Type 1 diabetes (T1D) is an autoimmune disease whereby the insulin-producing β-cells of the pancreas are destroyed by the immune system, possibly related to an inappropriate immune reaction to dietary antigens and/or microbes in the gut. We previously observed a deficit in gut-resident CD163+ M2 anti-inflammatory macrophages in BioBreeding diabetes-prone (BBdp) rats. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of the CD163 pathway and through the breakdown of toxic heme releases potent antioxidants. We hypothesized that the treatment of animals with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, would inhibit development of T1D through modulation of the CD163/HO-1 pathway and increase M2 macrophages. HO-1 expression was significantly increased in the pancreas and gut. T1D incidence was inhibited in CoPP-treated rats and these animals showed an unexpected increase in cells expressing CD68 (an M1 pro-inflammatory macrophage marker) in the pancreas and gut. CoPP induced the expression of cathelicidin anti-microbial peptide (CAMP) in the jejunum, which co-localized with CD163+ (M2) macrophages. KLF4, an M2 macrophage-specific transcription factor, was significantly upregulated in the pancreas and jejunum of CoPP-treated animals and co-localized with CD68 and HO-1 in the pancreas. We conclude that HO-1 induction prevented T1D through modulation of the gut immune system and potential recruitment of a unique population of anti-inflammatory M2 macrophages in the gut and pancreas

Resumo: O presente trabalho teve como objetivo desenvolver tecnologias para o uso de plantas como unidades de produção ou diretamente como fontes de agentes fotossensibilizantes do Ácido 5-Aminolevulinato (5-ALA), Protoporfirina IX (Proto IX) e precursores destes compostos em plantas. Os experimentos foram conduzidos no Núcleo de Pesquisas Avançadas em Matologia (NUPAM) - FCA - UNESP - Botucatu/SP, durante o ano de 2007. Foram realizados ensaios para seleção do melhor conjunto de condições para o acúmulo de protoporfirina IX e seus precursores em alface, milho e cana-de-açúcar, cujas variáveis analisadas foram: 1) seleção de comprimentos de onda; 2) duração dos períodos com e sem luz; 3) variedades (11 genótipos); 4) aplicação de antioxidantes (vitaminas C e E); 5) compostos que interferem na síntese de Proto IX (oxyfluorfen, carfentrazone e ácido levulênico); 6) adição de precursores da síntese de Proto IX (glutamato); 7) substituição da atmosfera com supressão do Oxigênio. Os resultados foram analisados sobre a dispersão das médias dos tratamentos em relação às parcelas testemunhas para os tratamentos e espécies estudadas. Utilizou-se a técnica de HPLC-MS para identificação e quantificação dos compostos. A partir dos resultados dos ensaios realizados foram selecionados três experimentos: I) Acúmulo de Proto IX e 5-ALA a partir de inibidores da protoporfirinogênio oxidase (Protox) e fontes seletivas de luz (Escuro, Sombrite 75%, Claro+Vitamina C+E, Claro, Filtro Azul, Filtro vermelho, Filtro Amarelo, Filtro Verde, Testemunha) em plantas de alface, pulverizadas com Oxyfluorfen + Glutamato monossódico + Vitamina C e E, portanto 9 tratamentos com 4 repetições. II) Seleção de genótipos de cana-de-açúcar (Saccharum officinarum) para acúmulo de Proto IX com uso de herbicidas inibidores da Protox (Oxyfluorfen e Carfentrazone)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work had as objective to develop technologies for the use of plants about units of production or directly as sources of agents photosensitizers, Acid 5- Aminolevulinate (5-ALA), Protoporphyrin IX (Proto IX) and precursors this compounds in plants. The experiments were conducted at the Center for Advanced Research in Weed Plant (NUPAM) - FCA - UNESP - Botucatu/SP, during the year 2007. Tests were conducted to select the best set of conditions for the accumulation of protoporphyrin IX and precursors in lettuce, corn and sugar cane, whose variables