingly recognized as an important hallmark of the aging process which in turn contributes to metabolic alterations, multi-organ damage, and a systemic pro-inflammatory state ('inflammaging'). On the other hand, obesity, the paradigm of adipose tissue dysfunction, shares numerous biological similarities with the normal aging process such as chronic inflammation and multi-system alterations. Accordingly, understanding the interplay between accelerated aging related to obesity and adipose tissue dysfunction is critical to gain insight into the aging process in general as well as into the pathophysiology of obesity and other related conditions. Here we postulate the concept of 'adipaging' to illustrate the common links between aging and obesity and the fact that, to a great extent, obese adults are prematurely aged individuals.

Cancer Risk Assessment Related to Anatomical Tissue Types

H. Strange, W. He, E. Denton and R. Zwiggelaar, 'Cancer Risk Assessment Related to Anatomical Tissue Types', Proceedings of the Twelfth Annual Conference on Medical Image Understanding and Analysis, 2008, p.138. Sponsorship: EPSRC

Matriz de colagénio suíno no recobrimento radicular

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

INVESTIGATION OF BUBBLE DYNAMICS AND HEATING DURING FOCUSED ULTRASOUND INSONATION IN TISSUE-MIMICKING MATERIALS

The deposition of ultrasonic energy in tissue can cause tissue damage due to local heating. For pressures above a critical threshold, cavitation will occur in tissue and bubbles will be created. These oscillating bubbles can induce a much larger thermal energy deposition in the local region. Traditionally, clinicians and researchers have not exploited this bubble-enhanced heating since cavitation behavior is erratic and very difficult to control. The present work is an attempt to control and utilize this bubble-enhanced heating. First, by applying appropriate bubble dynamic models, limits on the asymptotic bubble size distribution are obtained for different driving pressures at 1 MHz. The size distributions are bounded by two thresholds: the bubble shape instability threshold and the rectified diffusion threshold. The growth rate of bubbles in this region is also given, and the resulting time evolution of the heating in a given insonation scenario is modeled. In addition, some experimental results have been obtained to investigate the bubble-enhanced heating in an agar and graphite based tissue- mimicking material. Heating as a function of dissolved gas concentrations in the tissue phantom is investigated. Bubble-based contrast agents are introduced to investigate the effect on the bubble-enhanced heating, and to control the initial bubble size distribution. The mechanisms of cavitation-related bubble heating are investigated, and a heating model is established using our understanding of the bubble dynamics. By fitting appropriate bubble densities in the ultrasound field, the peak temperature changes are simulated. The results for required bubble density are given. Finally, a simple bubbly liquid model is presented to estimate the shielding effects which may be important even for low void fraction during high intensity focused ultrasound (HIFU) treatment.

THE ROLE OF ACOUSTIC CAVITATION IN ENHANCED ULTRASOUND-INDUCED HEATING IN A TISSUE-MIMICKING PHANTOM

A complete understanding of high-intensity focused ultrasound-induced temperature changes in tissue requires insight into all potential mechanisms for heat deposition. Applications of therapeutic ultrasound often utilize acoustic pressures capable of producing cavitation activity. Recognizing the ability of bubbles to transfer acoustic energy into heat generation, a study of the role bubbles play in tissue hyperthermia becomes necessary. These bubbles are typically less than 50μm. This dissertation examines the contribution of bubbles and their motion to an enhanced heating effect observed in a tissue-mimicking phantom. A series of experiments established a relationship between bubble activity and an enhanced temperature rise in the phantom by simultaneously measuring both the temperature change and acoustic emissions from bubbles. It was found that a strong correlation exists between the onset of the enhanced heating effect and observable cavitation activity. In addition, the likelihood of observing the enhanced heating effect was largely unaffected by the insonation duration for all but the shortest of insonation times, 0.1 seconds. Numerical simulations were used investigate the relative importance of two candidate mechanisms for heat deposition from bubbles as a means to quantify the number of bubbles required to produce the enhanced temperature rise. The energy deposition from viscous dissipation and the absorption of radiated sound from bubbles were considered as a function of the bubble size and the viscosity of the surrounding medium. Although both mechanisms were capable of producing the level of energy required for the enhanced heating effect, it was found that inertial cavitation, associated with high acoustic radiation and low viscous dissipation, coincided with the the nature of the cavitation best detected by the experimental system. The number of bubbles required to account for the enhanced heating effect was determined through the numerical study to be on the order of 150 or less.

