Diese Dissertation befasst sich mit den möglichen Folgen einer Kolonisation mit Helicobacter pylori (H. pylori) im Kindesalter. Von besonderem Interesse ist dabei die Entwicklung des Body-Mass-Index (BMI). Dazu werden die Daten einer populationsbezogenen Querschnittsstudie, welche 1998 und 2006 unter Leipziger Schulanfängern bzw. Achtklässlern durchgeführt wurde, analysiert. Insgesamt konnten 1349 bzw. 1161 Kinder, deren H. pylori-Status mittels 13C‑Harnstoff-Atemtest untersucht wurde, in die Untersuchung inkludiert werden. Dabei bestätigte sich, dass die Besiedlung mit H. pylori u.a. signifikant mit einem geringeren sozialen Status und einer geringeren Körpergröße der Kinder assoziiert ist. Der BMI der untersuchten Kinder unterscheidet sich jedoch nicht signifikant in Abhängigkeit von einer Kolonisation mit H. pylori. Auf diesen Ergebnissen basierend werden zwei Hypothesen diskutiert. Zum einen wird analysiert, inwieweit ein Einfluss einer Infektion mit dem Erreger auf den BMI der Kinder nicht sicher auszuschließen ist und dieser eventuell aufgrund der Komplexität der Gewichtsentwicklung in den Ergebnissen dieser Untersuchung nicht erkennbar ist. Zum anderen wird die geringere Körpergröße H. pylori‑besiedelter Kinder nicht auf mit der Infektion in Verbindung stehende Ernährungsstörungen in Zusammenhang gebracht, da sonst vor einer entscheidenden Größenminderung das Gewicht bzw. der BMI der Studienteilnehmer zurück bleiben müsste.

Seit 1983 ist das Konzept des Mukosa-assoziierten lymphatischen Gewebes (MALT) im Magen auf dem Boden einer chronischen Helicobacter(H.)-pylori-Infektion bekannt. Viele epidemiologische, biologische und molekulargenetische Studien haben die Rolle von H. pylori in der Lymphomgenese unterstützt. Bis heute wurden weltweit mehr als 650 Patienten mit gastralem MALT-Lymphom und H.-pylori-Infektion antibiotisch behandelt. Bei etwa 75% der Fälle kann mit Hilfe dieser Therapie eine komplette Lymphomremission induziert werden. Klinische prädiktive Faktoren helfen dabei, Patienten bezüglich ihres Risikos besser zu stratifizieren und damit die Probabilität des Ansprechens zu verbessern. Neue zytogenetische Erkenntnisse haben zudem dazu beigetragen, ein besseres Verständnis der Lymphomgenese zu erlangen. Mit der kürzlich beschrieben Translokation t(11;18) (q21;q21) könnte in Zukunft ein prädiktiver genetischer Faktor verfügbar sein. / The Role of Helicobacter pylori Infection for the Development and Treatment of Gastric MALT Lymphomas Since 1983, it is well known that mucosa-associated lymphoid tissue (MALT)-type lymphoma of the stomach is due to chronic Helicobacter pylori (H. pylori) infection. Many epidemiological, biological, and moleculargenetic studies have implicated the role of H. pylori in lymphomagenesis. Nowadays, more than 650 patients with gastric MALT lymphoma worldwide have been treated with antibiotics for H. pylori infection, achieving a complete remission in about 75% of cases. Clinical predictive factors help to stratify patients into risk groups, and help to predict the probability of lymphoma remission. New insights into cytogenetics have also contributed to the understanding of lymphomagenesis, and with the newly identified translocation t(11;18)(q21;q21) we might have also a genetic factor at hand to predict treatment response. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Helicobacter pylori est une bactérie qui colonise les cellules épithéliales gastriques humaines. Une des conséquences de cette infection est l'induction de cancers de l'estomac dans 1 à 3 % des cas, via l'injection d'une cytotoxine appelée CagA qui dérégule les voies de signalisation des cellules cibles. Cette injection, dont le mécanisme est encore inconnu, se fait grâce à un système de sécrétion de type IV (T4SS). Le pilus du cagT4SS est encore mal caractérisé. Les protéines CagI, CagL et CagH sont essentielles à la fonctionnalité du cagT4SS et à la biogenèse du pilus. De plus les trois protéines forment un sous-complexe dont les détails moléculaires n'ont pas encore été élucidés. Par conséquent mes études se sont focalisées sur ces trois protéines, leurs interactions et leur relation structure/fonction. J'ai mis en évidence que CagL interagissait directement avec CagI et CagH avec une affinité de l'ordre du micromolaire et que CagI et CagH n'interagissaient pas entre elles. La caractérisation de ces interactions a permis notamment d'identifier un complexe CagL-CagI. Afin de comprendre les détails structuraux de ce complexe, j'ai entrepris deux études structurales. La première consiste à déterminer les résidus de CagL impliqués dans l'interface d'interaction avec CagI par RMN. La seconde étude se focalise sur la détermination de la structure 3D du complexe CagI-CagL par microscopie électronique. Pour cela j'ai purifié le complexe CagI-CagL, monodisperse et stable en solution. Nous avons collecté des images du complexe par cryoEM et généré des classes 2D. Cette étude a permis pour la première fois de caractériser les interactions entre CagL-CagI-CagH et d'obtenir des informations structurales du complexe CagI-CagL / Helicobacter pylori is a bacterium that colonizes the human stomach in half of the world population. It is estimated that 20% and 3% of patients develop peptic ulcer and gastric cancer, respectively. For these