were analyzed: 1) selection of a wavelength, 2) duration of periods with and without light, 3) varieties (11 genotypes), 4) application of antioxidants to reduce the action of Proto IX (vitamins C and E), 5) compounds that interfere with the synthesis of Proto IX (oxyfluorfen, carfentrazone and levulênic acid), 6) addition of precursors for synthesis of Proto IX (glutamate), 7) Replacement of the atmosphere with suppression of the Oxygen. The results were analyzed on the dispersion of the averages of the treatments in relation to the portions witness for the treatments and studied species. It was used technique of HPLC-MS for identification and quantification of compounds. From the results of tests conducted three experiments were selected: I) Accumulation of Proto IX and 5- ALA from protoporphyrinogen oxidase (Protox) inhibitor selective and sources of light (Dark, Shade 75%, Light + Vitamin C + E, Light, Blue filter, Red filter, Yellow filter, Green filter, Witness) in lettuce plants, sprayed with Monosodium Glutamate + Oxyfluorfen + Vitamin C and E, so 9 treatments with 4 repetitions. II) Selection of genotypes of sugarcane for accumulation of Proto IX with the use of herbicides Protox inhibitors (Oxyfluorfen and Carfentrazone), precursors of Proto IX (Glutamate and Levulenic Acid) and antioxidants (vitamin C and E) in 8 genotypes... (Complete abstract click electronic access below) / Orientador: Edivaldo Domingues Velini / Coorientador: Fernando Gustavo Tonin / Coorientador: Maria Lúcia Bueno Trindade / Banca: Ana Catarina Catâneo / Banca:Robinson Antônio Pitelli / Mestre

A protoporfirina IX (PpIX) ocupa um lugar especial entre a família das porfirinas devido ao seu importante papel na natureza e diversas aplicações, inclusive em terapia fotodinâmica. Sob ação da luz visível, a PpIX transforma-se em fotoprodutos que podem ser citotóxicos ou fotocitotóxicos, e isto pode, de um lado, aumentar a eficiência do tratamento e, por outro lado, apresentar toxicidade no escuro, prejudicando o paciente. Como as características da sua fototransformação dependem do ambiente onde elas se encontram, tornam-se importantes os estudos dos efeitos do ambiente nas características da sua fototransformação. As propriedades espectroscópicas da PpIX sintética e endógena, extraída das glândulas harderianas de ratos da espécie Rattus novergicus albinus, e seus fotoprodutos e a atividade citotóxica foram estudadas no escuro e sob ação da luz visível em uma cultura de células malignas, em comparação com Photogem® e a porfirina TPPS4. A localização das protoporfirinas e seus fotoprodutos dentro da estrutura celular também foram observadas. Foi notado que existe uma diferença entre os espectros de absorção e fluorescência das PpIX endógena e sintética e também de seus fotoprodutos, e do grau de sua fototransformação. Associou-se estes efeitos à diferença do ambiente onde as porfirinas se encontravam, devido a interação da PpIX endógena e de seu fotoproduto com compostos celulares que poderiam estar presentes depois da purificação da PpIX obtida da glândula. A fotocitotoxicidade das PpIX sintética e endógena foram comparáveis. Os fotoprodutos, tanto da PpIX sintética quanto endógena não apresentaram fotocitotoxicidade ou mostraram fotocitotoxicidade menor do que a das porfirinas, resultado contrário ao encontrado na literatura. Em relação ao padrão Photogem®, as protoporfirinas apresentaram uma fotocitotoxicidade comparável a ele, mas uma citotoxicidade no escuro maior. A TPPS4 demonstrou tanto uma fototoxicidade, como uma citotoxicidade no escuro menor que o Photogem®. Da análise da distribuição intracelular das porfirinas PpIX e seus fotoprodutos com a de marcadores padrões de estruturas celulares, pode ser concluído que eles não penetraram no núcleo da célula, e sua distribuição nas demais partes do citoplasma foi difusa, sem nenhuma localização preferencial. / The protoporphyrin IX (PpIX) occupies a special