Kidney transplants from both living-related (LRD) and living unrelated (LURD) donors have superior function and survival than transplants from cadaver donors. This may be unsurprising as kidneys from living donors are procured under optimal conditions, from healthy donors with minimal ischaemia times. In contrast, cadaver kidneys are obtained from traumatised donors and may experience extended periods of cold ischaemic storage before transplantation. An immunohistochemical analysis has been performed on biopsies obtained before, and immediately after transplantation, to investigate the potential causes of early inflammatory events associated with cadaver renal transplantation that may influence subsequent graft outcome. An immunohistochemical analysis of biopsies obtained before transplantation demonstrated upregulated expression of endothelial E-selectin and proximal tubular expression of ICAM-1, VCAM-1 and HLA Class II antigens in cadaver donor kidneys. Analysis of donor parameters demonstrated that traumatic physiological events experienced in intensive care around the time of brain death were significantly associated with the induction of proinflammatory antigens. Antigen induction in cadaver donor kidneys before transplantation was significantly associated with early acute rejection. Furthermore, in cadaveric kidneys with long cold ischaemia times, glomerular neutrophil infiltration and deposition of activated platelets expressing P-selectin on intertubular capillaries were detected following reperfusion, in association with impaired short and long term graft function. Expression of inflammatory mediators were absent in all LRD renal allografts before and after reperfusion. A clinical trial was performed to determine whether ischaemia/reperfusion injury may be ameliorated by reflushing cadaver kidneys after cold storage to remove harmful products that may have accumulated in the vessel lumen. Reflushing did not prevent the inflammatory events observed after reperfusion or improve graft function. Therefore, a novel, oxygen free radical scavenger (lec-SOD) was obtained to assess its potential efficacy in preventing ischaemia/reperfusion injury. Lec-SOD bound with high affinity to macro- and microvascular endothelial cells under cold hypoxic conditions following incorporation into Marshall's preservation solution, significantly inhibiting cold hypoxia induced cell death, adhesion molecule induction and neutrophil adhesion. Furthermore, preservation of kidneys with lec- SOD for 18 hr in an experimental model of chronic renal allograft rejection, significantly attenuated neutrophil infiltration and MHC Class I induction day 1 post-transplant, with improved long term renal function. The results presented in this Thesis demonstrate that donor factors and cold ischaemia/ reperfusion injury elicit an early inflammatory response that may influence graft outcome of cadaver kidneys. Refinements in donor management and organ preservation may limit the deleterious effects of ischaemia/reperfusion injury in cadaver renal allografts, increasing graft survival to that observed in living donor transplantation.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Griffin, Paul; Committee Member: Butera, Robert; Committee Member: Halpern, Michael; Committee Member: Nichols, Richard; Committee Member: Vidakovic, Brani. Part of the SMARTech Electronic Thesis and Dissertation Collection.

In order to study the incidence of Estrogen Receptors (ER) in breast carcinoma, lung carcinoma and melanoma, an in situ hybridisation technique for ER mRNA (ER mRNA-ISH) was developed. Various technical aspects of the procedure including tissue fixation, hybridisation conditions, and demonstration technique were investigated in order to obtain an optimum technique for routine use. ISH results were compared with ER immunohistochemistry using the monoclonal antibodies ER1D5 and D5. Commercially available biotin labelled antisense oligonucleotides to ER, Poly A (total mRNA), and sense chromogranin (negative control) were applied to frozen and formalin-fixed paraffin sections of breast carcinomas. For frozen sections, various fixatives including formalin, alcohol, Schoobridge, Zamboni's and acetic- alcohol were compared. A direct streptavidin- eroxidase and an indirect demonstration method using anti-biotin were also compared. The effect of differing formamide concentrations and post hybridisation stringency washings were analysed. An optimised ISH technique was then applied to frozen sections of 21 cases of breast carcinoma and 11 cases of lung carcinoma. Results were compared to H222 staining on adjacent sections. The ISH technique was also optimised for use on formalin-fixed, paraffin-embedded sections of 28 breast carcinomas and 17 melanomas. The results were compared with ER1D5 and D5 immunohistochemistry done on adjacent sections. The occurrence of endogenous biotin was also studied on a range of normal tissues. Consistent ISH results were obtained when formamide was omitted from the hybridisation cocktail, high stringency post hybridisation washes were discarded, room temperature hybridisations and an indirect demonstration method were used. Fixation of frozen sections in acetic/ethanol gave more consistent results with good morphology and resulted in positive nucleolar staining in 90% of breast and 45% of lung carcinomas. Positive nucleolar staining was also present in frozen sections of one metastatic melanoma. In formalin fixed paraffin sections, acid hydrolysis and pronase treatment were required prior to ISH. Cytoplasmic and/or nucleolar ER mRNA-ISH staining was seen in 87% of breast carcinoma and 97% of melanoma studied. ER1D5 was present in 54% of breast carcinomas but was absent in all melanomas. D5, on the other hand, was found in 88% of the melanomas. In conclusion, ER mRNA-ISH can be successfully done on acetic/alcohol fixed frozen sections and formalin fixed paraffin sections. Formamide, high stringency washes and elevated hybridisation temperatures are detrimental to a successful ISH reaction and an indirect demonstration method (using anti-biotin) is preferred. Unfortunately, endogenous biotin can cause false positive ISH reactions and needs to be considered during interpretation. Results show that the localisation of ER mRNA in the nucleolus is specific. Both ER mRNA-ISH and ER immunohistochemistry indicate that melanomas and some lung carcinomas contain a receptor possibly similar to that in breast carcinomas. / Thesis (M.Sc.)--Department of Anatomical Sciences, 2004.

