Cobalt complexes bearing scorpionate ligands: Synthesis, characterization, cytotoxicity and DNA cleavage

A number of novel, water-stable redox-active cobalt complexes of the C-functionalized tripodal ligands tris(pyrazolyl)methane XC(pz)(3) (X = HOCH2, CH2OCH2Py or CH2OSO2Me) are reported along with their effects on DNA. The compounds were isolated as air-stable solids and fully characterized by IR and FIR spectroscopies, ESI-MS(+/-), cyclic voltammetry, controlled potential electrolysis, elemental analysis and, in a number of cases, also by single-crystal X-ray diffraction. They showed moderate cytotoxicity in vitro towards HCT116 colorectal carcinoma and HepG2 hepatocellular carcinoma human cancer cell lines. This viability loss is correlated with an increase of tumour cell lines apoptosis. Reactivity studies with biomolecules, such as reducing agents, H2O2, plasmid DNA and UV-visible titrations were also performed to provide tentative insights into the mode of action of the complexes. Incubation of Co(II) complexes with pDNA induced double strand breaks, without requiring the presence of any activator. This pDNA cleavage appears to be mediated by O-centred radical species.

Production, purification and characterisation of polysaccharides from pleurotus ostreatus with antitumor activity

Background: Mushroom polysaccharides play an important role in functional foods because they exhibit biological modulator properties such as antitumour, antiviral and antibacterial activities. The present study involved the production, purification and characterisation of intracellular and extracellular free and protein-bound polysaccharides from Pleurotus ostreatus and the investigation of their growth-inhibitory effect on human carcinoma cell lines. Results: Several fermentation parameters were obtained: batch polysaccharide productivities of 0.013 +/- 8.12 x 10-5 and 0.037 +/- 0.0005 g L-1 day-1 for intracellular and extracellular polysaccharides respectively, a maximum biomass concentration of 9.35 +/- 0.18 g L-1, Pmax = 0.935 +/- 0.018 g L-1 day-1, µmax = 0.218 +/- 0.02 day-1, YEP/X = 0.040 +/- 0.0015 g g-1 and YIP/X = 0.014 +/- 0.0003 g g-1. Some polysaccharides exhibited superoxide dismutase (SOD)-like activity of 50-200 units. Fourier transform infrared analysis of the polysaccharides revealed absorption bands characteristic of such biological macromolecules. Cytotoxicity assays showed that both intracellular and extracellular polysaccharides exhibited antitumour activity towards several tested human carcinoma cell lines in a dose-dependent manner. Conclusion - The polysaccharides of P. ostreatus exhibited high SOD-like activity, which strongly supports their biological effect on tumour cell lines. The extracellular polysaccharides presented the highest antitumour activity towards the RL95 carcinoma cell line and should be further investigated as an antitumour agent.

Redox-active cytotoxic diorganotin(IV) cycloalkylhydroxamate complexes with different ring sizes: Reduction behaviour and theoretical interpretation

Two series of new diorganotin(IV) cycloalkylhydroxamate complexes with different ring sizes (cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), formulated as the mononuclear [R2Sn(HL)(2)] (1:2) (a, R=Bu-n and Ph) and the polymeric [R2SnL](n) (1:1) (b, R=Bu-n) compounds, were prepared and fully characterized. Single crystal X-ray diffraction for [(Bu2Sn)-Bu-n{C5H9C(O)NHO}(2)] (3a) discloses the cis geometry and strong intermolecular NH center dot center dot center dot O interactions. The in vitro cytotoxic activities of the complexes were evaluated against HL-60, Bel-7402, BGC-823 and KB human tumour cell lines, the greater activity concerning [(Bu2Sn)-Bu-n(HL)(2)] [HL=C3H5C(O)NHO (1a), C6H11C(O)NHO (4a)] towards BGC-823. The complexes undergo, by cyclic voltammetry and controlled-potential electrolysis, one irreversible overall two-electron cathodic process at a reduction potential that does not appear to correlate with the antitumour activity. The electrochemical behaviour of [R2Sn(C5H9C(O)NHO)(2)] [R=Bu-n (3a), Ph (7a)] was also investigated using density functional theory (DFT) methods, showing that the ultimate complex structure and the mechanism of its formation are R dependent: for the aromatic (R = Ph) complex, the initial reduction step is centred on the phenyl ligands and at the metal, being followed by a second reduction with Sn-O and Sn-C ruptures, whereas for the alkyl (R=Bu-n) complex the first reduction step is centred on one of the hydroxamate ligands and is followed by a second reduction with Sn-O bond cleavages and preservation of the alkyl ligands. In both cases, the final complexes are highly coordinative unsaturated Sn-II species with the cis geometry, features that can be of biological significance.