place among the porphyrins family due their important role in nature and several applications, including Photodynamic Therapy. Under the action of visible light, PpIX becomes photoproducts that can be cytotoxic or photocytotoxic and these effects can, on one hand, increase the treatment efficiency and, on the other hand, be toxic in the dark, harming the patient. Since the characteristics of its phototransformation depend on the environment where they are finding, studies of the effects of environment on the characteristics of its phototransformation are important. The spectroscopic properties and cytotoxic activity were studied in the dark and under visible light action of synthetic and endogenous PpIX, extracted from mice harderian gland of the Rattus novergicus albinus species and its photoproducts, in a culture of malignant cells, compared with Photogem® and TPPS4 porphyrin. The cellular localization of protoporphyrin and its photoproducts inside cellular structure also were observed. It was observed that there is a difference between the fluorescence and absorption spectra of synthetic and endogenous PpIX and between its photoproducts and also the degree of its phototransformation. These effects can be linked to the difference of the environment where the porphyrins were, due the interaction of endogenous PpIX and its photoproducts with cellular components that could be present after purification of PpIX obtained from the gland. The photocytotoxic of synthetic and endogenous PpIX were comparable. The photoproducts of both synthetic and endogenous PpIX showed no photocytotoxic or were smaller than of porphyrins, a contrary result to that it was found in the literature. In relation to pattern composite Photogem®, the protoporphyrin showed a photocytotoxic comparable to it, but protoporphyrin a higher cytotoxicity in the dark. The TPPS4 demonstrated both a lower phototoxicity as a cytotoxicity in the dark than Photogem®. From analysis of intracellular distribution of the porphyrin PpIX and its photoproducts with markers patterns of cellular structures, may be concluded that they did not penetrate into the cell nucleus and its distribution in other parts of the cytoplasm was diffuse, without preferential location.

A cor vermelha brilhante característica do presunto Parma é resultante, principalmente, do pigmento Zinco-protoporfirina IX (ZnPP). A ZnPP é formada a partir da mioglobina por uma reação de transmetalação, catalisada pela enzima ferroquelatase (FECH), em que o íon de Fe(II) coordenado ao grupo heme é substituído pelo íon Zn(II). O presunto Parma apresenta uma maior estabilidade oxidativa em relação aos demais produtos cárneos curados além de não conter nitrito e nitrato, portanto, são considerados mais saudáveis. A utilização da FECH no processamento de carnes curadas pode permitir a produção de produtos cárneos curados mais saudáveis e em menor tempo. No presente trabalho a proteína ferroquelatase de Bacillus subtilis (BsFECH) foi expressa em células de E. coli BL21(DE3), purificada por cromatografia de afinidade ao níquel e exclusão por tamanho e caracterizada por dicroísmo circular, emissão de fluorescência do triptofano e cromatografia de exclusão por tamanho analítico. Em termos de estabilidade foi encontrado que altas concentrações de sal aumentam a estabilidade da proteína frente aos agentes denaturantes ureia e temperatura. A BsFECH produzida é capaz de ligar-se ao substrato modelo de porfirina (TPPS), conforme verificado por espectroscopia de UV-Vis, com uma Ka = 3,8x105 M-1 e é capaz de se associar à metamioglobina, conforme verificado por reação de cross-linking com dissuccinimidil suberato e avaliado por SDS-PAGE. A BsFECH aumenta significativamente a taxa de inserção de íons de zinco na TPPS e mostra uma cinética de saturação com uma constante de ligação aparente de Zn(II) ao complexo [BsFECH-TPPS] de 1,3x104 M e uma constante de primeira ordem de 6,6x10-1 h-1 para