HEATING IN VASCULAR TISSUE AND FLOW-THROUGH TISSUE PHANTOMS INDUCED BY FOCUSED ULTRASOUND

High intensity focused ultrasound (HIFU) can be used to control bleeding, both from individual blood vessels as well as from gross damage to the capillary bed. This process, called acoustic hemostasis, is being studied in the hope that such a method would ultimately provide a lifesaving treatment during the so-called "golden hour", a brief grace period after a severe trauma in which prompt therapy can save the life of an injured person. Thermal effects play a major role in occlusion of small vessels and also appear to contribute to the sealing of punctures in major blood vessels. However, aggressive ultrasound-induced tissue heating can also impact healthy tissue and can lead to deleterious mechanical bioeffects. Moreover, the presence of vascularity can limit one’s ability to elevate the temperature of blood vessel walls owing to convective heat transport. In an effort to better understand the heating process in tissues with vascular structure we have developed a numerical simulation that couples models for ultrasound propagation, acoustic streaming, ultrasound heating and blood cooling in Newtonian viscous media. The 3-D simulation allows for the study of complicated biological structures and insonation geometries. We have also undertaken a series of in vitro experiments, in non-uniform flow-through tissue phantoms, designed to provide a ground truth verification of the model predictions. The calculated and measured results were compared over a range of values for insonation pressure, insonation time, and flow rate; we show good agreement between predictions and measurements. We then conducted a series of simulations that address two limiting problems of interest: hemostasis in small and large vessels. We employed realistic human tissue properties and considered more complex geometries. Results show that the heating pattern in and around a blood vessel is different for different vessel sizes, flow rates and for varying beam orientations relative to the flow axis. Complete occlusion and wall- puncture sealing are both possible depending on the exposure conditions. These results concur with prior clinical observations and may prove useful for planning of a more effective procedure in HIFU treatments.

Novel coatings for hard tissue implants

A novel deposition process named CoBlastTM, based on grit blasting technology, has been used to deposit hydroxyapatite (HA) onto titanium (Ti) metal using a dopant/abrasive regime. The various powders (HA powder, apatitic abrasives) and the treated substrates were characterised for chemical composition, coating coverage, crystallinity and topography including surface roughness. The surface roughness of the HA surfaces could be altered using apatitic abrasives of different particle sizes. Compared to the standard plasma spraying process, the CoBlast surface produced excellent coating adhesion, lower dissolution, higher levels of mechanical and chemical stability in stimulated body fluid (SBF). Enhanced viability of osteoblastic cells was also observed on the CoBlast HA surfaces compared to the microblast and untreated Ti as well as the plasma HA coating. CoBlast offers an alternative to the traditional methods of coating HA implants with added versatility. Apatites substituted with antimicrobial metals can also be deposited to add functionality to HA coatings without cytotoxicty. The potential use of these coatings as an infection preventing strategy for application on hard tissue implants was assessed in vitro and also in vivo. Surface physicochemical properties and morphology were determined in addition to surface cytocompatibility assessments using a MG-63 osteoblast cell line. The antibacterial potential of the immobilised metal ion on the surface and the eluted ion to a lesser extent, contributed to the anticolonising behaviour of the surfaces against a standard bacteria strain (S. aureus) as well as a number of clinically relevant strains (MRSA, MSSA and S. epidermis). The results revealed that the surfaces coated with silver substituted apatites (AgA) outperformed the other apatites examined (apatites loaded with Zn, Sr and both Ag and Sr ions). Assessment of bacterial adherence on coated K-wires following subcutaneous implantation in a nude mouse infection model (S. aureus) for two days demonstrated that the 12% wt surface outperformed the 5% wt AgA coating. Lower inflammatory responses were activated with the insertion of the Ag loaded K-wires with a localised infection at the implantation site noted over the two day study period. These results indicated that the AgA coating on the surface of orthopaedic implants demonstrate good biocompatibility whilst inhibiting bacterial adhesion and colonising of the implant surface.