Malaria at Humaita county, Amazonas state, Brazil: XVII — immune response in patients with Plasmodium falciparum according to gametocytes

In August 1983 the Authors studied 36 patients with Plasmodium falciparum malaria and 14 normal individuals born in Humaita region who had never had malaria, had no spleen enlargement and had negative parasitemia as well as passive hemagglutination. Medical histories were obtained and complete physical examination were performed in all of them just as blood tests, parasite density and lymphocyte typing. The lymphocytes were separated and then frozen in liquid nitrogen for later typing by rosette formation. The patients were divided in two groups according to the presence (13 patients) or abscence (23 patients) of gametocytes before treatment. Severe malaria was predominant in the group without gametocytes. The results showed a decrease in the T-cell numbers in Plasmodium falciparum acute malaria patients both with or without gametocytes before the treatment, while B-cell numbers were normal only in the patients with gametocytes. These observations as like as those previously reported by the Authors, permit to associate the presence of gametocytes in peripheral blood and normal number of B-cells in patients with mild Plasmodium falciparum malaria.

Immunoperoxidase technique in experimental chronic chagasic myocarditis

Chagas'disease has been described as the commonest form of chronic myocarditis. An immunologic pathogenesis has been discribed for this form of the disease. So far, no immunoperoxidase technique has been used for the detection of immunological deposits in chronic experimental Chagas'myocardiopathy. Forty-one Swiss mice, three months old were inoculated intraperitoneally with doses between 10 and 10(5) Tulahuen trypomastigotes. Mice were reinoculated one month after with doses between 10² and 10(5) and sacrificed at 6 (n=21) and 9 months (n=9) after the first inoculation. ECGs were recorded before sacrifice. Immunoperoxidase technique (peroxidase-antiperoxidase method), immunofluorescence (direct and indirect) as well as histological studies were performed in myocardiums and skeletal muscles of the surviving animals. The most sensitive methods for detecting chronic chagasic infection were the routine histologic studies (73%) and the ECGs 83% and 89% on 6 and 9 mo. post-infected mice, respectively. Myocardial involvement varied from interstitial mild focal lymphocyte infiltrates up to replacement of myocytes by loose connective tissue. Atrial myocardiums (21/23, 91%) were more affected than ventricles (9/23, 39%). Typical chagasic nests were rarely found. Skeletal muscle involvement (11/18 and 7/9) varied from mild to extensive lymphocyte and plasmacell infiltrates, and necrotic fibers. The involved antigen were shown in skeletal muscles by the immunoperoxidase technique as diffusely arranged granular intracytoplasmatic deposit for both IgC and total immunoglobulins. The coincidence between this technique and histologic muscle lesions was 11/18 (61(%) in 6 mo. and 6/8 (75%) at 9 mo. post-infection. In heart, delicate granular deposits of total immunoglobulins were seen diffusely arranged within the ventricular myocytes; coincidence between immunoperoxidase technique anl histologic involvement increased from 36 to 66% in animals sacrifeced 6 and 9 mo. post-infection. This strongly stressed the increase of immunologic phenomena with the chronification of infection. Concerning sensitivity, immunoperoxidase and direct immunofluorescence were highly sensitive in skeletal muscle (100%, p < 0.01). Conversely, direct immunofluorescence technique showed poor results in heart while immunoperoxidase increased its sensitivity from 21.4% (at 6 mo.) to 66.6% (at 9 mo.) post-infection (p < 0.001). Considering the necessity of obtaining an adequate vaccine in order to prevent this disease an experimental model like this, rendering immunological reactions as revealed by the immunoperoxidase technique, would be useful.