a dissociação do complexo ternário. A reação de troca ferro/zinco na mioglobina catalisada pela BsFECH é facilitada pela proteólise limitada da mioglobina com pepsina que abre um caminho para a reação de troca metálica com base na interação proteína-proteína entre o fragmento globina da mioglobina e a BsFECH. / The bright red color, characteristic of the Parma ham, results mainly of the pigment Zinc-Protoporphyrin IX (ZnPP). The ZnPP is formed from myoglobin by the reaction, catalyzed by ferrochelatase enzyme (FECH), in which Fe(II) ions coordinated to the heme group is replaced by Zn(II) ions. Parma ham shows greater oxidative stability when compared to others cured meat products besides do not contain nitrite and nitrate and, therefore, is considered healthier. The use of FECH in the processing of cured meats may allow the production of healthier cured meat products in a shorter period of time. In this work, the ferrochelatase protein from Bacillus subtilis was expressed in E. coli BL21(DE3) cells, purified by nickel affinity chromatography and size exclusion, and characterized by circular dichroism, fluorescence emission of tryptophan and analytical size exclusion chromatography. In terms of stability, it was found that the high salt content enhances the protein stability against the denaturation agents urea and temperature. The BsFECH produced is able to bind to the porphyrin model substrate (TPPS), as verified by UV-Vis spectroscopy, with Ka = 3.8x105 M-1 and is capable to associate to metamyoglobin as verified by cross-linking reaction with dissuccinimidil suberato, as observed by SDS-PAGE. The BsFECH increases, significantly, the zinc ions insertion rate in TPPS and shows a saturation kinetics behavior with an apparent biding constant of Zn(II) to the [BsFECH-TPPS] complex of 1.3x104 M and a first order rate constant for the dissociation of ternary complex of 6.6x10-1 h-1. The Fe/Zn exchange reaction in the myoglobin as catalyzed by BsFECH is facilitated by myoglobin-limited proteolysis with pepsin that opens a reaction channel for the metallic exchange based on protein-protein interaction between the globin moiety of myoglobin and BsFECH.

Type 1 diabetes (T1D) is an autoimmune disease whereby the insulin-producing β-cells of the pancreas are destroyed by the immune system, possibly related to an inappropriate immune reaction to dietary antigens and/or microbes in the gut. We previously observed a deficit in gut-resident CD163+ M2 anti-inflammatory macrophages in BioBreeding diabetes-prone (BBdp) rats. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of the CD163 pathway and through the breakdown of toxic heme releases potent antioxidants. We hypothesized that the treatment of animals with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, would inhibit development of T1D through modulation of the CD163/HO-1 pathway and increase M2 macrophages. HO-1 expression was significantly increased in the pancreas and gut. T1D incidence was inhibited in CoPP-treated rats and these animals showed an unexpected increase in cells expressing CD68 (an M1 pro-inflammatory macrophage marker) in the pancreas and gut. CoPP induced the expression of cathelicidin anti-microbial peptide (CAMP) in the jejunum, which co-localized with CD163+ (M2) macrophages. KLF4, an M2 macrophage-specific transcription factor, was significantly upregulated in the pancreas and jejunum of CoPP-treated animals and co-localized with CD68 and HO-1 in the pancreas. We conclude that HO-1 induction prevented T1D through modulation of the gut immune system and potential recruitment of a unique population of anti-inflammatory M2 macrophages in the gut and pancreas

