Dissertations / Theses on the topic 'Human blood assay'

The oxidation of lipids and antioxidants has been extensively studied in human plasma but little attention has been given to how plasma proteins are oxidised. Proteins make up the majority of biomolecules in cells and plasma and therefore are the most likely reactants with oxidants and free radicals. Previous studies in the laboratory had shown that peroxyl radicals generated by the thermolytic decay of 2-azobis (2-amdinopropane) dihydrochloride (AAPH) generated significant amounts of protein hydroperoxides, but only after a six hour lag period. In this study the existence of the six hour lag period was confirmed and shown by dialysis of the plasma to be due to the presence of low molecular weight antioxidants. The addition of both uric acid and ascorbic acid to the dialysed plasma restored the lag phase suggesting that in vivo these antioxidants act to prevent protein hydroperoxide formation. Lipid oxidation was also observed in the plasma but only after a two hour lag phase. This was the first time lipid oxidation has been observed in the absence of protein oxidation. The lipid lag phase was also abolished by dialysis of the plasma and restored by the addition of ascorbic acid and uric acid. The kinetics of tocopherol loss suggests that the tocopherol radicals act to inhibit lipid oxidation by transferring the electrons to the water-soluble ascorbate. The loss of ascorbate appears to cause the formation of a tocopherol radical mediate the lipid peroxidation process. Overall the data shows ascorbic acid scavenging the peroxyl radicals while uric acid acts to reduce the overall AAPH generated radical flux. In a separate investigation, the production of protein-bound DOPA (PB-DOPA) on albumin during X-ray radiolysis and copper mediate Fenton oxidation was investigated using a fluorescence based derivatisation method (ED-DOPA), which was compared with the more specific acid hydrolysis and HPLC analysis method. The ED-DOPA method consistently gave a much higher reading that the HPLC based methods, suggesting that the ED-DOPA method was measuring DOPA plus DOPA oxidation products. This was confirmed by oxidising X-ray radiolysis generated PB-DOPA with Cu++ to cause DOPA oxidation. The Cu++ treatment drastically increased the level of signal given by the ED-DOPA assay while HPLC analysis showed all the DOPA had been oxidised.

Introduction: Rituximab is a monoclonal antibody used to treat hematological malignancies. The antibody depletes CD20+ B cells via cytotoxic immune mechanisms, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), which is mainly induced by natural killer (NK) cells. Rituximab is mostly well-tolerated but has been reported to induce the release of large amounts of cytokines in blood, thus causing systemic inflammatory response. Aim: To study rituximab-induced B cell depletion and cytokine release in blood from healthy volunteers and how this was affected by Fc modified versions of the antibody. Methods and materials: Fresh blood from healthy donors (n=3) was incubated with rituximab and Fc modified versions that influence the antibody’s target functions, namely ADCC and CDC, for 4 hours in a blood loop system. Results were measured using multicolor flow cytometry, except for cytokine release in plasma which was measured by enzyme-linked immunosorbent assay (ELISA). Results: Of all treatments, rituximab wild type (WT) showed superior B cell depletion than Fc mutant rituximab. The C1q knock-out variant (rituximab-P331S) and the variant with improved affinity to Fc receptor CD16 (rituximab-GASDALIE) did not differ in depletion. A cytokine release was not detected with the treatments, however, a cytokine stimulation in NK cells was observed. Rituximab-GASDALIE had the most prominent cytokine stimulation and CD107a (marker of NK cell functional activity) expression on NK cells. Rituximab-WT and rituximab-P331S had a minor and similar cytokine stimulation and CD107a expression between each other. Rituximab-IgG2 had minimal B cell depletion, CD107a expression and cytokine stimulation. Conclusions: Rituximab depleted B cells without inducing measurable cytokine release for healthy individuals. Among the treatments, Fc mutant rituximab seem to induce less B cell depletion. Moreover, rituximab-GASDALIE appear to elicit an enhanced NK cell activation. Further studies should include more donors as supplement and the results should be interpreted as complementary data to future data analyzed by performing the loop experiment using blood from patients.

This study shows that herbs can be effectively screened for potiential bio-activity using in vitro methods. Further studies will be needed to better explore Artemisia afra&rsquo
s effect on immunoregulation, particularly long term effects of the herb on the immune system and its effect on other disease states.