18F-FDG measurement in primary lung cancer: SUV normalization to different distribution volumes

Introduction: Standard Uptake Value (SUV) is a measurement of the uptake in a tumour normalized on the basis of a distribution volume and is used to quantify 18F-Fluorodeoxiglucose (FDG) uptake in tumors, such as primary lung tumor. Several sources of error can affect its accuracy. Normalization can be based on body weight, body surface area (BSA) and lean body mass (LBM). The aim of this study is to compare the influence of 3 normalization volumes in the calculation of SUV: body weight (SUVW), BSA (SUVBSA) and LBM (SUVLBM), with and without glucose correction, in patients with known primary lung tumor. The correlation between SUV and weight, height, blood glucose level, injected activity and time between injection and image acquisition is evaluated. Methods: Sample included 30 subjects (8 female and 22 male) with primary lung tumor, with clinical indication for 18F-FDG Positron Emission Tomography (PET). Images were acquired on a Siemens Biography according to the department’s protocol. Maximum pixel SUVW was obtained for abnormal uptake focus through semiautomatic VOI with Quantification 3D isocontour (threshold 2.5). The concentration of radioactivity (kBq/ml) was obtained from SUVW, SUVBSA, SUVLBM and the glucose corrected SUV were mathematically obtained. Results: Statistically significant differences between SUVW, SUVBSA and SUVLBM and between SUVWgluc, SUVBSAgluc and SUVLBMgluc were observed (p=0.000<0.05). The blood glucose level showed significant positive correlations with SUVW (r=0.371; p=0.043) and SUVLBM (r=0.389; p=0.034). SUVBSA showed independence of variations with the blood glucose level. Conclusion: The measurement of a radiopharmaceutical tumor uptake normalized on the basis of different distribution volumes is still variable. Further investigation on this subject is recommended.

Contributo para o estudo do tempo de espera para radioterapia em Portugal : adaptação de um instrumento de recolha de dados

RESUMO - É reconhecido o impacto negativo e prejudicial que o tempo de espera tem para radioterapia sobre o controlo tumoral e a taxa de sobrevida, bem como a importância de estabelecer tempos máximos para o início do tratamento, de forma a garantir o cumprimento de uma boa prática. O presente projecto de investigação tem o objectivo de construir e validar uma grelha de observação, como instrumento de recolha de dados, que se pretende no futuro aplicar, de forma a poder contribuir para o estudo sobre o tempo de espera para radioterapia em Portugal. Para alcançar o objectivo proposto, optou-se pela metodologia usada por Drinkwater e Williams na re-auditoria efectuada no Reino Unido pelo Royal College of Radiologists, em 2007, sobre os tempos de espera para radioterapia. A grelha de observação elaborada foi baseada na grelha utilizada por Drinkwater e Williams, na revisão da literatura, e tendo em consideração a realidade portuguesa. Após a análise das respostas dos peritos, ao questionário de avaliação e adequação do instrumento à realidade portuguesa, parece existir concordância na adequação do instrumento, o que nos permite afirmar a possibilidade da aplicação do mesmo nos centros de radioterapia de Portugal. --- --------------------------------ABSTRACT - It is recognized the negative impact that radiotherapy waiting time have in tumour control and survival, as well the importance of establish maximum waiting times for the start of the treatment, in the sense to guarantee a good practice. The present investigation project aim is to build and validates a data collection tool, which pretends to apply in the future, in the sense to contribute for the study of the radiotherapy waiting time in Portugal. To accomplish the project aim, we chose the method used by Drinkwater and Williams in the re-audit performed in United Kingdom for the Royal College of Radiologists, in 2007, about the radiotherapy waiting time. The data collection tool built was based on the data collection tool used by Drinkwater and Williams, on the literature review and taking in account the Portuguese reality. After the analyse of the experts answers, it seems to exist agreement about the adequacy of the data collection tool, which allow us to claim the possibility of the tool application at radiotherapy centres, in Portugal.

Telomerase promoter mutations in cancer: an emerging molecular biomarker?

João Vinagre, Vasco Pinto and Ricardo Celestino contributed equally to the manuscript.

P53 and Cancer-Associated Sialylated Glycans Are Surrogate Markers of Cancerization of the Bladder Associated with Schistosoma haematobium Infection

BACKGROUND: Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers. METHODOLOGY/PRINCIPAL FINDINGS: Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium. CONCLUSION/SIGNIFICANCE: This preliminary study suggests that p53 and sialylated glycans are surrogate biomarkers of bladder cancerization associated with S. haematobium, highlighting a missing link between infection and cancer development. Eggs of S. haematobium express sLea and sLex antigens in mimicry of human leukocytes glycosylation, which may play a role in the colonization and disease dissemination. These observations may help the early identification of infected patients at a higher risk of developing bladder cancer and guide the future development of non-invasive diagnostic tests.