Die Hämoxygenase 1 (HO-1) ist ein Stressprotein mit antientzündlichen, immunsupprimierenden und zytoprotektiven Eigenschaften, welche in vielen Tiermodellen nachgewiesen wurden. Die zugrunde liegenden Mechanismen sind wenig bekannt. Diese Arbeit demonstriert erstmalig, dass die physiologische Induktion von HO-1 wichtig für die Limitierung von T-Zell-abhängigen Hautentzündungen ist. So führt der HO-1-Inhibitor, Zinn-Protoporphyrin IX (Sn-PPIX), zu einer verstärkten Hautentzündung im Mausmodell. Die pharmakologische Induktion von HO-1 durch Kobalt-Protoporphyrin IX, Co-PPIX, hemmt dagegen die Entzündung in DNFB- bzw. TMA-induzierten murinen Kontaktallergiemodellen sowohl bei Verabreichung von Co-PPIX während der Sensibilisierung als auch vor der Auslösung. Bemerkenswerterweise hemmt eine Co-PPIX-Behandlung die Antigen-induzierte T-Zellproliferation ex vivo in Milzzellen von behandelten Mäusen und in vitro in humanen mononukleären Zellen des peripheren Blutes. Da eine HO-1-Induktion durch Co-PPIX nur in Monozyten und in aus Monozyten abgeleiteten myloischen Dendritischen Zellen (MDDC), nicht aber in T-Zellen, beobachtet wurde, fokussierten alle weiteren Untersuchungen auf Antigen-präsentierende Zellen. HO-1-Induktion durch Co-PPIX reduziert die Expression von MHC-Klasse II und akzessorischen Molekülen und steigert die Phagozytose und den oxidativen Burst von Monozyten. Die immunphänotypische Differenzierung und Maturierung von MDDC wird gehemmt. Funktionsteste zeigen eine Reduktion der Expression und Sekretion von proinflammatorischen und immunstimulatorischen Zytokinen, während die Sekretion des antientzündlichen Zytokins IL-10 gesteigert ist. Die Fähigkeit der MDDC zur Antigenpräsentation gegenüber T-Helferzellen ist für Allo- und Recallantigene stark herabgesetzt. Mittels adenoviraler HO-1-Transduktion von MDDC konnte die Spezifität der Effekte bestätigt werden. Diese Daten zeigen, dass eine verstärkte HO-1-Aktivität die Dendritischen Zellen zu einem unreifen und immunkompromittierten Phänotyp verändert und weisen darauf hin, dass die HO-1-Induktion einen wichtigen Ansatz für die Hemmung der zellulären Immunität und für die Behandlung von T-Zell-abhängigen Hautentzündungen darstellt. / Heme oxygenase 1 (HO-1) is an antiinflammatory stress protein. Its immunosuppressive and cytoprotective activities have been demonstrated in several animal models. The underlying mechanisms, however, are poorly understood. This study demonstrates for the first time that the physiological induction of HO-1 is important for the limitation and resolution of T cell-dependent skin inflammation. So, the HO-1 inhibitor, tin protoporphyrin IX (Sn-PPIX), augments cutaneous inflammation in mouse model. Moreover, pharmacologic HO-1 induction by the potent HO-1 inducer, cobaltic protoporphyrin IX (Co-PPIX), inhibits inflammation when applied around sensitization or before challenge in murine DNFB- and TMA-induced contact hypersensitivity models. Remarkably, Co-PPIX treatment inhibits antigen-driven T cell proliferation both ex vivo in murine splenocytes and in vitro in human peripheral blood mononuclear cells. Since induction of HO-1 mRNA and protein was found in monocytes and monocyte-derived myeloid dendritic cells (MDDC) but not T cells, further investigations focused on antigen-presenting cells. HO-1 induction by Co-PPIX depresses monocytic MHC class II and accessory molecule expression whereas phagocytosis and respiratory burst activities are augmented. Moreover, HO-1 induction inhibits the immunophenotypic differentiation and maturation of MDDC. Functional analysis revealed a decreased proinflammatory cytokine production whereas secretion of the antiinflammatory cytokine IL-10 is increased. Remarkably, the antigen-presenting capacity of MDDC for T-helper cells is diminished both for allo- and for recall-antigens. Adenoviral HO-1 transduction of MDDC confirmed that the effects are mediated by HO-1. These data indicate that an enhanced HO-1 activity switches myeloid DCs to an immature and functionally compromised phenotype and suggest that HO-1 induction represents an important approach for depressing T cell immunity and for the treatment of T cell-dependent skin inflammation.