碩士
國立中興大學
食品科學系
87
ABSTRACT A common method for measurement of MDA (malondialdehyde) levels in human plasma is the TBA (thiobarbituric acid) test. Both the protocols of TBA test and the MDA levels measured as TBARS in human plasma vary widely. The specificity of the TBA reaction has been strongly questioned by various laboratories. Hence, we used the plasma of 16 volunteers to study the correlation between four commonly used methods. Furthermore, we improved the procedure of TBA test and examined the adaptability of the modified procedure. In addition, we investigated the relationship of total MDA, bound MDA and free MDA in human plasma. Th four methods used were: (1) measurement of TBARS of plasma TCA-supernatant (Method A; Radiguez-Martinez and Ruiz-Torres 1992)；(2) measurement of lipid peroxides (Method B; Yagi 1989)；(3) measurement of total MDA (Method C; Chirico et al. 1993)；(4) measurement of MDA in plasma treated with KI prior to TBA reaction (Method D; Chow and Li 1994). The results show that there were positive correlations between TBARS determined by method A and method B (r = 0.740, P<0.02) and by method C and method D (r = 0.516, P<0.05). There was a negative correlation between TBARS by method B and method D (r = 0.548, P<0.05). The results show the need of standardization of the various TBA tests. Following the TBA test protocols of each of the four methods, TBARS (or TBA-MDA adducts) were detected by spectrophotometry, flourescence and HPLC/Vis. The results show that there were significant correlations among the values obtained by the three detectors within each protocol. The TBARS obtained from the four methods with spectrophotometric detection were higher than those detected with flourescence and HPLC/Vis detection. There was a significant correlation (r = 0.889, P<0.002) between TBARS determined by method A with spectrophotometric detection and HPLC/Vis detection. Hence, method A with spectrophotometric detection could be a simple method to determine free MDA in plasma. There also was a positive correlation in lipid peroxides determined by method B between flourescence and HPLC/Vis detection (r = 0.918, P<0.001). There was no significant difference in lipid peroxides obtained from method B by flourescence detection and HPLC/Vis detection (P>0.20). Thus, most materials measured by method B should be TBA-MDA adducts rather than lipid peroxides. In order to improve the TBA test procedure, we investigated the effects of alkaline hydrolysis, types of acids, acid-heating time, BHT (butylated hydroxytoluene) and KI on levels of TBA-MDA adduct in human plasma. Results show that alkaline hydrolysis was a critical step for measurement of total MDA in plasma, because this treatment led to release of MDA from plasma proteins. TCA was a better protein precipitating reagent than H3PO4 since the TBA-MDA adduct of TCA-whole plasma did not increase after 30 min. BHT was used to inhibit the formation of lipid peroxidation during TBA test. The levels of TBA-MDA adduct were decreased 23% by inclusion of 0.2% BHT in plasma. The addition of 1% KI resulted in 43% decrease in the TBA-MDA adduct. Therefore, both BHT and KI were included in the modified procedure. The total MDA obtained from 16 volunteers by the modified procedure and detected by spectrophotometry, flourescence and HPLC/Vis were 1.92±1.04 μM, 1.81±0.92μM and 1.54±0.72μM, respectively. The differences among the three values were not significant (P>0.05). The data shows that TBARS obtained by the modified procedure were essentially TBA-MDA adduct. The recovery (average: 82~106%), precision (within-assay C.V%: 2~4%, between-assay C.V%: 4~8%) and sensitivity of the modified procedure was comparable to other methods detected by HPLC. In addition, the modified procedure with HPLC/Vis detection compared favorably with other indices of lipid peroxidation. Furthermore, we measured total MDA, bound MDA and free MDA of plasma by the modified procedure with HPLC/Vis detection. The results show that total MDA (1.45±0.17μM) was equal to bound MDA (1.34±0.07μM) plus free MDA (0.07±0.02μM). There was no significant correlation between free MDA and bound MDA (r = 0.620, n=6, P>0.05) or between free MDA and total MDA (r = 0.587, n=6, P>0.05). By contrast, bound MDA and total MDA was highly correlated (r = 0.941, n=9, P<0.001). Thus, the results indicate that the measurement of total MDA or bound MDA is a better index of lipid peroxidation than the measurement of free MDA in plasma. In conclusion, the applicability of the modified procedure is good. However, the application of the modified procedure for assessing oxidative stress in various diseases remains to be examined. Keywords Human plasma, MDA (malondialehye), lipid peroxidation, HPLC