Implicações imunobiológicas da infecção a herpesvirus em tumores malignos : pesquisa e análise quantitativa de herpesvirus humanos no astrocitoma pilocítico

RESUMO: As infecções virais podem contribuir para o desenvolvimento do cancro, estando vários tumores malignos associados aos Herpesvirus (HHV). O vírus de Epstein-Barr (EBV) e o Herpesvirus 8, dois Herpesvirus, foram reconhecidos como agentes etiológicos de várias neoplasias. O astrocitoma pilocítico do cerebelo é um dos tumores cerebrais mais frequentes na criança, adolescentes e jovens adultos e a proliferação astrocitária ocorre geralmente após vários tipos de agressão, nomeadamente a infecção viral. Para investigar esta eventual interligação, estudámos 35 astrocitomas pilocíticos, pesquisando a presença dos 8 Herpesvirus. Neste estudo, foram utilizadas 10 amostras de biópsias do cerebelo de doentes que faleceram por doenças não relacionadas com infecção ou patologia tumoral. A maioria dos astrocitomas (33) eram tumores de baixa malignidade. As amostras foram analisadas por PCR (Polymerase Chain Reaction) quantitativa em tempo real (qPCR), com amplificação do gene da DNA polimerase viral. Treze astrocitomas e 7 controles revelaram pequenas quantidades de DNA viral (1-100 cópias/100ng DNA) de todos os Herpesvirus, com excepção do HHV6 A e B que estava ausente nas amostras. O EBV foi identificado em 9 dos 35 astrocitomas (26%) e em 7 dos 10 controles (70%) estando muito mais presente nos controles. As amostras positivas para o EBV foram também analisadas por imunohistoquímica, não tendo sido imunoreactivas para os anticorpos utilizados. A PCR com CODEHOP (consensus-degenerated hybrid oligonucleotide primers) foi utilizada para investigar a presença de um eventual Herpesvirus novo nestas amostras. Não foi identificada nenhuma sequência indicativa de um novo HHV por este método. 24. Em conclusão, os dados apontam para a presença de Herpesvirus, com particular relevância para o EBV, em tecido de cerebelo normal e em tumores cerebrais, embora em níveis demasiado baixos para poderem ser responsabilizados pela indução tumoral. A presença de sequências de DNA de Herpesvirus, nomeadamente do EBV, no Sistema Nervoso Central vem enriquecer a discussão sobre o significado da infecção viral na oncogénese humana, particularmente na neuro-oncogénese. ABSTRACT: Viral infections can contribute to the development of human cancer. Several human malignancies are linked with Human Herpesviruses (HHVs). Epstein-Barr virus and HHV8, two hHerpesvirus, have been recognized as etiologic agents of several neoplasms. Pilocytic astrocytoma of the cerebellum is one of the most common brain tumour in children, adolescents and young adults and astrocytary proliferation generally occurs after several types of injury, namely viral infection. To further explore this association, we have searched the tissue from 35 pilocytic astrocytoma, for all the 8 HHV. In this study, ten brain biopsies (cerebellum) from patients who died of unrelated diseases were used as controls. Most of the astrocytomas (33) were of low grade malignity. Samples were assessed by Real-time quantitative Polymerase Chain Reaction (q PCR) amplification of viral DNA polymerase gene. Thirteen astrocytoma and 7 controls showed low viral DNA levels (1-100 copies/100ng DNA) for all HHVs, with the exception of HHV6 that was absent. EBV was identified in 9 of the 35 astrocytoma (26 %) and in 7 of the 10 controls (70%) being more present in controls. EBV positive samples were also assessed by Immunohistochemistry (IHC) but none showed immunoreactivity for the antibodies used. PCR with consensus-degenerated hybrid oligonucleotide primers (CODEHOP) were also used to look for novel HHVs in these samples and no sequence indicative of a new HHV was detected. 26 Altogether the data indicate the presence of HHVs, with relevance for EBV in normal cerebellum tissue and also in brain tumours but at too low levels to be considered responsible for tumour induction. The presence of HHV DNA sequences, particularly EBV, in the studied brain tumours and control samples, further enriches the discussion about the relevance of viral infection in human oncogenesis, particularly neuro-oncogenesis.RÉSUMÉ: Les infections virales peuvent contribuer au développement du cancer. Les vírus de type Herpès sont associés à plusieurs néoplasies. Il est par exemple établi que les vírus Epstein-Barr et « human Herpesvirus 8 » (HHV-8) sont responsables de plusieurs tumeurs malignes. L´astrocytome pilocitique du cervelet est l’une des tumeurs les plus fréquentes chez les enfants, adolescents et adultes jeunes. En général la prolifération des astrocytes se produit en réponse à une agression. Posant l’hypothèse d’une agression d’origine virale, nous avons recherché la présence des 8 vírus Herpès dans les tissus de 35 astrocytomes. Dans cette étude, 10 échantillons de biopsie de cervelet de patients décédés suite à d’autres pathologies, ont été utilisés comme contrôles. La majorité des astrocytomes étaient de très basse malignité. Les échantillons ont été étudiés par PCR quantitative en temps réel, en amplifiant le gène de l’ADN-polymérase virale. Treize astrocytomes sur 35 (37%) et 7 contrôles sur 10 (70%) ont été trouvés positifs pour tous les HHV sauf l´HHV6, toujours avec un nombre de copies de polymérase virale bas (< 100 copies/100 ng d’ADN). Notamment l’EBV a été identifié 7 fois dans les contrôles (70%) et 9 fois dans les astrocytomes (26%). Les échantillons positifs pour l`EBV ont aussi été étudiés par immuno-histochimie. Aucun signal n’a été observé avec les anticorps utilisés. Enfin, une technique de PCR avec oligonucléotides dégénérés (CODEHOP ou consensus degenerated hybrid oligonucleotide primers) a été utilisée pour rechercher la présence d´un éventuel nouveau vírus Herpès dans les échantillons d’astrocytome. Aucun nouveau vírus n’a été identifié. 28 En résumé, nous avons établi la présence de vírus Herpès, en particulier l´EBV, dans le cervelet normal et dans les tumeurs du cerveau. Les quantités d’ADN viral retrouvées sont faibles et ne permettent pas d’attribuer à ces vírus la responsabilité de l’induction des tumeurs. Cependant, la présence d’ADN de vírus Herpès dans le cerveau sain ou pathologique vient enrichir la discussion sur le signification de l´infection virale dans les processus d´oncogenèse en général, et dans la neuroonco-genèse en particulier.