reasons, H. pylori was identified as a group 1 carcinogen by the World Health Organization (WHO) in 1994. The most virulent strains of H. pylori carry a type IV secretion system (Cag-T4SS) responsible for the injection of the oncoprotein CagA into gastric epithelial cells. One remarkable feature of the cagT4SS is its external pilus which composition is not clear. CagL, CagH and CagI proteins are critical components of the Cag-T4SS because these proteins are necessary for CagA translocation and are involved in pilus formation. Moreover CagL forms a sub-assembly with CagI and CagH but the molecular details of the complex are still to be discovered. Our objective is to better understand the molecular basis of CagLIH complex by interaction and structural study. CagL interacts with CagI and CagH with a with Kds of 5 µM. CagI does not interact with CagH. The structural study of CagL/CagI complex has been investigated by a two-pronged approach. First I have purified the CagL/CagI complex and collected cyo-EM micrographs. In parallel I have collected NMR spectra of CagL in the presence of CagI and identify the changes in the spectra to determine the residues involved in the interaction. In this study we have, for the first time, characterize the CagL-CagI-CagH interactions and obtained structural informations of the CagI-CagL complex

The work presented in this thesis describes the determination of structures of peptides and proteins at atomic resolution. Nuclear Magnetic Resonance (NMR) spectroscopy was used as the principal method of investigation. The thesis is divided into three parts. Part I of the thesis consists of chapters 1 to 4, and deals with structural studies of two novel conotoxins. Part II of the thesis consists of chapter 5 and deals with structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2. Part III of the thesis consists of Appendices A to D. Appendix A describes implementation of a novel pulse sequence for determination of disulfide connectivity using long-range 13 C–13 C scalar couplings across disulfide bonds. Appendices B, C and D contain supplementary infor- mation (acquisition parameters and chemical shifts) for the structural studies presented in parts I and II of the thesis. Part I: Structural studies of novel conotoxins from Conus monile Chapter 1 gives a brief overview of the conotoxins and their structural studies. The first half of the chapter describes biosynthesis, classification schemes, nomenclature, com- monly observed post-translational modifications and applications of conotoxins. The latter half of this chapter summarizes the challenges involved in the structural studies of conotoxins in light of the recent developments in integrated transcriptomic and venomic studies of conotoxins. The key homonuclear and heteronuclear NMR experiments that are employed for structural studies of conotoxins are summarized. Emphasis was laid on describing the spectral features and the structural information that can be gleaned from these experiments. Finally, the current mass spectrometric and NMR methods available for determination of disulfide connectivity are discussed Chapter 2 describes sample preparation and preliminary biophysical characteriza- tion of a conotoxin Mo3964 that contains a hitherto uncharacterized cysteine framework (C–CC–C–C–C). The sequence of Mo3964 was identified at the nucleic acid level as a cDNA clone. Analysis of the signal sequence revealed that the toxin belongs to the M-superfamily, while the cysteine framework bears more resemblance to O- and K- super- family of conotoxins. Structural studies were initiated to determine the disulfide connec- tivity, tertiary structure and biological activity. The gene corresponding to the mature toxin sequence was cloned in a bacterial expression vector pET21a(+) as a C-terminal tag to the cytochrome b5 fusion protein host system. The fusion protein was obtained by recombinant expression using the bacterial expression host E. coli BL21(DE3) and the mature toxin was obtained by either enzymatic or chemical cleavage of the fusion protein followed by size exclusion chromatography and reverse phase HPLC. Proton 1D NMR spectra of the purified peptide exhibited sharp lines and good spec- tral dispersion indicating that molecule was well folded. Formation of disulfide bonds in the mature toxin was ascertained by high resolution mass spectra of intact and chemically modified Mo3964. The peptide toxin exhibited remarkable stability to chemical denatu- ration and proteolytic digestion. Spectroscopic studies clearly showed that Mo3964 pos- sesses a very stable and well defined structure as long as its disulfide bonds are intact. Analytical size exclusion chromatography and Multi Angle Light Scattering (MALS) studies showed that Mo3964 exists in solution as monomer albeit with a non-globular structure. Electrophysiological studies showed that Mo3964 inhibits outward potassium currents in rat Dorsal Root Ganglion (DRG) neurons and increases the reversal potential of rat voltage gated sodium channel rNav 1.2 stably expressed on Chinese Hamster Ovary (CHO) cells at peptide concentrations as low as 10 nM. Chapter 3 describes the determination of disulfide connectivity and tertiary stricture of Mo3964. Initial attempts to determine disulfide connectivity using direct