The hexavalent Chromium [Cr(VI)] is a human carcinogen, which is still authorized for use in several industrial settings because it has been difficult to replace. This was the reasoning to select it as a priority chemical by the European Human Biomonitoring Initiative (HBM4EU, https://www.hbm4eu.eu/), which aims to bridge chemicals human exposure to their possible impact on health. For that purpose, not only exposure was evaluated, but also early effect biomarkers were done to reflect potential health outcomes in several countries across Europe. In Portugal, the study was developed in one aircraft maintenance company since the substitution of Cr(VI) is not expected in the near future. Following the company agreement and the volunteers informed consent, an individual questionnaire was filled in order to obtain personal information, as well as lifestyle habits and occupational issues. Personal air samples were collected in order to assess occupational exposure to Cr(VI) soluble and insoluble compounds. Sampling for effect biomarkers analyses involved blood samples from 50 workers and 26 healthy individuals (controls). Biomarkers of effect involving the analysis of chromosome alterations (micronucleus assay) and DNA damage (comet assay) were studied; the results were statistically compared. Cr(VI)-exposed workers display a significantly higher frequency of micronucleated binucleated cells (p < 0.001) and an increased level of DNA breaks (comet assay) (p < 0.001) when compared with the non-exposed group. Only in the workplaces dedicated to painting exterior surfaces the values (0.4 mg/m3) were higher than the Occupational Exposure Limit (OEL of 0.010 mg/m3) currently proposed by the Directive (EU) 2019/130, 16/01/2019. The present results suggest a potential health risk for this group of workers given that an association between an increased micronucleus frequency and cancer risk has been shown. Also, these findings should promote the investment in new risk management measures and the effective application of the ones already in place, such as adequate local ventilation and a frequent use of protective equipment.

Yes
The comet assay is a sensitive method used to detect DNA damage, measuring DNA breaks and alkali labile lesions in eukaryotic cells. Here, the use of whole blood in the alkaline gel electrophoresis method is described. Two hundred and seventy blood samples from individuals were examined: 120 healthy individuals, 65 suspected or pre-cancerous individuals and 85 cancer patients. Each sample was divided into two identical volumes in different falcon tubes. The blood was prepared and stored by adding the same amount of RPMI medium and 10% DMSO. Using the Student’s t-Test, the data showed a p value = 0.59 for Olive tail moment (OTM) and 0.16 for % tail DNA, and no statistically significant differences between the two methods, with or without treatment. In conclusion, using whole blood instead of isolated lymphocytes saves time, is still very sensitive and requires less than 20 µL of blood from each individual.

Infectious diseases caused by antibiotic resistant bacteria are difficult to treat using traditional antibiotics and have emerged as a global public health problem (Bartlett, Gilbert, & Spellberg, 2013). An alternative strategy to bypass antibiotic resistance in bacteria is to enhance the host immune system (Hancock, & Sahl, 2006), especially innate immunity (Ajesh, & Sreejith, 2009), in order to combat bacterial infections. To verify the augmentation of innate immunity under certain stimuli, a quantifiable measurement of bacterial killing ability is needed. A new method of using heparinized human peripheral whole blood and mild heated blood samples from the same individuals as comparisons has been developed. This assay was used to quantify the bacterial killing ability of phagocytic cells by measuring the bacterial load after co-culturing with the Gram negative bacteria Pseudomonas aeruginosa. The assay shows that peripheral whole blood of healthy humans is capable of being activated ex vivo upon bacterial challenge and suppressing the bacterial load, while blood gently heated at 48oC for one hour failed to do so. The morphology of the phagocytic cells was examined microscopically. Phagocytosis of bacteria in neutrophils is observed in intact blood, and no such changes were found in heated blood. The level of inflammatory cytokine interleukin 8 (IL-8) was elevated in intact blood after bacteria inoculation, while in the heated blood the cytokine level stayed at baseline, indicating a persistence of leukocyte viability in whole blood and damaged leukocyte activity in gently heated blood. The data show that the human peripheral whole blood assay is suitable as a method for ex vivo bacterial killing quantification with the mildly heated blood sample from the same individual as a comparison. When kept at 37oC ex vivo, human whole blood can be activated upon bacterial challenge, produce inflammatory cytokines, reduce bacterial load, and phagocytes in the whole blood can successfully maintain their cell viability and capacity to phagocytose bacteria.