In order to study the incidence of Estrogen Receptors (ER) in breast carcinoma, lung carcinoma and melanoma, an in situ hybridisation technique for ER mRNA (ER mRNA-ISH) was developed. Various technical aspects of the procedure including tissue fixation, hybridisation conditions, and demonstration technique were investigated in order to obtain an optimum technique for routine use. ISH results were compared with ER immunohistochemistry using the monoclonal antibodies ER1D5 and D5. Commercially available biotin labelled antisense oligonucleotides to ER, Poly A (total mRNA), and sense chromogranin (negative control) were applied to frozen and formalin-fixed paraffin sections of breast carcinomas. For frozen sections, various fixatives including formalin, alcohol, Schoobridge, Zamboni's and acetic- alcohol were compared. A direct streptavidin- eroxidase and an indirect demonstration method using anti-biotin were also compared. The effect of differing formamide concentrations and post hybridisation stringency washings were analysed. An optimised ISH technique was then applied to frozen sections of 21 cases of breast carcinoma and 11 cases of lung carcinoma. Results were compared to H222 staining on adjacent sections. The ISH technique was also optimised for use on formalin-fixed, paraffin-embedded sections of 28 breast carcinomas and 17 melanomas. The results were compared with ER1D5 and D5 immunohistochemistry done on adjacent sections. The occurrence of endogenous biotin was also studied on a range of normal tissues. Consistent ISH results were obtained when formamide was omitted from the hybridisation cocktail, high stringency post hybridisation washes were discarded, room temperature hybridisations and an indirect demonstration method were used. Fixation of frozen sections in acetic/ethanol gave more consistent results with good morphology and resulted in positive nucleolar staining in 90% of breast and 45% of lung carcinomas. Positive nucleolar staining was also present in frozen sections of one metastatic melanoma. In formalin fixed paraffin sections, acid hydrolysis and pronase treatment were required prior to ISH. Cytoplasmic and/or nucleolar ER mRNA-ISH staining was seen in 87% of breast carcinoma and 97% of melanoma studied. ER1D5 was present in 54% of breast carcinomas but was absent in all melanomas. D5, on the other hand, was found in 88% of the melanomas. In conclusion, ER mRNA-ISH can be successfully done on acetic/alcohol fixed frozen sections and formalin fixed paraffin sections. Formamide, high stringency washes and elevated hybridisation temperatures are detrimental to a successful ISH reaction and an indirect demonstration method (using anti-biotin) is preferred. Unfortunately, endogenous biotin can cause false positive ISH reactions and needs to be considered during interpretation. Results show that the localisation of ER mRNA in the nucleolus is specific. Both ER mRNA-ISH and ER immunohistochemistry indicate that melanomas and some lung carcinomas contain a receptor possibly similar to that in breast carcinomas. / Thesis (M.Sc.)--Department of Anatomical Sciences, 2004.

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Description based on contents viewed Oct. 3, 2008; title from PDF t.p. Includes bibliographical references (p. 64-67).

HER2 breast cancer is an aggressive disease that occurs in 20 - 30% of the breast cancer population. Treatment for HER2 breast cancer includes use of an anti-HER2 monoclonal antibody, trastuzumab. Testing for HER2 is of critical importance due to the adverse side effects and substantial costs associated with this anti-HER2 treatment. Currently, two kinds of tests, Fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC), are FDA approved for determination of HER2 status in breast cancers. Clinical and non clinical factors that affect the choice HER2 test and the use of anti-HER2 therapy in breast cancer were analyzed using a data set containing information from six outpatient oncology clinics in the United States. The analysis showed that geographic location, cancer stage, and diagnosis date (pre- or post-publication of testing guidelines) have significant effects on choice of test. With regard to trastuzumab prescription, geographic location and HER2 status have significant effects on the prescription of trastuzumab. In addition, there was a non-significant trend for certain Medicare patients not to receive trastuzumab therapy. These findings indicate that disparities are present in breast cancer care based on geography and cancer stage, and highlight the importance of testing guidelines. The cost effectiveness of FISH vs. IHC was determined, by considering the financial and health-related costs associated with testing and subsequent treatment as well as the accuracy of each test. The results show that FISH is the optimal choice for HER2 testing and is more cost-effective than IHC.