fragmenta- tion of the intact peptide in the mass spectrometer failed due to the relatively large size of the molecule and its resistance to endoproteases. Partial reduction alkylation based methods failed as the first stage of partial reduction gave rise to a mixture of various single disulfide bond reduced species which could not be separated from each other. Subsequently, information about the disulfide connectivity was obtained using a method that does not necessitate separation of such a mixture of single disulfide bond reduced species. This method involves partial reduction, cyanylation of the reduced cysteines and alkali mediated cleavage of the peptide backbone on the N-terminus of cyanylated cysteines. Structural studies were carried out using homonuclear and heteronuclear NMR meth- ods. The hydrogen bond network and hence topology of the molecule was determined with high accuracy using the long-range HNCO-COSY experiment that correlates hydrogen- bond donor-acceptor pairs. This experiment utilizes the three bond heteronuclear scalar coupling, i.e., the h3JN C O′ coupling across the hydrogen bonds. All these restraints proved crucial to the assignment of the disulfide connectivity in Mo3964, given its novel cysteine framework. The structure of Mo3964 was calculated using a total of 549 NOE distance restraints, 84 dihedral angle restraints and 28 hydrogen bond distance restraints. The tertiary structure was constructed from the disulfide connectivity pattern 1–3, 2–5 and 4–6, that is hitherto undescribed for the M–superfamily conotoxins. The ensemble of structures showed a backbone Root Mean Square Deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (φ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The remarkable stability and anomalous spectral properties exhibited by Mo3964 could be rationalized using the disulfide connectivity and the tertiary structure. The tertiary structural fold has not been described for any of the known Conus peptides. Further, a search for structures similar to that of Mo3964 using the web server DALI returned no hits indicating that the peptide scaffold of Mo3964 has no structural homologues. Hence, the conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules. The structure of Mo3964 was submitted to the Protein Data Bank (PDB ID: 2MW7)[1]. Chapter 4 describes the structural studies of a 17 residue, single disulfide containing conopeptide Mo1853. The samples for structural studies were obtained either by chemical synthesis or by recombinant expression methods. Structural studies using homonuclear solution NMR methods revealed that Mo1853 exists as two equally populated cis and trans X–Pro conformers which are in slow exchange regime, compared to the chemical shift timescale. Sequence specific assignments were obtained for both the conformers by analysis of homonuclear 2D 1 H,1H–DQF–COSY,1H,1 H–TOCSY, 1H,1 H–NOESY and 1H,1 H–ROESY spectra. Temperature dependence of chemical shifts was measured and coalescence was observed for two amide protons at 318 K. At this temperature, the rate of exchange and the free energy of activation were determined to be 59 Hz and ≈ 67.2 kJ mol−1 respectively. The evidence for this conformational equilibrium was also observed as exchange correlation peaks in the 2D- NOESY and ROESY spectra. Tertiary structures of both the cis and trans conformers were determined using distance restraints, backbone dihedral angle restraints, the disulfide bond restraint and the cis or trans conformation of the X–Pro peptide bond. Tertiary structures of both the conformers consist of a 29-membered macro-cyclic ring formed by 9 amino acid residues which are cyclized by side chain to side chain disulfide bond. The conformation of the X–Pro peptide bond which is located within this macro-cyclic ring causes the cis structure to be compact and the trans structure to be in an extended form. Analysis of the tertiary structures indicated that the trans conformer is stabilized by hydrogen bonds while the cis conformer is likely to be stabilized by hydrophobic interactions. This was further corroborated by the fact that at lower temperatures, the hydrophobic interactions became weaker reducing the population of the cis conformer with respect to that of the trans conformer. Preliminary electrophysiological studies carried out on rat DRG neurons indicate that Mo1853 transiently reduces late outward potassium currents. Part II: Structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2 Chapter 5 presents the solution NMR studies of the Smr domain from MutS2 of H. pylori , henceforth called as HpSmr. In H. pylori , MutS2 is involved in suppression of homologous recombination and its Smr domain was shown to be necessary for this activity. As of date, in spite of the availability of structural information for the Smr domain, unambiguous identification of the residues involved in metal binding, DNA binding and catalysis remains elusive. Structural studies were carried out on two different constructs of HpSmr viz., HpSmr– (His)6 and GSHM–HpSmr, with and without the hexahistidine tag respectively. Se- quence specific assignments of HpSmr–(His)6 were obtained at two different sample pH conditions viz., pH 8.0 and pH 5.35 using the standard suite of triple resonance NMR experiments. Since, valines and leucines