Kolorektale Karzinome weisen eine Rotfluoreszenz auf. Die Art des zugrunde liegenden Fluorophors und sein diagnostisches Potential waren Gegenstand der Arbeit. Protoporphyrin IX (PpIX) wurde als das prädominant vorkommende Fluororphor in Primärtumoren und ihren Metastasen identifiziert. Das Fluorophor wurde in Abwesenheit von Nekrose und in sterilen Lokalisationen nachgewiesen. Affymetrix-GeneChip und quantitative PCR untersuchungen der Enzyme der Häm-Synthese machen eine Minderexpression als Ursache der PpIX Akkumulation wahrscheinlich. In Nicht-vorbehandelten Fällen erlaubt die PpIX-Fluoreszenz eine Diskrimination der metastatisch befallenen Lymphknoten mit einer Sensitivität von 62% bei einer Spezifität von 78% (p / Colorectal cancers exhibit a red fluorescence. The nature of the responsible fluorophore and its eventual diagnostic potential were investigated. Protoporphyrin IX (PpIX) was identified as the predominant fluorophore in primary tumors and their metastases. The fluorophore occurred in the absence of necrosis and in sterile locations. Affymetrix-GeneChip and quantitative PCR investigations of the heme metabolic pathway enzymes suggest a reduced expression of the enzyme ferrochelatase to cause the PpIX accumulation. In untreated cases, PpIX fluorescence discriminates metastatically involved lymph nodes from all other palpable nodes with a sensitivity of 62% at a specificity of 78% (p

Background The potential dire consequences of iron deficiency (ID) and iron deficiency anaemia (IDA) on childhood development are of major public health concern. Many factors contribute to anaemia, ID being only one progressive factor. The prevalence of ID and IDA must be accurately determined before iron intervention strategies can be safely prescribed. There is continued uncertainty regarding the optimal approach to identifying and measuring ID, as indicators have different roles, explore different aspects of iron metabolism and cannot be directly compared. Furthermore, inflammation and infection have a confounding effect on the commonly applied indicator and acute phase reactant, serum ferritin (SF). In the public health setting, a suitable method to assess iron status in developing countries has to be inexpensive, standardised, established, easy to measure and its applications specific to identifying ID. Aim We conducted secondary analysis of screening data from three independent iron intervention studies to critically evaluate the indicators used to determine iron status in 6-11-year-old primary school children from three South African provinces. Study design and methods A cross-sectional descriptive analysis was performed on the screening data collected in 2009 and 2010 during iron intervention studies in KwaZulu-Natal (n=736), Northern Cape (n= 1045), and North West (n=546). The three distinct study sites were analysed independently and collectively. Children’s haemoglobin (Hb), SF, transferrin receptor (TfR), zinc protoporphyrin (ZPP), and C-reactive protein (CRP) concentrations were measured and body iron calculated. ID prevalence was compared using different methods (namely the single indicators SF, TfR and ZPP, body iron and the multiple criteria model), and the influence of inflammation on SF was considered. Literature suggests that the multiple criteria model provides a more complete assessment of iron status. The performance of single and body iron indicators were compared to the multiple criteria model (by assessing sensitivity, specificity and predictive values). Results Significant positive correlations between CRP (indicator of inflammation) and SF existed in all study sites and the combined sample (p < 0.01). The mean SF concentration was substantially higher in subjects with inflammation than those without. A different SF cut-off to identify ID was applied to subjects with inflammation. The percentage of ID subjects varied using different indicators (4.2 – 26.5% in KwaZulu-Natal; 4.1 – 13.4% in Northern Cape; 7.0 – 24.4% in North West; and 5.4 – 15.2% in the combined sample). The sensitivity, specificity and predictive values of alternate ID indicators varied within and between study sites, compared to the multiple criteria model. Conclusion Simply using Hb as an ID indicator is inaccurate. The vast differences between percentages identified as ID by different indicators is reason for concern. No consistent agreement appeared between single ID indicators, body iron and the multiple criteria model for ID identification after correcting for inflammation in primary school children. The global view of the multiple criteria model as the gold standard for estimating ID is debatable and potentially impractical at a public health level. Current evidence cautions against overestimating the prevalence of ID, as there is more associated harm than deficiency underestimation. This critical analysis has confirmed a need for research to identify a suitable, accurate and precise alternative to Hb as a tool in the South African public health setting. Furthermore, the impact of inflammation on iron status indicators, in particular SF, should be assessed in context to clearly set parameters for its use in nationally-representative nutrition surveys, the cornerstone of iron intervention strategies. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2015