No
Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. OBJECTIVES: To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses.

BACKGROUND: The human immune system is comprised of various cells that function to fight infection while also avoiding harmful inflammatory and autoimmune responses. The immune system consists of the innate and adaptive immune responses. While the adaptive immune response is involved in the late phase of infection and plays a role in generating classic T and B-cell based immunological memory, the innate immune response is the first line of defense and plays a role in early recognition of foreign substances and induction of an inflammatory response. Although the innate immune response is a natural and inborn response, it varies with age. Human newborns in particular are immunologically distinct due to their polarized T helper type 2 (Th2) cell response and increased anti-inflammatory cytokine production. While these adaptations protect the fetus from rejection by the mother, they lead to increased susceptibility of newborns to infection. While immunization is the most promising strategy to combat this increased susceptibility of newborns, the responses of newborns to different vaccines are impaired as a result of their functionally distinct immune system. For this reason, there have been efforts to develop and incorporate adjuvants into vaccines in order to enhance the immune response of newborns and young infants, who suffer the greatest burden of infectious diseases. OBJECTIVE: The objective of this thesis is to determine which compound(s) in a small molecule library (compound 037 family) that had been identified based on a high throughput TNF-α screen of human primary mononuclear cells activates immune cells and has the potential to act as effective vaccine adjuvants. METHODS: Human cord blood was collected from 7 healthy term newborns after Cesarean section and peripheral blood was collected from the arms of 15 healthy adult volunteers between the ages of 18 and 60. The blood was processed and the compounds of the small molecule library were tested via whole blood assays and enzyme-linked immunosorbent assays (ELISAs) to measure production of pro-inflammatory cytokines, specifically tumor necrosis factor (TNF) and interleukin-1β (IL-1β). The responses of the compounds were further characterized by measuring additional cytokines and chemokines via a 41-plex cytokine multiplex assay. RESULTS: It was determined that at the top two concentrations that were tested (3.7 μM and 11.0 μM), certain compounds of the 037 family induced TNF and IL-1β production comparable to that produced by R848, an already established adjuvant. The compounds with the most activity depended on which cytokine was being produced as well as the age group (newborn vs. adult). Results of the 41-plex cytokine multiplex showed that while there is no clear indication of which group of cytokines are produced after stimulation with these compounds, there was increased production of certain chemokines, especially in adults. CONCLUSIONS: The difference in activity of the compounds in the 037 family suggests that the functional groups might play a role in enhancing activity of certain compounds. The increased production of chemokines after stimulation with these compounds suggests that the compounds might activate pathways different from TLR7/8 but still results in an inflammatory response. More work needs to be done in order to identify the receptors and pathways that these compounds activate.

碩士
國立中興大學
環境工程學系所
101
Both estrone-2,3-quinone (E1-2,3-Q) and estrone-3,4-quinone (E1-3,4-Q) are reactive metabolites of estrogen that are thought to be responsible for the estrogen-induced genotoxicity. The objective of this research is to develop a biochemical assay using estrone (estrone, E1) quinone adducts as biomarker of exposure to assess the cumulative body burden of estrogen-derived quinones in target organs. This method ultilizes trifluoroacetic anhydride (TFAA) as catalysts to cleave cysteinyl adducts of estrogen-derived quinones on proteins. The cleaved adducts are recovered by organic solvent extractions and analyzed by gas chromatography-electron-impact/negative-ion-chemical-ionization-massspectrometer (GC-EI-MS/GC-NICI-MS). Time-course experiments suggested that both E1-2,3-Q-4-S-Alb and E1-3,4-Q-2-S-Alb rapidly reached maximum values at 5 min mark and remained constant thereafter. We applied this methodology to analyze the background levels of estrone quinone-derived adducts on bovine serum albumin (BSA) and human serum albumin (HSA). Results showed that cysteinyl adducts of E1-2,3-Q-4-S-Alb and E1-3,4-Q-2-S-Alb were detected in HSA (n=5) with mean levels at 71.5 and 265 (pmol/g), respectively. Similarily, we detected these adducts in BSA (n=5) with mean levels at 46.4 and 118 pmol/g for E1-2,3-Q-4-S-Alb and E1-3,4-Q-2-S-Alb, respectively. In human MCF-7 breast cancer cells, with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) pretreatment (10 nM for 72 h), production of estrogen quinone-derived protein adducts was detected in cells exposed to E1. Co-treatment of a catechol-o-methyl transferase (COMT) inhibitor further enhanced the production of estrogen quinone-derived adducts (~3 fold). Further investigation indicated that the background levels of these adducts in human albumin (Alb) derived from female breast cancer patients (n=10) were ~2 fold greater than those of healthy controls (n=10). Overall, we concluded that the methodology developed in this study may be applied to epidemiological study to serve as biomarkers of environmental exposure to modulators of estrogen homeostasis.