Ex vivo culture of patient tissue and examination of gene delivery

Gene therapy has emerged as a realistic prospect for the treatment of cancer due to its potential for selective tumour cell targeting. The greatest challenge gene delivery vectors face is the ability to safely and efficiently deliver genes into target cells. The overall objectives of this thesis are to evaluate the efficacy of various gene delivery methods in a clinically relevant tumour model and to also investigate potential strategies for tumour selective delivery. We began with the development of a tumour slice model system using patient waste tissue. This model involves the use of fresh human tumour tissue, cut into thin slices and maintained ex vivo and is universally applicable to gene delivery methods, using a real-time luminescence detection method to assess gene delivery. The nature of the ex vivo culture system permitted examination of specific physiological variables, the influence of intratumoural factors and tissue specific effects on vector expression. Adenoviral vectors under the control of the human CXCR4 promoter demonstrated a 'tumour on' and 'normal off' expression profile when compared with the ubiquitously active CMV promoter when tested in patient tumour tissue. In addition, we developed an ex vivo system of changing oxygenation using the hypoxia inducer, cobalt, to mimic the transient hypoxic conditions found in solid tumours. We found that Adenoviral transgene expression was robust in the cycling hypoxic conditions relevant to solid tumours and re-oxygenation of chronically hypoxic tissue enhanced transgene expression. Finally, we demonstrated an AAV-based tumour targeting strategy using a tumour-selective promoter allowing for the efficient targeting of AAV vectors to cancer cells and the sparing of normal tissue in both murine metastatic liver tumours models and patient tissue. The thesis highlights the importance of indepth preclinical assessment of novel therapeutics and may serve as a platform for further testing of novel gene delivery approaches.

Comparison of ELISA for tissue transglutaminase autoantibodies with antiendomysium antibodies in pediatric and adult patients with celiac disease.

BACKGROUND: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients. METHODS: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients. RESULTS: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively. CONCLUSION: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.

Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study.

OBJECTIVE: To investigate the value of serum antitissue transglutaminase IgA antibodies (IgA-TTG) and IgA antiendomysial antibodies (IgA-EMA) in the diagnosis of coeliac disease in cohorts from different geographical areas in Europe. The setting allowed a further comparison between the antibody results and the conventional small-intestinal histology. METHODS: A total of 144 cases with coeliac disease [median age 19.5 years (range 0.9-81.4)], and 127 disease controls [median age 29.2 years (range 0.5-79.0)], were recruited, on the basis of biopsy, from 13 centres in nine countries. All biopsy specimens were re-evaluated and classified blindly a second time by two investigators. IgA-TTG were determined by ELISA with human recombinant antigen and IgA-EMA by an immunofluorescence test with human umbilical cord as antigen. RESULTS: The quality of the biopsy specimens was not acceptable in 29 (10.7%) of 271 cases and a reliable judgement could not be made, mainly due to poor orientation of the samples. The primary clinical diagnosis and the second classification of the biopsy specimens were divergent in nine cases, and one patient was initially enrolled in the wrong group. Thus, 126 coeliac patients and 106 controls, verified by biopsy, remained for final analysis. The sensitivity of IgA-TTG was 94% and IgA-EMA 89%, the specificity was 99% and 98%, respectively. CONCLUSIONS: Serum IgA-TTG measurement is effective and at least as good as IgA-EMA in the identification of coeliac disease. Due to a high percentage of poor histological specimens, the diagnosis of coeliac disease should not depend only on biopsy, but in addition the clinical picture and serology should be considered.