Background The potential dire consequences of iron deficiency (ID) and iron deficiency anaemia (IDA) on childhood development are of major public health concern. Many factors contribute to anaemia, ID being only one progressive factor. The prevalence of ID and IDA must be accurately determined before iron intervention strategies can be safely prescribed. There is continued uncertainty regarding the optimal approach to identifying and measuring ID, as indicators have different roles, explore different aspects of iron metabolism and cannot be directly compared. Furthermore, inflammation and infection have a confounding effect on the commonly applied indicator and acute phase reactant, serum ferritin (SF). In the public health setting, a suitable method to assess iron status in developing countries has to be inexpensive, standardised, established, easy to measure and its applications specific to identifying ID. Aim We conducted secondary analysis of screening data from three independent iron intervention studies to critically evaluate the indicators used to determine iron status in 6-11-year-old primary school children from three South African provinces. Study design and methods A cross-sectional descriptive analysis was performed on the screening data collected in 2009 and 2010 during iron intervention studies in KwaZulu-Natal (n=736), Northern Cape (n= 1045), and North West (n=546). The three distinct study sites were analysed independently and collectively. Children’s haemoglobin (Hb), SF, transferrin receptor (TfR), zinc protoporphyrin (ZPP), and C-reactive protein (CRP) concentrations were measured and body iron calculated. ID prevalence was compared using different methods (namely the single indicators SF, TfR and ZPP, body iron and the multiple criteria model), and the influence of inflammation on SF was considered. Literature suggests that the multiple criteria model provides a more complete assessment of iron status. The performance of single and body iron indicators were compared to the multiple criteria model (by assessing sensitivity, specificity and predictive values). Results Significant positive correlations between CRP (indicator of inflammation) and SF existed in all study sites and the combined sample (p < 0.01). The mean SF concentration was substantially higher in subjects with inflammation than those without. A different SF cut-off to identify ID was applied to subjects with inflammation. The percentage of ID subjects varied using different indicators (4.2 – 26.5% in KwaZulu-Natal; 4.1 – 13.4% in Northern Cape; 7.0 – 24.4% in North West; and 5.4 – 15.2% in the combined sample). The sensitivity, specificity and predictive values of alternate ID indicators varied within and between study sites, compared to the multiple criteria model. Conclusion Simply using Hb as an ID indicator is inaccurate. The vast differences between percentages identified as ID by different indicators is reason for concern. No consistent agreement appeared between single ID indicators, body iron and the multiple criteria model for ID identification after correcting for inflammation in primary school children. The global view of the multiple criteria model as the gold standard for estimating ID is debatable and potentially impractical at a public health level. Current evidence cautions against overestimating the prevalence of ID, as there is more associated harm than deficiency underestimation. This critical analysis has confirmed a need for research to identify a suitable, accurate and precise alternative to Hb as a tool in the South African public health setting. Furthermore, the impact of inflammation on iron status indicators, in particular SF, should be assessed in context to clearly set parameters for its use in nationally-representative nutrition surveys, the cornerstone of iron intervention strategies. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2015

The inner surface of the stomach is lined by a mucous membrane known as the gastric mucosa. The integrity of the gastric mucosa is critical for protecting the stomach from the low pH and proteolytic environment within the lumen. Both clinically and experimentally, exposure of gastric mucosal cells to bile salts is known to cause injury. Bile salts present in duodenogastric reflux are thought to play a significant role in gastric ulcer formation and alkaline gastritis. In vitro, studies using physiologic concentrations of the secondary bile salt, deoxycholic acid, indicate that bile salts can induce apoptosis in cultured human gastric epithelial cells in a caspase-dependent manner. Therefore, there is interest in developing approaches that can protect gastric cells from bile salt-induced damage. It has been shown that induction of the stress protein, heme oxygenase-1, can provide protection against apoptosis. Therefore, the objective of this study was to test the hypotheses that heme oxygenase-1 expression could be induced in human gastric epithelial cells and that furthermore; this would provide protection from deoxycholic acid-induced apoptosis. Heme oxygenase-1 expression was induced pharmacologically or by introduction of a plasmid expressing heme oxygenase-1 into the gastric epithelial cell line, AGS. Induction of heme oxygenase-1 prior to challenge with deoxycholate reduced apoptotic-associated morphological changes, DNA fragmentation, the appearance of oligonucleosomes in the cytoplasm, and activation of caspase-3 and caspase-9. Based on these results, it was concluded that expression of heme oxygenase-1, or the introduction of its products, can provide protection to human gastric epithelial cells against sodium deoxycholic acid induced-apoptosis.