constitute about 25% of the total number of amino acid residues in HpSmr–(His)6 , stereospecific assignments were obtained for di- astereotopic methyl groups of these residues by preparing a fractionally 13C labeled sample of HpSmr–(His)6 . Solution structure of HpSmr–(His)6 at pH 8.0 was determined using 766 NOE restraints, 170 backbone dihedral angle restraints and 70 hydrogen bond distance restraints. The tertiary structure exhibits the canonical α/β sandwich fold ex- hibited by all the other known structures of Smr domains. Further, NMR studies and analytical gel filtration studies indicated the presence of pH dependent conformational exchange in HpSmr that involves strand to coil transition in the C-terminal β-strand. In order ascertain that the conformational equilibrium is not at an artifact caused by the C-terminal hexa-histidine-tag, HpSmr protein construct GSHM–HpSmr, which does not have the hexa-histidine-tag, was prepared. Conformational exchange was observed in this construct as well. The preliminary NMR evidence suggests that the conformational exchange is caused by pH dependent cis–trans isomerization of a semi-conserved Proline residue Pro66 . We have hypothesized that the pH dependent modulation of the activity of Smr domain of MutS2 can be advantageous to H. pylori . Such a regulation could help the bacteria to achieve optimal rate of homologous recombination in response to changes in pH, which is necessary for maintaining homeostasis and tiding over stress conditions. Part III: Appendix Appendix A describes an NMR pulse program LRCC_CH2 that was designed with the aim of determining disulfide connectivity using long-range 13C–13 C (C β –C β ′ ) couplings across the disulfide bond. This experiment is a modification of an earlier experiment pub- lished by Bax and co-workers designed to measure the side-chain χ3 dihedral angle in me- thionines. The experiment described here is optimized for the detection of 3 bond scalar coupled methylene carbons. The details of modifications introduced in LRCC_CH2, its product operator analysis, a representative spectrum acquired on [U-13C,15 N]–Mo3964, short-comings and future directions are described. The C programming code that was used to implement the pulse program is also included in the appendix. Appendices B, C and D contain the supplementary information (acquisition pa- rameters for the NMR experiments and chemical shifts) for the structural studies carried out on Mo3964, Mo1853 and HpSmr.

Made available in DSpace on 2015-03-26T13:46:44Z (GMT). No. of bitstreams: 1 texto completo.pdf: 854857 bytes, checksum: 0da19cfc06d4ca09fceb926374e1228d (MD5) Previous issue date: 2004-01-30 / The discovery of Helicobacter pylori in human beings and its relation with gastritis, peptic ulcer and gastric neoplasia, has renewed the interest on the establishment of the incidence and on the clinical meaning of this bacteria in pets, specifically in dogs. There are several researches that indicate the presence of Helicobacter ssp in dogs, even though the relation of these bacteria with gastric alterations in dogs has not been clarified yet. The aim of this dissertation was to evaluate the presence of Helicobacter ssp in dogs, to analyse the macro and microscopic findings, determine the correlation between the presence of the bacteria with alterations in the gastric mucosa, and of these with the dogs age, and additionally to investigate the correlation of the positiveness of the urease test with the presence of the bacteria. During the necropsy of 60 dogs, samples from cardia, fundus, body, and pylorus regions were collected from the dogs stomach for conducting the urease test and histopathologic examination. The urease test was positive in 95,12% of the animals. The histopathologic examination using Carbol-Fucsina coloration revealed Helicobacter ssp in 96,66% of the animals. The most frequent observed alteration in preparations colored by HE were infiltrated with inflammatory cells, predominantly mononuclear, lymphoid nodules hyperplasia and hiperemy. Correlation between the urease test and the histopathology for the diagnostic of Helicobacter ssp in dogs was found. Age was not found correlated with infection by Helicobacter ssp. It was not found any correlation between inflamatory alterations, in the presence of the bacteria, with the age of the animals. The fundus and body stomach regions presented more positive results in the urease test and the body and pylorus regions presented more positive results in the histopathologic examination. There was correlation between the number of Helicobacter ssp, the number of inflamatory cells and lymphoid nodules in those regions. The high percentage of dogs infected by Helicobacter ssp and the variation in the patogenicity among the species indicate the necessity of further investigation for the identification of species more pathogenic and mechanisms of patogenicity in dogs. Besides, due to the zoonotic potential, further research should be done on the helicobacterias. / A descoberta do Helicobacter pylori em seres humanos e a sua relação com gastrite, úlcera péptica e neoplasia gástrica, tem levado à renovação do interesse no estabelecimento da incidência e do significado clínico da bactéria em animais domésticos, especificamente cães. Existem vários