碩士
國立中正大學
化學暨生物化學研究所
100
Cigarette smoke contains many alkylating agents which damage DNA producing DNA adducts, such as 3-ethyladenine (3-EtA) and 7-ethylguanine (7-EtG). To quantify ethylated adducts on DNA, I used neutral thermal hydrolysis to release 3-EtA and 7-EtG from DNA. The adducts were purified by a reversed-phase solid-phase extraction column and analyzed by stable isotope dilution capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) under the highly selected reaction monitoring (H-SRM) mode. The amount of ethylated DNA adducts represents the steady-state levels of DNA adducts resulting from the ethylating agent after repair in vivo. The detection limit of 3-EtA and 7-EtG was 30.7 and 55.9 amol, respectively. Levels of 3-EtA and 7-EtG are 16.0 ± 7.8 and 9.7 ± 8.3 in 108 normal nucleotides, respectively, in white blood cells (WBC) DNA of 20 smokers, which are statistically significantly higher than those in 20 nonsmokers with 5.4 ± 2.6 3-EtA and 0.3 ± 0.8 7-EtG in 108 normal nucleotides (p < 0.0001). These DNA adducts are repaired in vivo and then released in human urine. The urine samples were purified by a mixed-mode cation exchange followed by a reversed-phase solid-phase extraction column and analyzed by capLC-NSI/MS/MS under the H-SRM. The urinary concentration of 3-EtA and 7-EtG are 120.9 ± 33.5 pg/mL and 61.0 ± 8.5 pg/mL in 5 smokers. The highly sensitive and specific stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically useful in assessing the possibility of measuring ethylpurines in human WBC DNA and in urine as risk biomarkers for smoking-related cancers.

The relevance of preconceptional and prenatal toxicant exposures for genomic stability in offspring is difficult to analyze in human populations, because gestational exposures usually cannot be separated from preconceptional exposures. To analyze the roles of exposures during gestation and conception on genomic stability in the offspring, stability was assessed via the Comet assay and highly sensitive, semiautomated confocal laser scans of gammaH2AX foci in cord, maternal, and paternal blood as well as spermatozoa from 39 families in Crete, Greece, and the United Kingdom. With use of multivariate linear regression analysis with backward selection, preconceptional paternal smoking (% tail DNA: P>0.032; gammaH2AX foci: P>0.018) and gestational maternal (% tail DNA: P>0.033) smoking were found to statistically significantly predict DNA damage in the cord blood of F1 offspring. Maternal passive smoke exposure was not identified as a predictor of DNA damage in cord blood, indicating that the effect of paternal smoking may be transmitted via the spermatozoal genome. Taken together, these studies reveal a role for cigarette smoke in the induction of DNA alterations in human F1 offspring via exposures of the fetus in utero or the paternal germline. Moreover, the identification of transgenerational DNA alterations in the unexposed F1 offspring of smoking-exposed fathers supports the claim that cigarette smoke is a human germ cell mutagen.

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The aim of the study was to use in vitro human whole blood cultures to screen the water samples collected from the Eerste/Plankenbrug river system for cytotoxicity and inflammatory activity and for the first time investigate the impact on the cell- mediated and humoral immune pathways. Water samples were collected fronm the sites during the dry summer season and rainy winter season. Blood was collected from the healthy male volunteers and diluted with RPMI 1640. For cytotoxicity and inflammatory activity 2.5ul of blood for 18-20 hrs at 37 C... This study shows that waster from the Plankenbrug River is heavily polluted by contaminants from both the agricultural area and informal settlement of Kayamandi. These contaminants can be potentially immunotoxic during the summer season and they can result in inflammatory diarrheal disease and immunosuppression in exposed individuals..."

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2- yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 +/- 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.