Immune reconstitution after autologous hematopoietic stem cell transplantation

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

Cell and humoral immunity in endemic pemphigus foliaceus

A study was conducted on 16 patients with pemphigus foliaceus, ten of them with the localized form (group G1) and six with the disseminated form (group G2). These patients were submitted to full blood counts, quantitation of mononuclear cell subpopulations by monoclonal antibodies, study of blastic lymphocyte transformation, and quantitation of circulating antibodies by the indirect immunofluorescence test, in order to correlate their clinical signs and symptoms and laboratory data with their immunological profile, and to determine the relationship between circulating autoantibody titers and lesion intensity and course of lesions under treatment. Leucocytosis was observed especially in group G2. All patients showed decreased relative CD3+ and CD4+ values and a tendency to decreased relative values of the CD8+ subpopulation. Blastic lymphocyte transformation indices in the presence of phytohemagglutinin were higher in patients (group G1+G2) than in controls. The indirect immunofluorescence test was positive in 100% of G2 patients and in 80% of G1 patients. The median value for the titers was higher in group G2 than in group G1. Analysis of the results as a whole permits us to conclude that cell immunity was preserved and that there was a relationship between antibody titers detected by the direct immunofluorescence test and extent of skin lesions.

Cyclophosphamide effect on paracoccidioidomycosis in the rat

Paracoccidioidomycosis is an endemic fungal disease widely distributed throughout Latin America. The potent immunosuppressor cyclophosphamide (CY) has been used to modulate host immune response to Paracoccidioides brasiliensis in an experimental model. Inbred male Buffalo/Sim rats weighing 250-300 g were inoculated with 5 x 10(6) P. brasiliensis cells of the yeast phase form by intracardiac route. One group of animals was treated with 20 mg/kg body weight at days +4, +5, +6, +7, +11 and +12 post-infection (pi.), while a control group was infected alone. No mortality was recorded in either group. Treated rats presented: a) a decrease in granuloma size, which contained less fungal cells; b) a lack of specific antibodies up to 35 days pi., and c) a significant increase in the footpad swelling test (DTH) against paracoccidioidin. Splenic cell transfer from CY-treated P. brasiliensis-infected donors to recipients infected alone led to a significant increase in DTH response in the latter versus untreated infected controls. Likewise, in treated infected recipients transferred with untreated infected donor spleen cells, footpad swelling proved greater than in controls. Thus, it would seem that each successive suppressor T lymphocyte subset belonging to the respective cascade may be sensitive to repeated CY doses administered up to 12 days pi.. Alternatively, such CY schedule may induce the appearance of a T cell population capable of amplifying DTH response.