relatos da presença de Helicobacter ssp em cães, mas a relação dessas bactérias com as alterações gástricas em cães ainda não está esclarecida. Os objetivos desse trabalho foram avaliar a presença de Helicobacter ssp em cães, analisar os achados macro e microscópicos, determinar a correlação entre a presença da bactéria e as alterações da mucosa gástrica, e destas com a idade, além de correlacionar a positividade do teste da urease à presença da bactéria. Durante as necropsias de 60 cães, foram coletadas amostras das regiões cárdica, fúndica, do corpo e pilórica dos estômagos para a realização do teste da urease e exame histopatológico. O teste da urease foi positivo em 95,12% dos animais. O exame histopatológico usando a coloração de Carbol- Fucsina revelou Helicobacter ssp em 96,66% dos animais. As alterações mais freqüentemente observadas em preparações coradas por HE foram infiltrados de células inflamatórias, predominantemente mononucleares, hiperplasia de nódulos linfóides e hiperemia. Houve correlação entre o teste da urease e a histopatologia para o diagnóstico de Helicobacter ssp em cães. Não houve correlação da infecção por Helicobacter ssp com a idade. Também não foi demonstrada correlação entre alterações inflamatórias, na presença da bactéria, com a idade dos animais. As regiões fúndica e do corpo do estômago apresentaram maior número de resultados positivos no teste da urease e as regiões do corpo e pilórica apresentaram maior número de resultados positivos à histopatologia. Houve correlação entre o número de Helicobacter ssp, o número de células inflamatórias e nódulos linfóides nestas regiões. O alto percentual de cães infectados por Helicobacter ssp e a variação na patogenicidade entre as espécies indicam a necessidade de estudos mais profundados para identificação de espécies mais patogênicas e de mecanismos de patogenicidade em cães. Além disto, devido ao potencial zoonótico, grande atenção deve ser dada às helicobactérias.

Made available in DSpace on 2014-12-17T14:13:45Z (GMT). No. of bitstreams: 1 SilvaEL_DISSERT.pdf: 2857936 bytes, checksum: 9a0ed46e2b6c06e351eff5810e5f24d8 (MD5) Previous issue date: 2010-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Magnetic targeting is being investigated as a means of local delivery of drugs, combining precision, minimal surgical intervention, and satisfactory concentration of the drug in the target region. In view of these advantages, it is a promising strategy for improving the pharmacological response. Magnetic particles are attracted by a magnetic field gradient, and drugs bound to them can be driven to their site of action by means of the selective application of magnetic field on the desired area. Helicobacter pylori is the commonest chronic bacterial infection. The treatment of choice has commonly been based upon a triple therapy combining two antibiotics and an anti-secretory agent. Furthermore, an extended-release profile is of utmost importance for these formulations. The aim of this work was to develop a magnetic system containing the antibiotic amoxicillin for oral magnetic drug targeting. First, magnetic particles were produced by coprecipitation of iron salts in alkaline medium. The second step was coating the particles and amoxicillin with Eudragit? S-100 by spray-drying technique. The system obtained demonstrated through the characterization studies carried out a possible oral drug delivery system, consisting in magnetite microparticles and amoxicillin, coated with a polymer acid resistant. This system can be used to deliver drugs to the stomach for treatment of infections in this organ. Another important finding in this work is that it opens new prospects to coat magnetic microparticles by the technique of spray-drying. / A vetoriza??o magn?tica tem sido investigada como uma forma de entrega local de f?rmacos combinando precis?o, m?nima interven??o cir?rgica e concentra??o satisfat?ria do f?rmaco na regi?o de interesse. Part?culas magn?ticas s?o atra?das a partir da aplica??o de um campo magn?tico e f?rmacos associados a essas part?culas podem ser direcionados ao seu s?tio de a??o atrav?s de uma aplica??o seletiva do campo na regi?o de interesse. Helicobacter pylori ? a mais comum causa de infec??o bacteriana cr?nica no est?mago. O tratamento padr?o ? a tripla terapia oral contendo dois antibi?ticos e um inibidor da bomba de pr?tons. Sendo assim, um perfil de libera??o prolongada ? de suma import?ncia para essas formula??es. O objetivo deste trabalho foi desenvolver um sistema magn?tico com potencial emprego na vetoriza??o de antibi?tico por via oral. Inicialmente, part?culas magn?ticas foram produzidas por co-precipita??o de sais de ferro em meio alcalino. Em seguida, as part?culas foram revestidas a partir da dispers?o da suspens?o magn?tica em uma solu??o contendo o pol?mero dissolvido e a amoxicilina, e ent?o submetido ? secagem por aspers?o (spray-drying). Atrav?s das caracteriza??es realizadas p?de-se verificar a obten??o de um potencial sistema para vetoriza??o de f?rmacos por via oral contendo micropart?culas de magnetita e amoxicilina revestidos por um pol?mero gastro-resistente. Adicionalmente, um importante aspecto nesse trabalho ? a abertura de novas perspectivas para o revestimento de micropart?culas magn?ticas atrav?s da t?cnica de spray-drying.

Made available in DSpace on 2014-12-17T14:16:32Z (GMT). No. of bitstreams: 1 DalianaMBOS_DISSERT.pdf: 1178965 bytes, checksum: a8c57235d95835138c537bc774c7af70 (MD5) Previous issue date: 2012-02-27 / The expression of human leukocyte antigen G (HLA-G) and human leukocyte antigen E (HLA-E) in physiological and pathological processes remains unknown, it is believed that these molecules play a fundamental role in the establishment and maintenance of immune tolerance by inhibiting the functions of immunocompetent cells. In literature we found no published study involving the bacterium Helicobacter pylori (H. pylori) with expression of HLA-G and HLA-E. The objective this study is investigated the expression of this protein in gastric biopsies of patients with the bacterium H. pylori. Sixty-four biopsies of the patients with diagnosis of infection by H. pylori were evaluated to expression of HLA-G and HLA-E. The samples were stratified according to the presence of carcinoma or peptic ulcers. Patients without H. pylori were used to control. To investigate the expression of this protein were used immunohistochemistry technique with monoclonal antibody anti-HLA-G and anti-HLA-E. Other criteria such as analysis of the inflammatory infiltrate (hematoxylin-eosin) and identification of H. pylori (Giemsa) were analyzed. We detected HLA-G and HLA-E molecules in the most samples containing ulcer and gastric carcinoma. In negative control group was not detected the presence of HLA-G and HLA-E. The presence of H. pylori seems modulate the expression of HLA-G and HLA-E, favoring the evolution of infection, giving different degrees of gastric lesion in epithelium of these patients / A express?o do ant?geno leucocit?rio humano G (HLA-G) e do ant?geno leucocit?rio humano E (HLA-E) em processos fisiol?gicos e patol?gicos permanece pouco conhecida. Acredita-se que essas mol?culas desempenham papel fundamental no estabelecimento e manuten??o da toler?ncia imunol?gica, inibindo as fun??es das c?lulas imunocompetentes. Na literatura internacional, at? o momento, n?o foi encontrado nenhum estudo publicado correlacionando Helicobacter pylori (H. pylori) com express?o de HLA-G e HLA-E. O presente trabalho tem como objetivo correlacionar a express?o dessas prote?nas em bi?psias g?stricas de pacientes acometidos com H. pylori. Sessenta e quatro esp?cimes g?stricas de pacientes acometidos com H. pylori foram avaliados para express?o de HLA-G e HLA-E. As amostras foram estratificadas de acordo com a presen?a de carcinoma ou de ?lcera p?ptica. Como controle foram analisados esp?cimes g?stricas de pacientes vivos sem H. pylori. Para detectar a express?o dessas mol?culas utilizou-se a t?cnica de imunohistoqu?mica com os anticorpos monoclonais anti-HLA-G e anti-HLA-E. Outros crit?rios como an?lise do infiltrado inflamat?rio (hematoxilina-eosina) e identifica??o do H. pylori (Giemsa) foram analisados. As mol?culas de HLA-G e HLA-E foram detectadas em grande parte das amostras contendo ?lcera e carcinoma g?strico. No grupo controle n?o foi detectada a presen?a de HLA-G e HLA-E, indicando que a bact?ria H. pylori modula a express?o dessas mol?culas no grupo dos pacientes que apresentaram ?lcera ou carcinoma g?strico. A presen?a da bact?ria H. pylori parece modular a express?o do HLA-G e do HLA-E, favorecendo assim a evolu??o da infec??o, o que confere diferentes graus de les?o do epit?lio g?strico desses pacientes

Orientador: Helena Zerlotti Wolf Grotto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T08:24:50Z (GMT). No. of bitstreams: 1 Alvarenga_ElianadaCosta.pdf: 3519502 bytes, checksum: cc024f9bd1176e4c4f56d989bba5a487 (MD5) Previous issue date: 2009 / Resumo: A deficiência de ferro é provavelmente o distúrbio nutricional mais freqüente no mundo. O ferro é um componente essencial da molécula de hemoglobina, da mioglobina e de diversas enzimas. Tem papel fundamental no transporte de oxigênio, na transferência de elétrons e atua como cofator em muitos processos enzimáticos, incluindo a síntese de DNA. Diversos estudos têm mostrado a contribuição da infecção pelo Helicobacter pylori (H. pylori) no desenvolvimento da anemia ferropriva e a associação entre o H. pylori e a diminuição do estoque de ferro. O objetivo do presente trabalho foi verificar a possível associação entre a infecção pelo H. pylori e a deficiência de ferro em um grupo de pacientes adultos. Desse modo pretendeu-se conhecer melhor as alterações hematológicas presentes nos pacientes infectados pelo H. pylori, principalmente as relacionadas ao metabolismo do ferro. Foram estudados 156 pacientes adultos de ambos os sexos que foram submetidos à endoscopia digestiva alta para esclarecimento diagnóstico. Desses 156 pacientes, 125 apresentaram alterações à endoscopia, que justificaram a realização da biópsia gástrica. A avaliação da presença de anemia foi feita pelos dados hematimétricos e pelo conteúdo de hemoglobina dos reticulócitos (Ret-He) e o estado do ferro foi avaliado pelas dosagens de ferro sérico, capacidade de ligação do ferro à transferrina e ferritina sérica. A possível participação da atividade inflamatória na eritropoiese foi verificada pela correlação entre os parâmetros hematimétricos e do estado do ferro com os níveis de proteína C reativa (PC-R). O diagnóstico do H. pylori foi realizado através da análise histológica, teste da urease e teste sorológico (IgG anti- H. pylori). Os 125 pacientes foram separados em H. pylori positivo (n= 75) e negativo (n= 50). Não houve diferença significativa nos valores dos parâmetros hematológicos e bioquímicos, tendo sido diferentes somente os valores de IgG anti-H. pylori e de gastrina sérica, significativamente superiores no grupo H. pylori positivo. Foi observada uma correlação inversa fraca, mas significativa entre os níveis de PC-R e Ret-He e entre gastrina sérica e Ret-He no grupo H. pylori positivo. De acordo com os critérios laboratoriais, dos 125 pacientes apenas 17 (13,6%) mostraram níveis de Hb indicativos de anemia, sendo que desses, 7 (5,6%) mostraram ser positivos para a infecção pelo H. pylori. Não foi observada diferença na freqüência da deficiência de ferro entre os grupos de pacientes infectados pelo H. pylori e os não infectados pelo H. pylori. Dentre os pacientes estudados a gastrite foi confirmada em 110 pacientes, sendo que 74 (67,2%) eram H. pylori positivo. A gastrite foi classificada de acordo com o sistema de Sidney em: moderada/intensa (36.63%), leve (63,37%) e inespecífica (8,1%). Quando os pacientes com gastrite moderada/intensa foram comparados com os com gastrite leve, não houve diferença entre os parâmetros estudados, exceto os valores da titulação de IgG, que foram significativamente superiores no grupo com gastrite moderada/intensa. Portanto, podemos concluir que no grupo de indivíduos estudados a infecção pelo H. pylori e a intensidade da gastrite não foram fatores determinantes ao desenvolvimento da deficiência de ferro. / Abstract: Iron deficiency is probably the most common nutritional disorder in the world. Iron is an essential component of the molecule of hemoglobin, myoglobin and various enzymes. It has a fundamental role in oxygen transport, in electrons transference and acts as a cofactor in many enzymatic processes, including synthesis of DNA. Several studies have shown the contribution of Helicobacter pylori (H. pylori) infection in the development of anemia and the association between H. pylori and reduction of iron store. The objective of this study was to investigate the association between H. pylori infection and iron deficiency in a group of adult patients. Thus we intend to study the hematological alterations in H. pylori infected patients, mainly those related to iron metabolism. We studied 156 adult patients of both sex undergoing routine upper endoscopy. Of these 156 patients, 125 had gastric biopsies stained for H. pylori identification. The evaluation of the presence of anemia was performed by erythrocyte indeces and reticulocyte hemoglobin content (RET-He) and the iron status was assessed by measurements of serum iron, iron binding capacity of the transferrin and serum ferritin levels. Inflammatory activity influence on erythropoiesis was verified by the correlation among erythrocyte parameters and the state of the iron with C-reactive protein (CR-P) levels. The diagnosis of H. pylori was performed by histological, urease and serological (IgG anti-H. pylori) methods. Patients were divided into H. pylori positive (n = 75) and negative (n = 50). There was no significant difference in hematological and biochemical parameters, except for IgG anti-H. pylori values and serum gastrin, significantly higher in H. pylori positive group. There was a weak but significant inverse correlation between CR-P and Ret-He levels and between serum gastrin and Ret-He in H. pylori positive group. According to laboratory criteria, only 17 of 125 patients (13.6%) showed levels of hemoglobin indicative of anemia, 7 of 17 patients were positive for H. pylori infection. There was no difference in the frequency of iron deficiency anemia between H. pylori positive and negative groups. Gastritis was confirmed in 110 patients, of these 74 (67.2%) were H. pylori positive. The gastritis was classified according to the Sydney system as: moderate / severe (36.63%), mild (63.37%) and nonspecific (8.1%). There was no difference between groups with moderate/ severe gastritis and mild gastritis concerning hematologic and biochemical parameters, except the values of the titers of IgG, which were significantly higher in the group with moderate / severe gastritis. Therefore, we conclude that infection by H. pylori and the intensity of gastritis were not determining factors for the development of iron deficiency in the studied group. / Universidade Estadual de Campi / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T23:14:22Z (GMT). No. of bitstreams: 1 Oliveira_TelmaBarbosade_M.pdf: 1175004 bytes, checksum: 5912fb5d3dc25609414bb097190091e2 (MD5) Previous issue date: 2008 / Resumo: O He/icobacter pylori é uma bactéria gram-negativa que está relacionada ao desenvolvimento de doenças gástricas e extragástricas. Dentre as doenças gástricas incluem-se o câncer gástrico, a gastrite crônica, a úlcera péptica e linfoma tipo MALT. Com relação às doenças extragástricas essa bactéria recentemente foi relacionada com a anemia por deficiência de ferro e com algumas doenças autoimunes, como a artrite reumatóide e a púrpura trombocitopênica idiopática. A PCR tem sido uma importante ferramenta para a análise de pequenos fragmentos de DNA, os quais podem, inclusive, ser armazenados por um tempo maior em amostras emblocadas em parafina ou obtidas a fresco à temperatura de -80°C. Dessa maneira, foram estudados 33 pacientes com púrpura trombocitopênica idiopática (PTI) e H.pylori positivo. No que diz respeito à detecção da bactéria pela nested PCR, foi obtido um resultado de 100% de concordância em comparação aos resultados de histologia e teste da urease, usados rotineiramente. Além dos primers para detecção do He/icobacter pylori foram utilizados primers para as regiões do gene urease C e do gene cagA, sendo os fragmentos obtidos analisados em gel de 2% agarose observados sob luz ultravioleta. Foi obtida também uma concordância de 100% para a região do gene urease C. Em relação ao gene cagA, 30,3% desses pacientes apresentaram esse. Dos 33 pacientes positivos para H pylori, após tratamento específico, 27,7% tiveram remissão completa do quadro clínico, 33% remissão parcial e 40% não tiveram remissão. Para o gene cagA positivo, 9,0% dos pacientes com PTI tiveram remissão completa e em 21,2% não houve remissão. Em relação ao gene cagA negativo, 21,2% tiveram remissão completa e em 48,4% não houve remissão. Foram aplicados testes estatísticos para observar a relação do gene cagA com a PTI, (teste exato de Fisher, Box-plot média e desvio padrão da contagem das plaquetas e Mann-Withney). Os resultadoS' obtidos não foram estatisticamente significantes neste grupo estudado. Portanto o gene cagA não está relacionado com o desenvolvimento da PTI / Abstract: Helicobacter pylori is a gram-negative bacterium that is related to the development of gastric and extragastric diseases. Gastric diseases include gastric cancer, chronic gastritis, peptic ulcer and MALT lymphoma. Extragastric diseases include iron deficiency anemia and some auto-immune conditions such as reumathoid arthritis and idiopathic thrombocytopenic purpura. PCR has been an important tool for the analysis of small fragments of DNA that may be stored for a longer time inserted in paraffin blocks or fresh tissue at -80ºC. Here we studied 45 patients with ITP and 33 of these ones had positive tests, with agreement of 100% of histological and rapid urease test. Besides the primers for H. pylori detection, primers for urease C region and cagA gene were used and the fragments obtained were analysed in agarose gel by ultraviolet radiation. We obtained 100% of agreement for urease C region and for detection of H. pylori and 30% of agreement for cagA gene. In these 33 patients which were H. pylori positive, 27,7% had complete remission, 33,0% partial remission and 40,0% had no remission. In positive patients for cagA gene, 9,0% had complete remission and 21,2% had no remission. In negative patients for cagA gene, 21,2% had complete remission and 48,4% had no remission. We used statistical analysis (Exact Fishers test, Box-plot and Mann-Withney) to relation cagA gene and thrombocytopenic idiopathic purpura Our results don't suggest or correlate the presence of cagA gene with idiopathic thrombocytopenic purpura development / Mestrado / Mestre em Farmacologia